The Cytokine Handbook, Fourth Edition, Two Volume Set

The Cytokine Handbook Fourth edition VOLUME I

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The Cytokine Handbook Fourth edition VOLUME I Edited by

Angus W. Thomson Starzl Transplantation Institute University of Pittsburgh School of Medicine, USA

Michael T. Lotze Molecular Medicine Institute University of Pittsburgh School of Medicine, USA

Amsterdam • Boston • London • New York • Oxford • Paris San Diego • San Francisco • Singapore • Sydney • Tokyo

This book is printed on acid-free paper Copyright © 2003 Elsevier Science Ltd except for Chapters 5 and 8 which are a US Government work in the public domain and not subject to copyright First edition published 1991 Second edition published 1994 Third edition published 1998 Fourth edition published 2003 All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Academic Press An Imprint of Elsevier Science 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com Academic Press An Imprint of Elsevier Science 525 B Street, Suite 1900 San Diego, California 92101–4495, USA http://www.academicpress.com ISBN 1–12–689663–1 Library of Congress Catalog Number: 2002114101

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British Library cataloguing in Public Data Vol.I Cytokines – Handbooks, manuals, etc. 2. Chemokines – Handbooks, manuals, etc. I. Thomson, Angus W. II. Lotze, Michael T. 611’.0185

Typeset by J&L Composition, Filey, North Yorkshire Printed and bound in Great Britain by The Bath Press, Avon

Contents

Preface to the First Edition Preface to the Second Edition Preface to the Third Edition Preface to the Fourth Edition Foreword Joost J. Oppenheim

xiii xv xvii xix xxiii

Volume 1 SECTION I. BASIC CYTOKINE BIOLOGY 1 2 3 4

The cytokines: an overview Jan Vilcˇek Cytokine genetics: Polymorphisms, functional variations and disease associations Grant Gallagher, Joyce Eskdale, Jeff L. Bidwell The phylogeny of cytokines Chris J. Secombes and Pete Kaiser Cytokine signaling Brian E. Szente

3 19 57 85

SECTION II. THE CYTOKINES AND CHEMOKINES Subsection A. The Hematopoietin Family Members 5 Growth hormone Lisbeth A. Welniak and William J. Murphy 6 Prolactin Hallgeir Rui and Marja T. Nevalainen 7 Erythropoietin Christoph Kasper

103 115 149

vi 8 9 10 11

12 13 14 15 16 17 18 19 20 21 22

CONTENTS

Interleukin-2 Jian-Xin Lin and Warren J. Leonard Interleukin-3 John W. Schrader Interleukin-4 Hideho Okada, Jacques Banchereau and Michael T. Lotze Interleukin-5 Chee Choy Kok, Gretchen T. Schwenger, Ron I.W. Osmond, Debra L. Urwin and Colin J. Sanderson Interleukin-6 Family Tadamitsu Kishimoto Interleukin-7 Peter K.E. Trinder, Markus J. Maeurer Interleukin-9 Jean-Christophe Renauld and Jacques Van Snick Interleukin-11 James C. Keith, Jr. Interleukin-12 Family: [IL-12, 23, 12RA and 27] Pawel Kalinski, Walter J. Storkus, Angus W. Thomson, and Michael T. Lotze Interleukin-13 Gabriele Grünig, Jan E. de Vries and Rene de Waal Malefyt Interleukin-15 and 21 Michael R. Shurin, Irina L. Tourkova, Holger Hackstein and Galina V. Shurin Interleukin-16 Kevin C. Wilson, David M. Center, William W. Cruikshank Interleukin-17 Family [IL-17, IL-25] Mary A. Antonysamy and Muneo Numasaki Granulocyte-macrophage colony-stimulating factor Thomas Enzler and Glenn Dranoff Granulocyte colony-stimulating factor Scott M. White and David J. Tweardy

Subsection B. The Interferon Family Members 23 Type I interferons [IFNa, b, d, j, x, s, IL-28A (IFNk2) IL-28B (IFNk3) and IL-29 (IFNk1)] Jeanne M. Soos and Brian E. Szente 24 Interferon-c Gregory H. Schreiber and Robert D. Schreiber 25 Interleukin-10 YaoZhong Ding, Shuang Fu, Dmitriy Zamarin, Jonathan Bromberg 26 The IL-10 Family [Interleukins -19, -20, -22, -24 and -26] Holger Hackstein, Grant Gallagher, Sergei Kotenko, and Angus W. Thomson Index

167 201 227 263

281 305 347 363 383 409 431 465 475 503 525

549 567 603 627

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CONTENTS

Volume II Subsection C. The Beta Trefoil Cytokines and Proinflammatory Signaling Receptors 27 Interleukin-1 Family [IL-1F1, F2] Charles A. Dinarello 28 Interleukin-1 receptor antagonist [IL-1F3] William P. Arend and Christopher H. Evans 29 Interleukin-18 [IL-1F4] Haruki Okamura, Michael T. Lotze, Hiroko Tsutsui, Shin-ichiro Kashiwamura, Haruyasu Ueda, Tomohiro Yoshimoto and Kenji Nakanishi 30 Interleukin-1 Family [F5 to F10] Sanjay Kumar 31 The fibroblast growth factors Barbara Ensoli, Cecilia Sgadari, Giovanni Barillari and Paolo Monini 32 Hepatocyte growth factor and its receptor, MET Wendy M. Mars, Youhua Liu and Satdarshan P. Singh Monga 33 Toll-like receptors Bruce Beutler Subsection D. The TNF Family, Receptors, and Related Molecules 34 Lymphotoxins Steve W. Granger and Carl F. Ware 35 Tumor necrosis factor Haichao Wang, Christopher J. Czura and Kevin J. Tracey 36 TRAIL/Apo2L David H. Lynch and Avi Ashkenazi 37 RANKL (Receptor activator of NFjB ligand) Simon Blake and Ian James 38 CD95L/FasL and its receptor CD95 (APO-1/Fas) Marcus E. Peter, Bryan C. Barnhart and Alicia Algeciras-Schimnich 39 HMGB-1 Haichao Wang, Christopher Czura and Kevin J. Tracey Subsection E. The Tyrosine Kinase Receptor Signaling Molecules 40 Macrophage Colony Stimulating Factor [CSF-1] Michael T. Lotze and John A. Hamilton 41 EGF family ligands David C. Lee, C. Leann Hinkle, Leslie F. Jackson, Shunqiang Li, and Susan Wohler Sunnarborg 42 FLT3 ligands Stewart D. Lyman and Hilary J. McKenna 43 Stem cell factor Ian K. McNiece and Robert A. Briddell 44 Vascular endothelial growth factor Dmitry I. Gabrilovich and Mikhail M. Dikov

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CONTENTS

Subsection F. The Chemokines and other Cell Migration Regulating Factors 45 Macrophage migration inhibitory factor (MIF) Douglas A. Arenberg, and Richard Bucala 46 Interleukin-8 and other CXC chemokines Naofumi Mukaida, Sergey A. Ketlinsky, and Kouji Matsushima 47 CC chemokines Alberto Mantovani, Massimo Locati, and Silvano Sozzani 48 Fractalkine Violetta Zujovic and Jeffrey K. Harrison Subsection G. The TGF Beta Family Members 49 Transforming growth factor b Philip H. Howe 50 Inhibin/activin family David J. Phillips 51 Bone morphogenic proteins A. Hari Reddi

SECTION III. CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION 52 53 54 55 56 57 58 59 60 61

Index

Autoimmunity and cytokines: pathogenesis and therapy Evangelos Th. Andreakos and Marc Feldmann Cytokines and cancer Megan A. Cooper and Michael A. Caligiuri Interferon a: biology, pharmacology, and therapy for chronic viral hepatitis Geoffrey M. Dusheiko Haemopoietic cytokines John L. Lewis and Myrtle Y. Gordon Antiangiogenesis and proangiogenic Andrew L. Feldman, Steven K. Libutti Cytokines and transplantation Geetha Chalasani, Fadi G. Lakkis Cytokines and asthma Timothy B. Oriss and Anuradha Ray Cytokine gene transfer Paul D. Robbins, Walter J. Storkus, and Andrea Gambotto Antisense therapy Stanley T. Crooke Assays for cytokines Theresa L. Whiteside

Dedications For Robyn, Andrew, Natalie and Emma Angus Thomson

For Joan, Thomas, Anna, Mac and Jenny Michael Lotze

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Editorial Meeting, Stockholm/International Immunology Congress, July 2001. Drs. Lotze (left), Read (center), and Thomson (right).

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Preface to the First Edition Cytokines feature at the forefront of biomedical research. An understanding of their properties is now essential for the immunology student, researcher and teacher and for today’s medical practitioner who needs to understand immunologic disease and immunological approaches to therapy. The pace with which this ever-expanding field has developed has been rapid enough to exceed the most optimistic expectations and to bewilder the most assiduous student. Cytokine research is expected to provide the key to pharmacological manipulation of the immune response and commands the attention of a massive and highly focused biotechnology industry. The chapters in this book are a good representation of the areas to which molecular biology has been most successfully applied. Biotechnology companies provide most of the pure, well-characterized cell growth regulatory and effector molecules used in academic and industrial laboratories or in clinical medicine as diagnostic tools or therapeutic agents. Cytokines represent a sought-after symposium theme in immunology, molecular biology and molecular genetics. The cytokine literature ranges from the most basic to the applied. Unfortunately, technical advances, rapid expansion and diversification have prompted narrower specialization and reduced the ease of communication. The aim of this book is to inform and to provide detailed information and reference material on the many aspects of pure and applied cytokine science. These include the molecular characteristics of cytokines, their genes and receptors, the cellular sources and targets of cytokines, their biological activities and as best can presently be defined, their mechanisms of action. Confronted with such a vast amount of new information, up-to-date coverage is an almost unattainable goal. The scope of cytokine research could only be effectively covered in a multi-authored volume and it is indeed fortunate that each chapter is written by a leading authority(ies). Although many chapters focus on individual cytokines, it is also apparent that aspects such as cell sources, molecular structure, purification and bioassay have many features in common. A certain amount of duplication is, therefore, inevitable. The cytokine network, cytokine interactions, the roles of cytokines in disease pathogenesis and the therapeutic applications of cytokine research are dealt with in detail. In attempting to provide comprehensive coverage, a chapter on phylogeny has been included. The last chapter was commissioned to provide both perspective and a somewhat sobering view of the future. I am indebted to the many authors around the world who have so generously devoted their knowledge, energy and time to the creation of this book. I also wish to acknowledge the support of Dr Susan King, and her staff at Academic Press in London, whose skill and energy were essential in the genesis of The Cytokine Handbook. Angus W. Thomson University of Pittsburgh

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Preface to the Second Edition In the Preface to the First Edition, it was stated that to produce a book that provided up-to-date coverage of all aspects of the cytokine field was an ‘almost unattainable goal’. Since the First Edition went to press in the spring of 1991, advancement both in our knowledge and understanding of the cytokine network has been predictably rapid, fully justifying the publishers’ faith in a Second Edition of The Cytokine Handbook within 3 years. The original chapters have been revised and updated, and, with few exceptions, this has been undertaken by the original authors. Synthesis of information in the context of the cytokine network has again been a key objective. Every effort has been made to maintain currency and it is in promptly fulfilling this goal that the contributing authors deserve great credit. ‘New’ cytokines have, of course, emerged and continue to ‘appear’. Thus, this new edition features individual chapters on IL-9, IL-10 and IL-12, with a ‘stop-press’ overview of IL-13 and even newer molecules that are candidates for designation as interleukins IL-14 and IL-15. What constitutes a new interleukin? The assignation of a new designation depends on several clearly defined criteria, recently established by a sub-committee of the nomenclature committee of the International Union of Immunological Societies (Paul et al., 1992). The criteria laid down include molecular cloning and expression, a unique nucleotide and inferred amino acid sequence, and the availability of a neutralizing monoclonal antibody. Furthermore, the granting of a new interleukin designation requires that the candidate molecule be a natural product of cells of the immune system (defined loosely as lymphocytes, monocytes and other leukocytes). The new interleukin must also mediate a potentially important function in immune responses and exhibit an additional function(s) so that a simple, functional name might not be adequate. Finally, these characteristic features should have been described in a peer-reviewed publication. This new edition incorporates separate chapters on G-CSF, M-CSF and GM-CSF, whereas in the first edition, only one chapter – on ‘colony stimulating factors’ – covered the properties of these molecules. A chapter is also now afforded to TGF-b, which exhibits both inhibitory and stimulatory effects on a variety of cell types and is a potent immunosuppressant. Another significant and substantial new chapter concerns ‘chemokines’. IL-8 (the ‘highest’ interleukin designation afforded a separate chapter in the first edition) was the first member of the chemokine family to be identified. More recently, other low molecular weight chemotactic polypeptides have been discovered that play a key role in cell activation and chemotaxis during the inflammatory response. These include RANTES (regulated upon activation, normal T expressed and secreted!), macrophage chemotactic and activating factor (MCAF), macrophage inflammatory protein (MIP)-la and MIP-1b. All share a conserved, four cysteine motif and can be further subclassified on the spacing of the conserved cysteine residues.

PREFACE TO THE SECOND EDITION

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Evaluation of the clinical potential of cytokines is one of the most exciting challenges of contemporary medicine. In addition, and in contradistinction to cytokine or cytokine gene therapy, the emergence of a new class of therapeutic agents, comprising soluble cytokine receptors (e.g. soluble (s) IL-IR, sTNFR, sIFN-cR), receptor antagonists (e.g. IL-1ra) and counter-regulatory cytokines, represents one of the most important developments in the cytokine field in recent years. Successes in the sequencing, cloning and expression of cytokine receptors has facilitated production and evaluation of their therapeutic utility in several acute and chronic cytokine-mediated diseases. This exciting and challenging development is one of the themes covered under the several chapters devoted to therapeutic aspects of cytokine biology. The last few years have seen the advent of new approaches to interrogating the roles of cytokines in vivo. Thus cytokine gene ‘knockout’ mice and mice transgenic for cytokine gene reporter constructs are likely to provide new knowledge about the in vivo role(s) of cytokines, especially in experimental autoimmune disorders, cancer or infectious diseases that may be difficult to mimic satisfactorily in vivo. The molecular genetics of cytokines (reviewed in Chapter 2) is yet another new and exciting aspect of this key field of contemporary molecular biology and medicine that has provided the impetus for the second edition of The Cytokine Handbook. Those who acquire knowledge by reading this book, or who are stimulated by the implications of the recent developments described herein, owe thanks to the many experts around the world who have so generously given of their valuable time, energy and expertise. The cordial and enthusiastic support of these scientists and clinicians has been an impelling influence that would be difficult to overrate. I am indebted to my colleague Dr Mike Lotze for constructive suggestions, Ms Shelly Conklin for valuable secretarial help and to Dr Tessa Picknett and her colleagues at Academic Press in London for their resolute support and guidance in ensuring that the notion of a second edition became a tangible reality. Angus W. Thomson

REFERENCE Paul, W.E., Kishimoto, T., Melchers, F. et al. (1992). Clin. Exp. Immunol. 88: 367.

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Preface to the Third Edition As the Third Edition goes to press, the stream of cytokine discovery flows unabated. We are confronted by description after description of novel cytokine-controlled systems of cell differentiation and proliferation and of the role of cytokines in immune regulation. In order to keep pace with these developments, this new edition of The Cytokine Handbook has been completely revised and updated. The contributors are largely the same body of devoted international authorities, with several notable new additions. Amongst the newcomers to the extensive panoply of cytokines covered in this edition are the recently designated interleukins (IL)-16 (formerly lymphocyte chemotactic factor), IL-17 (identified originally as cytotoxic T lymphocyte antigen-8) and IL-18 (interferon-c inducing factor). The daunting expansion of new information is perhaps better illustrated by the ‘explosion’ of chemokines. About sixty distinct human chemokine gene products have been identified to date. Regarded historically as regulators of leukocyte trafficking, chemokines have recently made a major impact in the area of infectious disease, including dramatic new understanding that chemokine receptors are at the center of HIV pathogenesis. Just two examples of chemokines that have recently come to prominence are eotaxin (the CCR-3 receptor-specific, eosinophil-selective chemokine) and the structurally unique fractalkine, with intrinsic adhesion and chemotactic activities. Two chapters are now devoted to the vastly expanding area of chemokines and their receptors. The Third Edition sees individual chapters afforded to IL-1 through IL-15, with the exception of IL-14, reflecting the current uncertainty about the identity of the IL-14 molecule. The TNF-related cytokine-receptor superfamily of secreted and membrane-bound ligands that includes TNF/lymphotoxin (a/b, CD40, Fas and 4–1BB systems, now appears to have important functional roles in the immune response. Each member is paired with a specific cell surface receptor(s) that, together, form a corresponding family of receptors. Recent studies have revealed important and unique roles of the TNF- related ligands and receptors that are covered in an additional new chapter. In addition to coverage of the colony-stimulating factors G-, M- and GM-CSF, two new chapters in this edition are afforded to individual hematopoietic cytokines – stem cell factor (SCF) (c-kit ligand) and flt-3 ligand – that have many biological functions in common. SCF is a potent costimulatory or synergistic factor in cytokine cocktails for manipulation of hematopoietic cells. It appears to be of value in the combination of ex vivo ‘expansion’ technologies with gene transfer methods for the correction of genetic disease or the support of multiple chemotherapy cancer patients. The recently cloned cytokine flt-3 ligand has been shown to dramatically increase the numbers of functional dendritic cells and also to increase natural killer cells in vivo. Moreover it exerts anti-tumor activity, raising expectation of a future role of this molecule as a human immunotherapeutic agent.

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Therapeutic applications of cytokines are now covered in detail in several chapters, including a new chapter on cytokine gene therapy. The first therapeutic cytokine gene transfer study began several years ago in patients with advanced malignancy; now numerous trials have been approved for the transfer of cytokine genes to tumor cells, tumor-infiltrating lymphocytes, blood lymphocytes, or fibroblasts. A few studies involve the use of cytokine genes for the treatment of non-malignant conditions, such as rheumatoid arthritis. A new chapter in this edition reviews progress in cytokine gene therapy. As in the two previous editions, valuable up-to-date practical information is included in the extensive account of cytokine assays. The phylogeny of cytokines has become such a growth area that the information can barely be contained in a single chapter. I am again immensely indebted to the many experts around the world who have made this new edition possible. On the home front, my colleague Dr Mike Lotze has been a constant source of creative suggestions and lively discussion. My thanks are due once again to Dr Tessa Picknett, senior editor, and also to Mr Duncan Fatz and Ms Emma White at Academic Press in London, and to Ms Shelly Conklin for invaluable secretarial assistance. Angus W. Thomson

Preface to the Fourth Edition It is also said that Sisyphus, being near to death, rashly wanted to test his wife’s love. He ordered her to cast his unburied body into the middle of the public square. Sisyphus woke up in the underworld. And there, annoyed by an obedience so contrary to human love, he obtained from Pluto permission to return to earth in order to chastise his wife. But when he had seen again the face of this world, enjoyed water and sun, warm stones and the sea, he no longer wanted to go back to the infernal darkness. Recalls, signs of anger, warnings were of no avail. Many years more he lived facing the curve of the gulf, the sparkling sea, and the smiles of earth. A decree of the gods was necessary. Mercury came and seized the impudent man by the collar and, snatching him from his joys, led him forcibly back to the underworld, where his rock was ready for him. The Myth of Sisyphus, Albert Camus; 1940.

Camus was awarded the Nobel Prize for Literature in 1957 for his existentialist contributions to finding order and meaning for man living in a world absurd and, at the time he wrote it, wracked by war. The notion of a mortal committed to daily striving to move his rock uphill serves as a suitable metaphor for cytokines, literally cell movers. At some level it also mirrors the profound labors of our contributors who, in each edition, find the energy to move their chapters a bit closer to the apogee of fuller understanding and bounded exposition. If this work is at all successful, it is because of them and their professorial stance to organizing what is becoming almost an impossibly large set of contributions to the literature. Astute observers will find this fourth edition substantially altered, now in two volumes, organized around cytokine families, many of them revealed by the availability of new sequence data arising from the human genome project. It also introduces quotes chosen in most part by the author of each chapter and denoting some of the personal affectations of each contributor. It has striven to be complete and still readable allowing access to the considerable literature now available for many of these factors which, Mercury-like, bring and move cells to their appointed fate. The final section knits together the individual cytokines into the clinical implications and, in some instances, applications of cytokines. The hope is that some of them might find their way singly, or most often, in pairs, into useful recombinant pharmaceutical agents. Since the last edition, interleukin 1 receptor antagonist (IL-1RA) has

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been approved for the therapy of rheumatoid arthritis (Schiff, 2000). The successful application of TNF antagonists in chronic inflammatory conditions continues to expand with what we suspect will be an increasing number of indications moving beyond arthritis (Criscione and St Clair, 2002) and inflammatory bowel disease to other difficult clinical problems. Up to the moment of press, we have been cognizant of new molecules and new insights and have incorporated these with assistance and support of our editor up to the recently identified IL-28 and IL-29! (Kotenko et al., 2002; Sheppard et al., 2002). Some of the major advances captured in this edition beyond those made possible by the availability of the full human and mouse genome represent the increased power of new technology. These include application of quantitative polymerase chain reaction (PCR) assessment of cytokines (Walker, 1998; Gulietti et al., 2001; Rajeevan et al., 2001), availability of measurement of multiple cytokines in ELISpot assays (Bennouna et al., 2002; Leong et al., 2002; Meidenbauer et al., 2002; Scheibenbogen et al., 2002; Shmitz et al., 2002) as well as microarrays (Benson et al., 2002; Cappellen et al., 2002) allowing careful evaluation of the downstream messages induced by cytokines and chemokines. The proteomic analysis and patterning of individual cytokines in vitro and in vivo is less fully developed (Petricoin et al., 2002; Schweitzer et al., 2002) but by the time of the fifth edition is expected to allow greater analysis of the complexity of cell communication mediated by cytokines. Also the deeper understanding of individual cells or cell mixtures has been made possible by the development of so-called high content screening using integrated high resolution fluorescence microscopy, data algorithm development and display and automation (Ghosh et al., 2000; Taylor et al., 2001). We anticipate that studying cells interacting with each other will provide a wealth of additional information, as yet only modestly pursued in the available literature. We would like to dedicate this edition of The Cytokine Handbook to our colleagues, the scientists laboring within academe and the biotechnology/pharmaceutical industries. Like Sisyphus and his spouse, they have to work long hours, often together, to move their chosen cytokine or chemokine closer to a fuller understanding and ultimate utility in discerning the biology writ large by their production, in full display within the public square. It is our expectation that within the bounds of creating new knowledge and creating value, that application of this knowledge might improve human health and the common weal. Finally to Shelly Conklin who organized our manuscripts dutifully as they each arrived, to Bridget Colvin for her scholarly contribution to the quotes, and to Jacqueline Read who together with Tessa Picknett as our editors, made the task of developing this edition more joyful than if it were in less able hands, we offer our humble appreciation and thanks. Michael T. Lotze Angus W. Thomson

REFERENCES Bennouna, J., Hildesheim, A., Chikamatsu, K. et al. (2002) Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses. J. Immunol. Methods 261(1–2): 145–156. Benson, M., Svensson, P.A., Carlsson, B. et al. (2002) DNA microarrays to study gene expression in allergic airways. Clin. Exp. Allergy 32(2): 301–308. Cappellen, D., Luong-Nguyen, N.H., Bongiovanni, S. et al. (2002) Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony-stimulating factor and the ligand for the receptor activator of NFkB. J. Biol. Chem. 277: 21971–21982. Criscione, L.G. and St Clair, E.W. (2002) Tumor necrosis factor-alpha antagonists for the treatment of rheumatic diseases. Curr. Opin. Rheumatol. 14(3): 204–211. Ghosh, R.N., Chen, Y.T., DeBiasio, R. et al. (2000) Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking. Biotechniques 29(1): 170–175.

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Gulietti, A., Overbergh, L., Valckx, D. et al. (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25(4): 386–401. Kotenko, S.V., Gallagher, G., Baurin, V.V. et al. (203) IFN- mediate antiviral protection through a distinct class II cytokine receptor complex. Nature Immunology 4, 69–77. Leong, S.P., Peng, M., Zhou, Y.M. et al. (2002) Cytokine profiles of sentinel lymph nodes draining the primary melanoma. Ann. Surg. Oncol. 9(1): 82–87. Meidenbauer, N., Gooding, W., Spitler, L. et al. (2002) Recovery of zeta-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer. Br. J. Cancer 86(2): 168–178. Petricoin, E.F., Ardekani, A.M., Hitt, B.A. et al. (2002) Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359(9306): 572–577. Rajeevan, M.S., Ranamukhaarachchi, D.G., Vernon, S.D. and Unger, E.R. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25(4): 443–451. Scheibenbogen, C., Sun, Y., Keilholz, U. et al. (2002) Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL-2-based treatment. Int. J. Cancer 98(3): 409–414. Schiff, M.H. (2000) Role of interleukin 1 and interleukin 1 receptor antagonist in the mediation of rheumatoid arthritis. Ann. Rheum. Dis. 59(Suppl 1): i103–i108. Schweitzer, B., Roberts, S., Grimwade, B. et al. (2002) Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20(4): 359–365. Sheppard, P., Kindsvogel, W., Xu, W. et al. (2003) IL-28, IL-29 and their class II cytokine receptor IL-29R. Nature Immunology 4, 63–68. Shmitz, M., Rohayem, J., Paul, R. et al. (2002) Quantification of antigen-reactive T cells by a modified ELISPOT assay based on freshly isolated blood dendritic cells. J. Clin. Lab. Anal. 16(1): 30–36. Taylor, D.L., Woo, E.S. and Giuliano, K.A. (2001) Real-time molecular and cellular analysis: the new frontier of drug discovery. Curr. Opin. Biotechnol. 12(1): 75–81. Walker, K.B. (1998) Detection and analysis of cytokine mRNA in tissues and cell lines. J. Immunol. Methods 212(1): 113–123.

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Foreword to the Fourth Edition What are cytokines? As so aptly discussed by Jan Vilcek in the introductory chapter to this book, The Cytokine Handbook, cytokines are regulatory peptides that can be produced by every nucleated cell type in the body. Cytokines have pleiotropic regulatory effects on haematopoietic and many other cell types that participate in host defense and repair processes. Cytokines therefore include lymphocyte-derived factors known as ‘lymphokines’, monocyte-derived factors called ‘monokines’, haematopoietic ‘colony stimulating factors’, connective tissue ‘growth factors’, and chemotactic chemokines. Why do cytokines exist? The evolution of large multicellular organisms require the development of intercellular messengers such as hormones, neuropeptides and cytokines to permit marshalling of co-ordinated cellular responses. It has been proposed that the structural homology between adhesion proteins that mediate cell-contact-dependent interactions and cytokine ligands, suggests that soluble cytokines evolved from cell-associated signals (Grumet et al., 1991). In fact, a number of cytokines and their receptors exist in both soluble (shed) and cell-associated form and can have different functional consequences in each state. How are cytokines different from hormones? Endocrine hormones, which are generally produced by specialized glands, are present in the circulation and serve to maintain homeostasis. In contrast, most cytokines usually act over short distances as autocrine or paracrine intercellular signals in local tissues and – with the exception of macrophage colony-stimulating factor (M-CSF), stem cell factor, erythropoietin and a latent form of the transforming growth factor b (TGFb) – only occasionally spill over into the circulation and initiate systemic reactions. Except for the above, cytokines generally are not produced constitutively, but are generated in response to danger signals to contend with challenges to the integrity of the host. The functions of cytokines are distinct from those of hormones, since they serve to maintain homeostasis by regulating innate host defense and the immune system, through damage control and by promotion of reparative processes. How did lymphokines come to be discovered? The possibility that cell-derived factors mediate biological activities was first suggested by experiments carried out by Rich and Lewis (1932). They observed that migration of neutrophils and macrophages in

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cultures of tuberculin-sensitized tissues was inhibited by antigen. Waksman and Matoltsy (1958) observed that macrophages in monolayer cultures were actually stimulated rather than damaged by exposure to tuberculin antigens. George and Vaughan (1962) improved the technique of evaluating migration of mononuclear cells using capillary tubes. The study of ‘lymphokines’ was initiated concurrently by David et al. (1964) and Bloom and Bennet (1966). They used this technique to show that antigens could stimulate sensitized lymphocytes in cultures to produce macrophage migration inhibitory factors (MIF). At about the same time, supernatants of mixed leukocyte cultures were found by Kasakura and Lowenstein (1965) to be ‘blastogenic’ for lymphocytes. This ‘blastogenic factor’ (BF) was subsequently called ‘lymphocyte mitogenic factor’ (LMF). This was followed by the discovery of a variety of lymphocyte-derived biological activities in culture supernatants. Ruddle and Waksman as well as Kolb and Granger described a cytotoxic lymphocyte-derived mediator called ‘lymphotoxin’ (LT) in 1968. In vitro monocyte migration in response to supernatants of antigen activated lymphocytes was attributed to a lymphocyte-derived chemotactic factor (LCF) (Ward et al., 1969) and was correlated with the recruitment of mononuclear cells to in vivo inflammatory sites. MIF and the macrophage aggregation factor (MAgF) (Lolekha et al., 1970) presumably served to retain cells at inflammatory sites. The necrotic centers of some granulomas could be attributed to the cytodestructive activity of LT (Kolb and Granger, 1968; Ruddle and Waksman, 1968), and the presence of lymphoblasts and frequent mitotic figures was mediated by LMF (Kasakura and Lowenstein, 1965). Moreover, identification of macrophage-activating factor (MAF) (Nathan et al., 1971) and lymphocyte-derived immune interferon (IFN-c) (Green et al., 1969) provided a biological basis for acquired resistance to infectious organisms. Consequently, the various biological activities secreted by cultured antigen-stimulated lymphocytes provided in vitro models for the pathogenesis of in vivo delayed hypersensitivity reactions. These biochemically undefined, lymphocyte-derived activities were termed ‘lymphokines’ in 1969 by Dumonde et al. Discovery of the lymphokines revolutionized the conceptual basis of cell-mediated immunity and these biological activities were considered ‘in vitro correlates’ of cell-mediated immunity. What led to the recognition that lymphokines were members of the cytokine family? In 1971–1974 Gery and coworkers showed that the lymphocyte-activating factor (LAF) was produced by adherent monocytes and macrophages (Gershon and Kondo, 1971; Gery et al., 1971; Gershon et al., 1974). This was the first demonstration of the existence of non-lymphocyte-derived ‘monokines’. Based on this information and on his own observations that some replicating non-lymphoid cell lines, as well as virally infected non-lymphoid cells, could also produce lymphokine-like MIF and chemotactic factors, Cohen proposed that all these mediators including lymphokines should therefore be called ‘cytokines’ (Cohen et al., 1974). This resulted in the conceptual transformation of lymphokines from subjects of interest to a minor subset of immunologists, to cytokines that function as bidirectional intercellular signals between somatic and myeloid as well as lymphoid cells, with potential impact on a great number of biological disciplines. What developments galvanized the study of cytokines? The development of tissue culture techniques in the 1960s enabled immunologists to detect the presence of factors in tissue culture supernatants and enabled them to perform in vitro studies of the mobility, proliferation, differentiation and functional capabilities of lymphocytes and other leukocytes. Cytokines are very potent and active at pM to nM concentrations. Thus, they are active at only trace levels. This makes it particularly difficult to isolate and identify the biochemical structure of these peptides. These factors, which were originally disparagingly termed ‘lymphodrek’, could be purified only with the development of improved chromatography and microsequencing techniques in the late 1970s. The fortuitous development of molecular biology and monoclonal antibody technologies accel-

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erated the identification of cytokines in the 1980s ensuring the availability of abundant quantities of recombinant cytokines over the past decade. This has resulted in an information explosion that is reflected by The Cytokine Handbook. What was the origin of the interleukin terminology? By 1978, the confusing plethora of eponyms in existence for monocyte and lymphocyte-derived activities motivated investigators at the Second International Lymphokine Workshop – held near Interlaken, Switzerland – to propose more inclusive ‘neutral’ terms for these biological activities. The researchers recognized that the numerous monokine and lymphokine activities which they were detecting, along with a variety of bioassays, actually had numerous properties in common. This resulted in the erroneous impression that these cytokine activities, each with their own name, could all be attributable to one or two molecular entities. Paetkau proposed that monocyte-derived LAF/BAF/MCF be renamed interleukin-1 (IL-1), while lymphocyte-derived LMF/BF/TCGF should be called IL-2 (Mizel and Farrar, 1979). The interleukin terminology symbolized the broader roles of these cytokines and, with the progressive increase in the number of interleukins, now up to 27, has led to an explosive increase in the interest of investigators from a variety of disciplines in these mediators of inflammation and immunity. Why is the cytokine nomenclature so chaotic? Of the more than 200 cytokines that have been cloned to date, many retain their initial names, which usually denote only the functional activity that led to their discovery. Chaotic as this may be, such names are easier to recall than an interminable number of interleukins or chemokines. A minority of investigators request an interleukin designation to focus greater attention on their discovery. To qualify as an interleukin, the cytokine must be documented to have a unique amino acid sequence and functional activity involving leukocytes. The evidence is evaluated by the nomenclature and standardization committee of the International Cytokine Society and the Union of Immunological Societies who make a recommendation to the World Health Organization. Despite this rather straightforward process, committees have had to resolve conflicts concerning simultaneous claims to the IL-4 number and even a fraudulent claim (e.g. IL-4a). More recently, an error in the nucleotide sequence of the precursor region of IL-16 led to a re-evaluation of the IL-16 designation. Fortunately, the error did not involve the region of the gene coding for the mature protein and the translated amino acid sequence proved to be correct and exhibited the proposed functions of IL-16. In contrast, the nucleotide and consequent amino acid sequence of IL-14 have proven to be incorrect. Unfortunately, the correct protein sequence of IL-14, if any, remains unknown. It is therefore unclear whether IL-14 exists; hence its omission from this Handbook. Perhaps such errors can be prevented by requiring that novel cytokine sequences be independently confirmed to be eligible for an interleukin designation. Structurally distinct interleukins that use the same receptor have been given a number followed by a Greek letter, as for example IL-1a and IL-1b. Is there any order to the cytokine chaos? Despite the identification of many structurally distinct cytokines, they can be organized into groups that exhibit functional similarities based on shared receptor utilization. This holds for IL-1a and IL-1b as well as TNFa and LT which use shared and unshared receptors. Receptor chains are shared by a number of the cytokine groups. The IL-2c receptor chain is shared by IL-2, 4, 7, 9, 15 and 21. The gp130 chain of the IL-6 receptor is shared by IL-6, IL-11, LIF, oncostatin-M, CNTF and cardiotrophin-1. Members of the TNF family of ligands share receptors with homology to the TNF receptors. This includes TNFa, LTa, LTb complex, Fas ligand, CD70, CD40 ligand, CD30 ligand, nerve growth factor and more. A receptor b chain is shared by IL-3, IL-5 and GM-CSF. Homologous G-protein-coupled receptors are used by IL-8 and the chemokine family. The receptors for IFNa, b, x, c and IL-10 also

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show homology, as do those for the TGFb family of cytokines. Since IL-12 and IL-18 have overlapping activities, their receptors also may be related. These observations permit a rational organization of cytokines into families. The advent of additional information concerning the relationship and location of cytokine genes, shared signal transduction pathways and the tertiary structure of cytokines may enable us to further classify newly discovered cytokines. Although we have always presumed that the most important cytokines and receptors have been discovered, we are repeatedly surprised by identification not only of novel cytokines and receptors, but whole families. Undoubtedly many more cytokines will be identified now that the genome has been mapped. Why are cytokines important? Recombinant cytokines provide useful laboratory probes for studying the cell biology of innate and adaptive immunity and inflammation. Cytokines are the major orchestrators of host defense processes and, as such, are involved in responses to exogenous and endogenous insults, repair and restoration of homeostasis. Microbial pathogens have operated on these principles far longer than immunologists and have been shown to produce variants of proinflammatory cytokines, their receptors and chemokine antagonists that subvert and suppress the host immune and inflammatory defenses. Deletion of these products reduces the pathogenicity of these viruses. In addition to their role in host defense, cytokines appear to play a major role in development and some of them may account for as yet unidentified embryonic inductive factors. The study of cytokines is also elucidating the mechanisms underlying pathophysiological processes. Cytokines mediate not only host responses to invading organisms, tumors and trauma, but also maintain our capacity for daily survival in our germ-laden environment. In fact, the development of more sensitive methods of detection is revealing the presence of detectable, but low levels of cytokines associated with a variety of binding proteins in the serum. This probably reflects the production of cytokines in response to the many nonpathogenic stimulants present in our conventional environment. Detection of cytokines in disease states may provide useful diagnostic tools. The therapeutic administration of pharmacological doses of cytokines or at times cytokine gene therapy is being used in a wide variety of infectious diseases and in immunocompromised patients with genetic defects, AIDS, autoimmune diseases and neoplasias. This has led to the development of numerous biotechnology firms which are also evaluating antagonists of cytokines for their anti-inflammatory effects. Why a Cytokine Handbook? Studies of cytokines have drawn scientists from a multiplicity of fields including immunologists, hematologists, molecular biologists, neurobiologists, cell biologists, biochemists, physiologists, and others. Consequently, the burgeoning field of cytokine research is unique and interdisciplinary. The chapters in this Handbook cover the structure and functions of cytokines, their genes, receptors, mechanisms of signal transduction and clinical applications. This fourth edition of The Cytokine Handbook is required, at this relatively early date, to keep up with rapid developments in these dynamic disciplines. All the chapters, and even this foreword, have been updated and numerous new chapters by internationally renowned experts have been added including the new Interleukins, 19–27. The new edition has been greatly reorganized with the addition of chapters to cover the more general topics of molecular genetics, signal transduction and common structural features of cytokines and their receptors. The cytokine chapters have been grouped based on the nature of their receptors. All the hematopoietin family members including growth hormone, prolactin, erythropoietin, ciliary neurotrophic factor and nerve growth factor as well as all of the interleukins are now included. There are now separate sections with multiple chapters on the interferon family, IL-1 family, TNF family, growth factors, the chemokine family and transforming growth factor family. Finally, there is an expanded section on the therapeutic

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applications of cytokines and cytokine inhibitors. Thus, this fourth edition has been considerably expanded based on progress in cytokine research and on the fact that many additional intercellular mediators should be considered cytokines and included here. Consequently, The Cytokine Handbook provides us with the opportunity to keep up with the rapidly evolving studies of cytokines. Acknowledgements I am grateful for the critical input of Drs R. Neta, M. Grimm and S. Durum and the secretarial assistance of Ms Cheryl Fogle. Joost J. Oppenheim Laboratory of Molecular Immunoregulation National Cancer Institute Maryland, USA

REFERENCES Bloom, B.R. and Bennet, B. (1966). Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 153: 80–82. Cohen, S., Bigazzi, P.E. and Yoshida, T. (1974). Similarities of T cell function in cell-mediated immunity and antibody production. Cell. Immunol. 12: 150–159. David, J.R., Al-Askari, S., Lawrence, H.S. and Thomas, L. (1964). Delayed hypersensitivity In vitro. J. Immunol. 93: 264–273. Dumonde, D.C., Wolstencroft, R.A., Panayi, G.S. et al. (1969). Lymphokines: non-antibody mediators of cellular immunity generated by lymphocyte activation. Nature 224: 38–42. George, M. and Vaughan, J.H. (1962). In vitro cell migration as a model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111: 514–521. Gershon, R.K. and Kondo, K. (1971). Infectious immunological tolerance. Immunology 21: 903–914. Gershon, R.K., Gery, I. and Waksman, B.H. (1974). Suppressive effects of in vivo immunization on PHA responses in vitro. J. Immunol. 112: 2215–221. Gery, I., Gershon, R.K., and Waksman, B.H. (1971). Potentiation of cultured mouse thymocyte responses by factors released by peripheral leucocoytes. J. Immunol. 107: 1778–1780. Green, J.A., Cooperland, S.R. and Kibnick, S. (1969). Immune specific induction of interferon production in cultures of human blood lymphocytes. Science 164: 1415–1417. Grumet, M., Mauro, V., Burgoon, M.P. et al. (1991). Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules. J. Cell. Biol. 113: 1399–1412. Kasakura, S. and Lowenstein, L. (1965). A factor stimulating DNA synthesis derived from the medium of leucocoyte cultures. Nature 208: 794–798. Kolb, W.P. and Granger, G.A. (1968). Lymphocyte in vitro cytotoxicity: Characterization of human lymphotoxin. Proc. Natl. Acad. Sci. USA 61: 1250–1255. Lolekha, S., Dray, S. and Gotoff, S.P. (1970). Macrophage aggregation in vitro: A correlate of delayed hypersensitivity. J. Immunol. 104: 296–304. Mizel, S.B. and Farrar, J.F. (1979). Revised nomenclature for antigen non-specific T cell proliferation and helper factors. Cell. Immunol. 48: 433–436. Nathan, C.F., Karnovsky, M.L. and David, J.R. (1971). Alterations of macrophage functions by mediators from lymphocytes. J. Exp. Med. 133: 1356–1376. Rich, A.R. and Lewis, M.R. (1932). Nature of allergy in tuberculosis as revealed by tissue culture studies. Bull. Johns Hopkins Hosp. 50: 115–131. Ruddle, N.H. and Waksman, B.H. (1968). Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. J. Exp. Med. 128: 1267–1279. Waksman, B.H. and Matoltsy, M. (1958). The effect of tuberculin on peritoneal exudate cells of sensitized guinea pigs in surviving cell culture. J. Immunol. 81: 220–234. Ward, P.A., Remold, H.G. and David, J.R. (1969). Leukotactic factor produced by sensitized lymphocytes. Science 163: 1079–1081

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INDEX

(Note: page numbers in italics refer to figures and tables, those in bold refer to main discussions of topics. ‘Plate’ refers to illustrations in Plate Section) A-SMase, 845–6 acromegaly, 106 activation-induced cell death (AICD) lymphocytes, 719, 890, 891, Plate 38.1 multiple sclerosis, 894 activin, 1123, 1153–69 angiogenesis, 1159 anti-inflammatory actions, 1165, 1166–7 apoptosis, 1159–61, 1164, 1166 binding proteins, 1155–7 biologic activity, 1155 branching morphogenesis, 1162–3 cancer, 1161 cell proliferation, 1159, 1161, 1166 cyclin/CDK pathway, 1160 differentiation, 1161–2 fibrosis, 1163 follistatin, 1159 functions, 1158 genes, 1154 hematopoiesis, 1164–5 hepatocyte growth factor, 1159, 1163 IL-1b, 1166–7 IL-6 function blocking, 1166 immune system, 1161–8 inflammation, 1165, 1166–7 clinical syndromes, 1167–8 interactions with signaling pathways, 1159

lymphocytes, 1165 MAPK pathway, 1159 molecular characterization, 1154–5 p53 expression, 1160 proinflammatory actions, 1165–6 receptors, 1157–8 type I, 1157 type II, 1157 signaling, 1156, 1157, 1158 intracellular components, 1158–9 TGFb cross-regulation, 1159 Smads, 1158–9, 1163, 1164 TGFb synergism, 1163 wound healing, 1163–4 activin receptor interacting protein 1 (ARIP1), 1159 acute phase response, IL-6, 284–5 Ad-5, 1338 ADAM, 970–1 ADAM17 see TNF-a-converting enzyme (TACE) adaptor proteins, CSF-1, 935 ADAR family, 555, 1236, 1238 adeno-associated virus (AAV), 1340 adenosine deaminase, 555 RNA dependent (ADAR1), 555, 1236, 1238 adenoviruses, 1338–9 adrenal adenoma, cortisol-secreting, 661

adrenal gland, 713 adrenocorticotrophic hormone (ACTH), 285, 1037 adult respiratory distress syndrome (ARDS) CXC chemokines, 1067 IL-1Ra, 681 MIF, 1041–2 TNF, 848 agranulocytosis, drug-induced, 510 AIDS, progression to, 1376 airway diseases eosinophilia, 418, 1320 IL-17, 495–6 see also adult respiratory distress syndrome (ARDS); asthma AK-155 see IL-26 alcohol abuse, IL-17, 496 ALK1, 1122 allergic disease contact dermatitis IL-1Ra, 681 IL-17, 497 IL-4 animal models, 245–6 IL-4Ra gene variants, 234 IL-5, IL-4 and IL-13 coexpression, 265 IL-12 therapy, 398 IL-15, 444 lung and IL-13, 417, 418 MI-L, 486 allograft acceptance, 1301–5

xxx allograft acceptance (continued) gene transfer, 1343 allograft rejection, 489–90, 1297 acute, 1297–9 human studies, 1305 CD95L, 892 chronic, 1299–301 human studies, 1306 gene expression studies, 1306 gene polymorphism studies, 1306–7 MIF in renal transplant, 1042 Alzheimer’s disease, IL-1Ra, 680 amino acid, 550 amniotic fluid, IL-1Ra, 678 cAMP, 937 amphibians, 57 chemokines, 68 FGF, 75 IL-1, 62 IL-2, 64 interferons, 58 TGFb, 74 amphiregulin (AR), 959, 960, 967–9 receptors, 968–9 amphoterocin see HMGB1 Anakinra, 691–2 ancestin, 1014 anemia aplastic and GM-CSF, 511, 513 of chronic disease (ACD), 157–8 Flt3, 996–7 hemolytic, 1191 angiogenesis, 1279–90 activin, 1159 anti-TNFa therapy, 1199 biology, 1280–2 bone formation, 1179 cancer, 1281 cardiovascular disease, 1282 CXC chemokine inhibition, 1063 FGFs, 755, 757–8, 758, 761 fracture healing, 1281 HGF, 791 IL-8, 1057 IL-10, 610–11 IL-12, 393 IL-15, 446 IL-18 inhibition, 721–2 inflammatory response, 1281 inhibitors, 1220 endogenous, 1281, 1282, 1283, 1284, 1285, 1286–8 synthetic, 1285, 1288–9 ocular disease, 1282 pathologic situations, 1280 physiologic, 1280–1 reproduction, 1280–1

INDEX

rheumatoid arthritis, 1282 tissue repair, 1281 tumor growth, 1220 VEGFs, 1017, 1019, 1024–5 wound healing, 1281 see also antiangiogenesis angiopoietin-2, 1281 angioproliferative disease, 1066 angiostatin, 1220, 1282, 1283, 1284, 1286 ankylosing spondylitis, 1192 antagonism, xxv, 12–13 anti-CD2, 1303 anti-CD3, 169, 170, 1303 anti-CD20 antibody, 1226 anti-cytokine antibodies, 1387 anti-cytokine therapy autoimmune disease, 1200 cancer, 1222–3 anti-IL-5 antibody, 1327 anti-IL-6 receptor antibody, 294 anti-inflammatory therapy in autoimmune disease, 1200–1 anti-TNF therapy, 852 anti-TNFa therapy Crohn’s disease, 1199, 1201 mechanism of action, 1199 methotrexate combination, 1197, 1198, 1199, 1201 psoriasis, 1199–200 rheumatoid arthritis, 1197, 1198, 1199, 1201, 1202, Plate 52.6 spondylarthropathy, 1199 anti-VEGF antibody, 1285, 1289–90 antiangiogenesis, 244, 1279–91 cancer, 1281 gene therapy, 1290 antiangiogenic agents, clinical trials, 1285, 1287–90 antibodies anti-cytokine, 1387 ELISA, 1382, Plate 61.2 immunoassay, 1383 immunohistochemistry, 1386, Plate 61.4 TAG-labeled, 1385 antibody-dependent T cell cytotoxicity, 248 antigen-induced cell death (AICD), 886–7 antigen presenting cells (APC) IFN-c, 582–3 IL-12, 384, 385, 386, 393 IL-12RA, 387 IL-15, 433 lung, 1324 antigens in autoimmunity, 1192

antisense therapy, 1349–68 drugs molecular mechanisms, 1350–3 splicing inhibition, 1352–3 antiviral activity, 551 AP-1, 827, 839, 840 CD95L, 887 IL-8, 1057 AP-2, 839 Apo2-ligand (Apo2L), 861, 862 apoptosis, 10, 861 activin, 1159–61, 1164, 1166 B cell protection by IL-4, 238 CD95-induced, 891–2, 893 CD95 signaling, 896, 897–8, 899, 900, Plate 38.2 CD95L, 890, 892, Plate 38.1 defective, 1192 FGF regulation, 765 hepatocytes, 865 HIV infection, 893–4 IFN-c, 586 IFN type I regulated, 556 IL-3, 215 IL-7 protection of DTEC, 321 IL-7Ra inhibition, 313 IL-15 inhibition, 442 induction, 12 keratinocytes, 444, 445, 447 multiple myeloma, 449 multiple sclerosis, 895 suppression, 215 T cells, 447 TGFb-mediated, 1135, 1136–40, Plate 49.6 prostate epithelium, 1137–8 thymocytes, 891 TNFa, 1215, Plate 53.2 TRAIL/Apo2L, 864, 865–6 apoptotic activating factor-1 (Apaf-1), 1138 N-arginine dibasic convertase (NRDc), 971 arresten, 1286 arthritis gene therapy, 693–4, 696, 698, 1343 IL-1Ra, 679–80, 687–8, 691–2, 698 gene therapy, 693–4, 696, 698 IL-18, 722 see also osteoarthritis; rheumatoid arthritis ARTS protein, 1137 Ask1/MKK6/p38, 889 assays for cytokines, 1375–92 bioassays, 1377, 1378, 1379–81 cytokine transcript detection, 1387–8

INDEX

ELISA, 1378, 1382–3, Plate 61.2 ELISPOT frequency analysis, 1388–9, 1390, Plate 61.6, Plate 61.7 enhanced chemoluminescence technique, 1385 flow cytometry, 1386, 1387, 1390, Plate 61.5 multicolor, 1387, Plate 61.5 gene arrays, 1385 immunoassays, 1377, 1378, 1382–4 sandwich-type, 1384 two-site principle, 1382, Plate 61.2 immunohistochemistry, 1386–7, Plate 61.4 immunometric assay (IRMA), 1382 intracytoplasmic cytokine (ICC) assays, 1390 microplate-based, 1385 multiplex, 1384–5 PBMC, 1385–6, Plate 61.3 pyrogen for IL-1, 1377 quality control, 1391 reference reagents, 1391 RNase protection, 1387, 1388 sample preparation, 1390–1 secretion, 1386 selection, 1390 in situ hybridization (ISH), 1387, 1388 staining for proteins, 1386–7, Plate 61.4 types, 1376–8 asthma, 22, 1313–28 adult-onset, 1314 atopic, 1314–24 CC chemokines, 1087 CXC chemokines, 1068 cytokine-directed therapies, 1326–7 diagnosis, 1314 GATA-3, 1324–5 GM-CSF production, 510 IFN-c, 1318, 1322–3, 1324 IL-1Ra, 681 IL-2, 1317, 1323–4 IL-3, 1314 IL-4, 1314, 1316, 1317, 1319–20, 1323, 1325 animal models, 245–6 IL-5, 1314, 1315, 1316, 1317, 1319–20 IL-9, 354–5, 1314, 1317, 1320–1 IL-10, 1317, 1321–2 IL-12, 1324, 1325 therapy, 398

IL-13, 417–18, 1314, 1316, 1317, 1319–20, 1322–3 IL-15, 444 IL-16, 470 IL-17, 495 IL-18, 722, 1318, 1323–4 incidence, 1314 MI-L, 486 MIF, 1041 pathogenesis, 1314–15 T cells, 1324–6 astroglial cells, 527 atherosclerosis CX3CL1, 1104 inflammation, 681 atopic disease, 22, 1376 eczema and IL-15, 447 IL-12 therapy, 398 see also allergic disease dATP, 1138 autoantibody production prevention, CD95/CD95L, 891 autoimmunity/autoimmune disease, 1189–204 anti-cytokine therapy, 1200 anti-inflammatory therapy, 1200–1 anti-TNFa therapy, 1197, 1198, 1199–200 antigens, 1192 CD95/CD95L, 894–5 experimental neuritis (EAN), 495 gene therapy, 1342–3 HGF, 791 HMGB1, 920 IL-1Ra, 677, 679–80, 680, 687–8 IL-1RI, 661 IL-4 animal models, 245–6 IL-5, 272–3 IL-6, 292 IL-10, 605, 609 gene therapy, 1342, 1343 IL-13, 418–19 IL-15, 447–9, 451 IL-17, 491–5 IL-18, 722 initiation, 1191, 1193 lymphoproliferative disorders, 861 MHC, 1189–90, 1192 models, 1192–3 perpetuation, 1191, 1193 prolactin, 136 sex hormones, 1190 stages, 1191, 1193 susceptibility, 851–2, 1190 T cells, 1191, 1192 tissue damage, 1190–1, 1193 TNF, 851–2

xxxi B-cell malignancies chronic lymphocytic leukemia, 324 IL-6, 1222 IL-10, 1217–19 lymphoma, 323–4 IL-2, 170 B cells, 4 apoptosis protection by IL-4, 238 CD40-dependent activation, 239–40 CD95, 891 differentiation, 239–40, 314 IL-7 role, 315, 316 fate, 314 Flt3, 996 GH effects, 107, 108 IFN-c, 585 IL-1 family, 647 IL-2, 171, 185 IL-3, 203, 204, 205–6 IL-4, 230 biologic effects, 237–40 signaling, 235 IL-5 activity, 272–3 IL-6, 273, 282 IL-7, 314–18 IL-7R, 314 IL-9, 349 IL-12, 392–3 IL-13, 413–14 IL-15, 443 inflammation, 292 Ly-1 positive, 23 lymphopoiesis, 316 lymphotoxin, 830 maturation, 315 blockade, 317 ontogeny, 237 osteoprotegerin, 875 proliferation, 238 RANK, 875 TGFb-mediated apoptosis, 1139–40 B-lymphocyte chemoattractant/B cell attracting chemokine 1 see CXCL13 bacille Calmette–Guérin (BCG), 587, 589 Bambi (BMP and activin membranebound inhibitor), 1124–5, 1156 basic fibroblast growth factor (bFGF), 132, 1164 basophils IL-4 production, 233 IL-5 activity, 272 bc expression, 209–10 Bcl-2 activin, 1161

xxxii Bcl-2 (continued) apoptosis, 898, 1138 CD95-induced apoptosis, 898 erythropoietin effects, 155 expression regulation by IL-2, 181–2 by IL-4, 238 by IL-15, 444 FGF regulation of expression, 765 FGFR signaling, 767 IL-7, 313 IL-12, 392 TGFb-induced up-regulation, 1136 TGFb-mediated apoptosis, 1138 TGFb signaling, 1137 Bcl-XL erythropoietin effects, 155 expression regulation by IL-2, 181–2 by IL-15, 444 TGFb-induced up-regulation, 1136 TGFb-mediated apoptosis, 1138 TGFb signaling, 1137 BCRF1, 606, 611, 1218 behavior, prolactin effects, 133 betacellulin (BTC), 959, 972–4 activities, 973–4 gene, 973 precursor processing, 973 protein, 972 receptors, 973 betaglycan, 1121, 1122, 1157 see also TGFb receptors, type III BFU-E (burst-forming uniterythrocyte), 152, 153, 154, 1164, 1165 big-big prolactin, 117 big-prolactin, 117 bIL-3, 209, 210–11 bindarit, 1094–5 bioassays, 1377, 1378, 1379–81 advantages, 1381 cell function induction, 1380–1 cytotoxicity assays, 1380 pitfalls, 1381 proliferation measurement, 1380 specificity, 1381 synergism of cytokines, 1381 two-step procedure, 1381 types, 1379–81 birds chemokines, 68, 69, 70 FGF, 76 IL-1, 62–3 IL-2, 64–5 IL-3, 65–6 IL-4Ra, 71

INDEX

IL-6, 66–7 IL-8, 68, 69, 70 IL-11Ra, 71 IL-13Ra, 71 IL-15, 70 IL-16, 71 IL-17R, 71 IL-18, 70–1 IL-21R, 71 IL-22R, 71 interferons, 59–61 PDGF, 76 stem cell factor, 65–6 TGFb, 74 TNF, 72 birth, IL-1Ra, 678 blood IL-1Ra, 677 umbilical cord and stem cell factors, 1013 blood transfusion, 1267 platelets, 374 BMP, 1179–83 clinical applications, 1182–3 embryogenesis, 1182 fractures, 1183 isolation, 1180 limb morphogenesis, 1182 pathophysiology, 1182 pleiotropy, 1181–2 signaling, 1124, 1125 Smads, 1182 structure, 1180–1 BMP receptors, 1159, 1182, Plate 51.1 signaling, 11, 1182, Plate 51.1 BMP2, 1180, 1181, 1182 recombinant, 1182–3 BMP3, 1180, 1181, 1182 BMP4, 1180, 1181 osteosarcoma, 1182 BMP7 see osteogenic protein 1 (OP-1) BMP11, 1181 BMS2.4, 550 bone formation, 1179, 1180 fractures, 1183, 1281 giant cell tumor, 879 heterotopic formation, 1182 IL-1Ra, 689–90 estrogen action, 682 gene therapy, 695 IL-11 effects, 367–8 IL-18, 713 loss in osteoporosis, 878–9 metabolism and IL-5, 273 metastatic disease, 879–80 non-union, 1183

osteoclastic resorption, 494 RANK, 876 RANK-associated disorders, 877–8 resorption, 494 tumor-associated osteolysis, 879–80 VEGF in formation, 1025 bone gla protein see osteocalcin bone marrow Flt3, 994–5 G-CSF, 527, 530 IL-3, 215 IL-5, 269, 270 IL-7, 322 IL-11, 368 stem cell factor, 1012 stromal cells, 527, 550 bone marrow transplantation acute myeloid leukemia, 512 G-CSF, 1225 GM-CSF, 510, 511, 512, 513, 1225 IL-7, 327, 1226 IL-10, 611 rhIL-11, 375 IL-12, 396–7 bone morphogenetic protein see BMP Bottazzo–Feldmann hypothesis, 1192 brain CX3CL1, 1106–7 erythropoietin function, 159 IL-1Ra, 680, 688 IL-2R, 436 IL-3, 205 IL-7, 323 IL-15, 436 IL-17, 497 M-CSF, 945 TGFa expression, 966 brain injury IL-1Ra gene therapy, 694–5 ischemic, 497, 847 TNF, 847–8 breast, HGF in development, 789 breast and kidney/B cell- and monocyte-activating chemokine see CXCL14 breast cancer angiogenesis, 1220 CXC chemokines, 1069–70 human cell line, 125 IL-18, 722 prolactin, 136 PTHrP, 880 stem cell factor, 1014 breast tumors, GH overexpression, 106

INDEX

brefeldinA, 1387 bronchogenic carcinoma, IL-1Ra, 683 bullous pemphigoid, 419 C chemokines, 1049 cachexia, 1221, 1223 inhibins, 1155 calcium IL-18, 712 metabolism and prolactin, 134 calpain, 181 cancer, 1213–26 activin, 1161 angiogenesis, 1281 animal models, 1221–2 antiangiogenesis clinical trials, 1285, 1286–9 cachexia, 445, 1155, 1221, 1223 counter current invasion theory, 892, 1093, Plate 38.1 CSF-1, 943 CXC chemokines, 1069–70 CXCL10, 1063 cytokine gene transfer, 1341–2 cytokine inhibitors, 1222–3 cytokine polymorphisms, 1219 cytokine relationship, 1214–16 cytokine signaling blocking, 1222–3 cytokine therapy novel strategies, 1226 FGF, 758, 759–60, 763–4 FGFR, 766 Flt3, 1000–1 GM-CSF, 1269 immunotherapy, 512 HGF, 789–91 IFN-c in tumor rejection, 585–6 IL-1F7 antitumor activity, 742 IL-1Ra, 683 IL-6, 1220, 1222–3 IL-7, 323–8 IL-10, 1220 IL-11, 369 use, 1225–6 rhIL-11 pharmacology, 373–4 IL-12 therapy, 395, 397–8, 585 IL-13, 419 IL-15, 449, 451, Plate 53.4 antitumor therapy, 452 IL-17, 490–1 IL-18, 721–2 immunotherapy, 324, 328 inflammation, 1214–15, 1221–2 inhibin, 1155, 1161 M-CSF, 947 MCP-1, 1222 MET, 795, 796, 797

MIF, 1042, 1043, 1215–16, Plate 45.4 NFjB, 1215, Plate 53.2 p53, 1215–16 prognosis and MET, 795, 797 proto-oncogenes, 1219–20 stem cell factors, 1014–15 supportive therapy, 1225–6 T cells, 1224 TGFa, 966 TGFb, 1220 therapy, 1224–6 TNF, 849–50 susceptibility, 851 TNF-a, 1215, 1222, Plate 53.2 TRAIL/Apo2L, 865 therapeutic use, 866 tumor-associated osteolysis of bone, 879–80 tumor progression, 1216–20 vaccines, 1224, 1226 VEGF, 1026–7 see also chemotherapy; metastases; named cancers; radiotherapy cancer cells CC chemokines, 1092–4 CD95 downregulation, 893 CD95L, 892–3 epiregulin, 975 GM-CSF, 1269 HB-EGF, 971–2 IL-4, 244–5 IL-4R, 250 IL-8, 1057 IL-11, 367 IL-15, 433, 434, 436, 449 IL-18, 719 M-CSF, 945–6 metastasis, 1220–1 canstatin, 1286 carboxyamido-triazole (CAI), 1285, 1289 carcinogenesis, 1213–14 classification, 1214 cytokines, 1216–20 treatment strategies, 1214 cardiac hypertrophy, HB-EGF, 972 cardiac myxoma, IL-6, 291, 292 cardiotrophin (CT-1), 1263 cardiovascular disease angiogenesis, 1282 IL-1Ra, 681–2, 689 gene therapy, 695 cardiovascular system, TNF, 848 cartilage IL-17, 493 IL-18, 713

xxxiii caspase-1 see ICE caspase-3, 1138 activation, 1140 CD95, 899 substrate, 1140 caspase-8, 865–6 activation, 900 CD95, 897, 898, 899, 900, Plate 38.2 inhibition, 900 inhibitor, 891–2 caspase-9, 1138 caspase-10, 865–6 CD95, 897 caspase cysteine protease, 1140 caspase inhibition, 899–900 caspase recruitment domains (CARD), 844 Castleman’s disease anti-IL-6 receptor antibody, 294 IL-6, 1222–3 abnormal production, 292 caveolin 1 (Cav-1), 1125 c-Cbl, CSF-1, 935 Cbl insulin receptor substrate, 129 CC chemokine(s), 1049, 1083–95 agonists, 1084, 1086–7 chicken, 70 dendritic cells, 1090–1 inflammation, 1087 inflammatory, 1084, 1086 lymphocyte traffic, 1091–2, Plate 47.2 production regulation, 1087–8 small antagonists, 1095 structure, 1086 therapeutic targets, 1094–5 transcriptional activation, 1094 tumor cells, 1092–4 CC chemokine receptor, 8, 9, 1084, 1085, 1086–7 antagonists, 1095 cancer, 1092 coupling, 1089–90 expression regulation, 1089–90 CC21, 1084 CCL1, 1088 CCL2 see monocyte chemotactic protein 1 (MCP-1) CCL3, 1084, 1088 dendritic cell production, 1091 CCL3L1, 1084, 1089 CCL4, 1084, 1088 CCL5, 1084, 1088, 1089 antagonist action, 1095 cancer, 1092 dendritic cell production, 1091 transcriptional activation, 1094

xxxiv CCL7, 1084 antagonist action, 1095 dendritic cell attraction, 1094 CCL8, 1084 CCL9, 1091 CCL10, 1091 CCL11, 1084, 1086, 1088, 1089 CCL13, 1084 CCL14, 1088 CCL15, 1088 CCL16, 1088 CCL17, 1088, 1102 cancer, 1092 T cell traffic, 1091, 1092 CCL18, 1088, 1091 CCL19, 1084, 1088, 1091 CCL20, 1084, 1090 CCL21, 1088, 1091 dendritic cell attraction, 1094 CCL22, 1088, 1089, 1102 cancer, 1092 dendritic cell production, 1091 T cell traffic, 1092 CCL24, 1086 CCL26, 1086 CCL27, 1091 CCR1, 1086, 1089 dendritic cell expression, 1090, 1091 expression regulation, 1089 CCR2, 1086, 1089–90 cancer, 1092 dendritic cell expression, 1090 CCR3, 1086, 1087, 1088, 1089 TH2 cell associated, 1091–2, Plate 47.2 CCR4, 1086, 1087, 1088 TH2 cell associated, 1091–2, Plate 47.2 CCR5, 894, 1086, 1087, 1089 dendritic cell expression, 1090, 1091, 1092 expression regulation, 1089–90 HIV infection, 1087 TH1 cell associated, 1091–2, Plate 47.2 CCR6, 1087, 1090 dendritic cell expression, 1091 CCR7, 1087, 1090–1, 1091 cancer, 1069–70, 1093 CCR8, 1086, 1087, 1088 TH2 cell associated, 1091–2, Plate 47.2 CCR9, 1087 CCR10, 1087 CCR11, 1087 CD4, 468, 469, 894

INDEX

CD4+ T cells autoimmune disease, 1191 cancer, 1224 gene therapy, 1342 CD95/CD95L, 893, 894 differentiation, 314 HIV infection, 893–4 IFN-c, 568, 570 IL-2, 169, 1304 IL-4, 231, 240–1, 246 IL-7, 318 IL-7R, 311, Plate 13.3 IL-8, 1061–2 IL-12 cancer therapy, 395 IL-13, 413 IL-15, 450, 452 IL-16, 466, 467, 468–9 IL-17, 478 IL-18, 718 immune response, 187 T-cell homeostasis, 332 CD8 T cells autoimmune disease, 1191 cancer gene therapy, 1342 differentiation, 314 IFN-c, 568, 570 IL-2, 1304 IL-4, 232, 246 IL-4 expression, 240–1 IL-7, 318 IL-7R, 311, Plate 13.3 IL-8, 1061–2 IL-12 cancer therapy, 395 IL-15, 442, 450, 452 IL-16, 466, 467 IL-18, 718 T-cell homeostasis, 332 tumor eradication, 327–8 tumor resistance, 1224 CD8ED, 210 CD14, 811, 815 CD16, 450 CD16ED, 211 CD19, 317 CD23, 238 CD25, 650 CD26, 1089, 1095 CD26/DPP IV, 1088–9 CD28, 1303 CD30, 232 CD34, 317 CD34 stem cells, 327 G-CSF, 532 IL-15, 441 CD40, 232, 238 B cell activation, 239–40 monocyte inflammatory pathway, 615

signaling pathway, 239 TGFb-mediated apoptosis of B cells, 1139–40 CD40–CD40L interaction, 386 CD40L, 242, 243, 386, 1303 CD44, 445, 797 CD56, 450 CD69, 271 CD80, 231 CD86, 231, 238 CD95 antisense oligonucleotides, 1364 apoptosis, 891–2, 893, 896, 897–8, 900, Plate 38.2 FADD, 896–7, 900, 901 signaling, 893 T cell receptor, 890–1 T cells, 891 CD95/CD95L autoantibody production prevention, 891 autoimmune disease, 894–5 CD4 T cells, 893, 894 diabetes mellitus type 1, 895 experimental autoimmune encephalomyelitis, 895 Hashimoto’s thyroiditis, 895 hepatocytes, 892 liver, 892 multiple sclerosis, 894–5 negative selection, 891 signaling, 888 IL-18, 719, 722 CD95 receptor, 885, 888–902 apoptosis, 892, 893, 897–8, 899 B cells, 891 caspase-8, 897, 898, Plate 38.2 caspase-10, 897 down-regulation in cancer cells, 893 expression regulation, 888–9 FADD, 896–7, 900 function, 889–902, Plate 38.1 HIV infection, 893–4 immune system, 889–92 internalization, 900 signaling, 896–901, Plate 38.2 activation, 900 alternative pathways, 899 inhibition, 899–900 inhibitor, 893 initiation, 896–7, Plate 38.2 Stat proteins, 889 stimulation-induced clustering, 900 structure, 888–9 T cells, 889, 893–4, 900–1 thymocytes, 891

INDEX

CD95L, 711, 885–902 AP-1, 887 apoptosis, 890, 892, Plate 38.1 cancer cells, 892–3 CTL, 888, 890, Plate 38.1 expression IL-18, 719 regulation, 886–7 function, 889–902, Plate 38.1 immune system, 889–92 infection, 892 membrane-bound, 887–8 NFjB, 887 signaling, 888 soluble, 887–8 structure, 886 T cells, 887 TILs, 893 transplant rejection, 892 CD154, 448 celiac disease IL-15, 448 TNF, 851 cell adhesion molecules, 1269 cell-associated signals, xxiii cell cycle repressors, 556 cell proliferation, activin, 1159, 1161, 1166 cellular immunity, IL-12, 392 central nervous system (CNS) CX3CL1, 1106–7 CX3CR1 expression, 1108–9 FGFs, 757, 762 IL-1Ra, 678 gene therapy, 694–5 IL-18, 713 TNF, 846–8 tumors and IL-4, 249 cerebral palsy IL-9, 355 IL-18, 724 cervical cancer IL-17, 490 TNF, 851 CFR, 749, 750 CFU-E, 152, 153, 1164, 1165 Chagas disease, 1192 chemokine(s), xxiii amphibians, 68 antisense oligonucleotides, 1366 birds, 68, 69, 70 CD26/DPP IV, 1088–9 constitutive, 1087–8 fish, 67–8 IL-15, 442, 444 IL-17 induction, 486 inducible, 1087–8

lymphoid microarchitecture formation, 830 nomenclature, 5, 7, 1050 phylogeny, 67–8, 69, 70 production stimulation, 13 protein structure, 1051, 1053 receptors, 1050 T cells, 1091–2, Plate 47.2 see also named subgroups chemokine receptors, 8, 9, 1052 antisense oligonucleotides, 1366 functional decoy, 1090 human, 1058–60 nomenclature, 5, 7, 1050 signal transduction, 1058–9 subclasses, 8, 9 chemotactic and angiogenic factor (CAF), 68 chemotherapy Flt3, 997 G-CSF, 532, 1225 GM-CSF, 513, 1225, 1269 management of neutropenia, 510, 511 IL-11, 1225–6 rhIL-11 pharmacology, 373, 374 mucositis, 512 cholesterol metabolism, 1094, 1095 cholinergic anti-inflammatory pathway, 853 chondroblasts, 1179 chondrocytes IL-1Ra gene therapy, 694 proteoglycan synthesis reduction, 1364 chordin, 1181, 1182, Plate 51.1 choriomammosomatotropin genes, 103 ciliary neurotrophic factor (CNTF), 385 ciliary neurotrophic factor receptor (CNTFR), 1263 circadian regulation, TGFa, 966 CIS (cytokine-inducible SH2containing protein), 90–1 IL-2 signaling, 183 IL-9 signaling, 358 prolactin effects, 129–30 CLL22, 1089 clotting inhibition, phosphorothioate oligonucleotides, 1356, 1357–8 CNS-1 (conserved noncoding sequence 1), 229 COL-3, 1285, 1288 colitis, IL-7, 322 collagen fragments, 1286 colon/colorectal cancer angiogenesis, 1220

xxxv CXC chemokines, 1069 Flt3 treatment, 1000 IL-1Ra, 683 IL-7, 322, 323, 326 NSAID protection, 1215 colony-forming unit-granulocyte macrophage (CFU-GM), 351 colony-forming unit-macrophage (CFU-M), 283 colony-stimulating factor(s) (CSFs), xxiii, 3, 4 phylogeny, 65–6 colony-stimulating factor 1 (CSF-1) adaptor proteins, 935 cAMP, 937 biochemical responses, 929–37 cancer, 943 c-Cbl, 935 cleaving, 991 clinical detection, 942–3 cytoskeleton, 937 delivery models, 945–6 exons, 992 c-Fms, 937–41 G protein, 933 inflammation, 942–3, 945 JAK–STAT pathway, 936 Na/H antiport activity, 933–4 phosphatases, 935–6 phospholipase, 932–3 PI3K, 934–5 PKC, 933 Ras activation, 930–1 S6 kinase, 933 signaling, 941–2 structure, 992 tyrosine phosphorylation, 934–7 ubiquination, 935 Vav proto-oncogene, 937 see also macrophage colonystimulating factor (M-CSF) colony-stimulating factor 1 receptor (CSF-1R), 929–30, 937–42 colorectal cancer see colon/colorectal cancer complement activation, phosphorothioate oligonucleotides, 1356, 1357 condyloma acuminatum, 557 connective tissue growth factor (CTGF), 1024 contact dermatitis IL-1Ra, 681 IL-17, 497 coronary artery disease, CX3CR1, 1110 corticosteroids, MIF expression, 1038 counter current invasion theory, 892, 1093, Plate 38.1

xxxvi CPT-11, 446 CRF2-4, 612 CRF2 orphan receptors, 612, 613, 614 IL-22, 632 Crohn’s disease activin, 1167–8 anti-TNFa therapy, 1199, 1201 CX3CL1, 1104, 1106 cytokine system, 1196 IL-1Ra, 683 IL-4 gene, 230 IL-6, 293 IL-10, 609 IL-15, 448 IL-16, 470 IL-18, 713 ISIS 2302, 1358–9 cryptic self hypothesis, 1192 CTLA-8, 475, 476, 477, 550 gene, 475, 476 murine, 477 Cushing’s syndrome, 661 CX3C chemokine(s), 1049 CX3C chemokine receptor, 8, 9, 67 CX3CL1, 1088, 1101–13 chemokine domain, 1103, 1104, Plate 48.3 chemokine receptor-dependent cell adhesion, 1112 chromosomal localization, 1102–3 CNS, 1106–7 dendritic cell production, 1091 expression, 1104, 1105, 1106–7, 1112 function, 1110–11 gene structure, 1102–3, Plate 48.1 protein sequence/structure, 1103–4, Plate 48.2 role, 1112 virally encoded, 1111 CX3CR1, 1101–2 antagonists, 1111 expression, 1107–9 function, 1110–11 gene, 1103 polymorphisms, 1110 regulation, 1107–9 role, 1112–13 signaling pathways, 1109–10, 1112, Plate 48.4 CX3CR1-binding proteins, 1111 CXC chemokine(s), 1049–70 birds, 68, 69, 70 cancer, 1069–70 ELR, 1050, 1063–4 genes, 1050, 1051 glomerulonephritis, 1068

INDEX

human biologic properties, 1060–3 production, 1056–8 infection, 1065–6 MIF induced, 1043 modifications in mice, 1063–4 pathology role, 1065–70 pulmonary disease, 1067–8 reperfusion injury, 1066–7 rheumatoid arthritis, 1068–9 skin disease, 1067 CXC chemokine receptor, 8, 9 birds, 68, 69, 70 fish, 67, 68 human, 1052, 1058–60 CXCL1, 1055, 1057, 1061, 1062 cancer, 1069 contact hypersensitivity, 1067 glomerulonephritis, 1068 infection, 1066 psoriasis, 1067 rheumatoid arthritis, 1068 CXCL2, 1055, 1057, 1061, 1062 cancer, 1069 contact hypersensitivity, 1067 glomerulonephritis, 1068 psoriasis, 1067 rheumatoid arthritis, 1068 CXCL3, 1055, 1057, 1061 psoriasis, 1067 CXCL4, 1054, 1057 biologic properties, 1062 cancer, 1069 CXCL5, 1057–8, 1062 cancer, 1069 glomerulonephritis, 1068 rheumatoid arthritis, 1068 CXCL6, 1055, 1058, 1061, 1062 cancer, 1069 cleaving, 1089 CXCL7, 1054–5, 1057, 1061, 1062 cancer, 1069 infection, 1066 CXCL8 see IL-8 CXCL9, 1055, 1058, 1062–3, 1286, 1287 allograft rejection, 1306 cancer, 1069 contact hypersensitivity, 1067 psoriasis, 1067 CXCL10, 1055–6, 1058, 1062–3 antisense oligonucleotides, 1367 cancer, 1069 contact hypersensitivity, 1067 infection, 1066 modifications in mice, 1064 psoriasis, 1067

pulmonary fibrosis, 1068 CXCL11, 1056, 1062–3 CXCL12, 1056, 1058, 1060, 1089 biologic activity, 1063 cancer, 1069, 1070 metastases, 1220 modifications in mice, 1064–5 pulmonary fibrosis, 1067 rheumatoid arthritis, 1068–9 CXCL13, 1056, 1058, 1063 dendritic cell production, 1091 modifications in mice, 1065 rheumatoid arthritis, 1069 CXCL14, 1056 CXCL16, 1091, 1111 CXCL18, 1056 CXCL25, 1055 CXCR genes, 68 IL-13, 414 CXCR1, 1052, 1059, Plate 47.2 cancer, 1069 CXCR2, 1052, 1059, Plate 47.2 agonists, 1094 cancer, 1069, 1094 infection, 1065, 1066 modifications in mice, 1064 CXCR3, 1052, 1059, Plate 47.2 agonists, 1088 ligands, 1062–3 modifications in mice, 1064 T-cell accumulation in skin disease, 1067 TH1 cell associated, 1091–2, Plate 47.2 CXCR4, 894, 1052, 1059–60, Plate 47.2 cancer, 1069–70, 1093 expression, 1063 infection, 1066 metastases, 1220 modifications in mice, 1064–5 rheumatoid arthritis, 1068–9 CXCR5, 1052, 1060, Plate 47.2 modifications in mice, 1065 CXCR6, 1052, 1060 CXCR8, Plate 47.2 CXL16, 1112 cyclin-dependent kinase (CDK), 1132, 1160 cyclin-dependent kinase inhibitors (CKIs), 556, 581 cyclin pathway, activin, 1160 cyclo-oxygenase 2 (COX-2), 494 HGF stimulation, 791 IL-1, 643 cysteine-rich FGF receptor see CFR cytochrome c, 1138 cytokine(s)

INDEX

activity, xxiv antagonism, 12–13 bioactivity, 1377–8 cell surface receptor binding, 1376 definition, 5 detection in cells/tissues, 1386–9 diagnostic tools, xxv diversity generation, 288, Plate 12.5 features, 5–6 gene therapy, xxvi importance, xxv inhibitors, 1384 inhibitory actions on cytokine production, 13 measurement, 1376–8 in body fluids, 1378–84 cellular supernatants, 1378–84 molecular philosophy of actions, 12 monocyte-derived, 4 networks, 11–14, 1195 nomenclature, xxv, 5 phylogeny, 57–76 pleiotropy, 12, 20, 1376 polymorphisms, 1219 production, 6, 20 profile, 1376, 1384–5 profiling, 1378 recombinant, xxiv, xxvi redundancy, 288, 1378 signaling, 85–94 staining for proteins, 1386–7, Plate 61.4 stimulatory actions on cytokine production, 13 structural features, 7 synergism, 12–13, 1381 therapeutic administration, xxvi transcript detection, 1387–8 in vitro production, 1385–6 cytokine gene transfer, 1335–45 therapeutic applications, 1341–5 vectors, 1336–41 cytokine receptors chains, xxiv–xxv class I, 8, 9 Box 1/Box-2 regions, 8, 9 signaling, 10 subfamilies, 8, 9 class II, 8, 9 families, 8, 9 trans-signaling, 14 transmodulation, 13–14 cytomegalovirus (CMV) IL-8, 1062 IL-10, 606, 607

IL-18, 720 TRAIL/Apo2L therapeutic use, 866 cytoskeleton, CSF-1, 937 cytotoxic T cells (CTL), 232 CD95L signaling, 888, 890, Plate 38.1 ELISPOT frequency analysis, 1389 IL-6-induced differentiation, 283 IL-7-induced, 326 IL-12, 392 cytotoxic T lymphocyte-associated antigen-8 see CTLA-8 cytotoxicity assays, 1380 D6, 1087 Dab2, 1131 Daf-1, 1122–3 DAN, 1182, Plate 51.1 DARC, 1087 Daxx protein, 899 DC3, 325 death-associated protein (DAP) kinase, 844 death-induced signaling complex (DISC), 865, 897, 898, 900, Plate 38.2 decoy receptor 3 (DcR3/TR6), 889 defensins, 1090 dendritic cells CC chemokines, 1087, 1090–1, 1094 cross-priming, 1342 CSF-1, 943–4 CX3CL1, 1106 Flt3, 999–1000, 1001, 1002 follicular, 322 GM-CSF, 507, 509 IL-1 family, 647 IL-2, 170 IL-4 activation, 249 signaling, 236, 242 tumor site, 248 IL-10, 1322 IL-15, 443–4 IL-18, 712 lung, 1324 RANK, 876–7 RANKL, 873 T cell differentiation, 1319, 1324–6 dendritic epidermal T cells (DTEC), 320, 321 diabetes mellitus, insulin-dependent CD95/CD95L, 895 IL-1Ra, 683, 690 gene therapy, 695 IL-1RI, 661

xxxvii IL-6, 292 IL-10, 610 IL-18, 722 pathogen association, 1192 TNF, 852 disease development, 19 cytokine role, 1344 infectious, 20–1 severity differences, 21 susceptibility differences, 21 in vitro disease association studies, 32–52 DNA non-viral vectors, 1340–1 vaccines, 1344 DR3, 24 Drosophila, Toll, 813–14, 816, Plate 33.3 dwarfism, 106 E-cadherin, 797 E2F-1, 556 E2F-4, 556 early response genes (EGR) 1–3, 887 ECRF3, 1066 ELISA, 1378, 1382–3 conjugated antibody, 1383 matrix effects, 1383 sandwich, 1382, Plate 61.2 ELISPOT frequency analysis, 1388–9, 1390, Plate 61.6, Plate 61.7 embryogenesis BMP, 1182 FGFs, 755, 756 HGF–MET interactions, 794–5 VEGF, 1024–5 embryonic implantation, IL-1Ra, 678 endocrine hormones, xxiii endoglin, 1121, 1122, 1282 see also TGFb receptors, type III endoplasmic reticulum LSP–IL-15, 437 SSP–IL-15, 436, Plate 18.1 endostatin, 1282, 1283, 1284 gene therapy, 1290 endothelial cell-derived neutrophil attractant-78 see CXCL25 endothelial cells CX3CL1, 1104, 1106 CX3CR1 expression, 1108 FGF2 release, 750 GM-CSF, 507 IL-4 effects, 243–4 IL-15, 445 endothelial leukocyte adhesion molecule 1 (ELAM-1), 243

xxxviii endotoxin tolerance, 818 see also lipopolysaccharide (LPS) enhanced chemoluminescence technique, 1385 enzyme-linked immunosorbent assay see ELISA eosinophil-derived neurotoxin (EDN), 271 eosinophil differentiation factor (EDF), 264 eosinophilia, 269–70 airway, 418, 1320 biological specificity, 269 eosinophils activation, 271 asthma, 1315 GM-CSF, 507 IL-3 production, 206 IL-4, 233, 243 IL-5 role in production, 269–70 IL-9, 351–2 IL-13, 415 IL-17, 496 MIF expression, 1038 production in vitro, 269–70 in vivo, 270 tissue localization, 271–2 eotaxin, 272, 1086, 1092 eotaxin-2, 272 eotaxin-3, 272 epidermal growth factor (EGF), 3, 4, 322 activities, 962, 963–4 discovery, 962 family, 959–76 genes, 961, 963 precursor processing, 960–1 proteins, 959–60, 962–3 receptors, 963 structure, 963 TGFb-mediated apoptosis, 1138 TGFb signaling, 1132, 1134 transmembrane precursor protein, 962 see also named members epidermal growth factor receptor (EGFR), 797, 959–60, 961 amphiregulin, 969 BTC binding, 973 cell migration, 971 epigen binding, 976 epiregulin binding, 975 inhibitors in cancer therapy, 1222 knockout mouse studies, 962 TGFa, 966

INDEX

epigen, 959, 975–6 receptors, 976 epiregulin, 959, 974–5 receptors, 975 epithelial cells airway and IL-16, 467 CX3CL1, 1106 IL-4 effects, 244 IL-9, 352 IL-13, 415, 416 Epstein–Barr virus (EBV)-associated lymphoma, 1217, 1218–19 Epstein–Barr virus (EBV) and IL-10, 606, 611, 629, 1217, 1218–19 Epstein–Barr virus-induced gene 3 (EBI-3), 385, 388, 394–5 Erb signaling network, 959 Erb1, 963 Erb2, 963 Erb4, 963 ErbB, 961 ErbB receptors, 961 ErbB2, 961 TGFa, 966 ErbB2/neu receptor, 1220 ErbB3, 961 BTC binding, 973 ErbB4, 961 BTC binding, 973 epiregulin binding, 975 HB-EGF, 971 TGFa, 966 ERK (extracellular regulated kinases), 129 IL-3-induced activation, 213 IL-17, 481 MIF signaling, 1044 TGFb-induced activation, 1136 TNF production, 840 VEGFR1 signaling, 1023 VEGFR2 stimulated, 1024 Erk-1, 213, 1132 Erk-2, 213 erythrocytes, 152, 153 erythroid burst-forming units (BFU-E), 1164, 1165 erythroid colony-forming units (CFU-E), 152, 153, 1164, 1165 erythroid progenitor cells, 351 erythropoiesis GATA, 156–7 inhibition, 152 erythropoietin, xxiii, 149–59 alpha-form, 151 beta-form, 151 cancer/cancer therapy, 1225 clinical studies, 157–8

clinical use, 151, 158, 1225 FGF2 combination, 763 function, 153 gene, 149–50 expression, 151–2 kidneys, 151 c-kit down-regulation, 208–9 liver, 151 misuse by athletes, 1267 nervous system, 159 production, 151–2 recombinant human, 150–1, 157–8 red cell production stimulation, 1267 side effects, 158 site of action, 152 stem cell factor synergy, 1012 structure, 149–51 erythropoietin mimetic peptides (EMPs), 154 erythropoietin receptor, 10, 153–4 active sites, 154 inactive dimers, 126 signaling, 154–7 Stat proteins, 155 structure, 153 estrogens bone action and IL-1Ra, 682 prolactin receptor upregulation, 125 etanercept, 852, 1199–200 expansile skeletal hyperphosphatasia, 878 experimental autoimmune encephalomyelitis (EAE) antisense oligonucleotides, 1367 CD95/CD95L, 895 CX3CL1, 1106–7 CX3CR1, 1109 IL-1Ra, 688 IL-1RI, 653 IL-10, 609–10 IL-18, 722 extracellular matrix FGF actions, 747, 748, 750 FGF binding, 753–4 hematopoietic progenitor cell cytokine response modulation, 1265 eye, TGFa expression, 966 eye disease angiogenesis, 1282 neovascular and FGFs, 758, 763 eye infection CD95L, 892 MIF, 1042

INDEX

familial expansile osteolysis, 878 Fanconi anemia Flt3, 997 IL-6 deficiency, 284 Fas see CD95 Fas-associated death domain protein (FADD), 827, 844 activin, 1161 CD95, 896–7, 900, 901 Fas-associated phosphatase 1 (FAP1), 899 FasL see CD95L Fcc receptor type I (FcgR-1), 579 Felty’s syndrome, 532 fetal liver kinase 2 see flk-2 FGF1, 750 angiogenesis, 757–8, 761 disease associations, 759, 763 embryogenesis, 755–6 expression, 754 features, 759 FGFR signaling, 767–8 neovascular disease, 763 rheumatoid arthritis, 759, 763 wound healing, 761–2 FGF2, 750, 751–3, Plate 31.1 angiogenesis, 757–8, 761 apoptosis regulation, 765 Bcl-2 expression regulation, 765 cancer, 758, 759, 763–4 disease associations, 759, 763, 769 ECM binding, 753, 754 embryogenesis, 755–6 expression, 754, 755 inhibition, 769 features, 759 FGFR signaling, 768 GM-CSF, 763 hematopoiesis, 762–3 HIV infection, 764–5 integrin expression, 761 MAPK activation, 761 MMPs, 754, 761, 762 neovascular disease, 758, 763 rheumatoid arthritis, 759, 763 VEGF synergy, 764, 765 wound healing, 761–2 FGF3, 749–50, 751, Plate 31.1 angiogenesis, 758 embryogenesis, 755–6 features, 759 FGF4 angiogenesis, 758 embryogenesis, 755–6 features, 759 hematopoiesis, 763 organogenesis, 756

FGF5, 759 FGF6, 759 FGF7 angiogenesis, 758 ECM binding, 753 features, 760 wound healing, 762 FGF8, 754 features, 760 organogenesis, 756 FGF9, 760 FGF10 features, 760 organogenesis, 756 wound healing, 761–2 FGF11–14, 760 FGF12, 756 FGF15, 760 FGF16–19, 760 FGF17, 756, 760 FGF20, 760 FGF21, 760 FGF22, 760 FGF23, 760 FGFR1, 752, 765 skeletal development abnormalities, 766 FGFR2, 765 skeletal development abnormalities, 766 FGFR3, 765 skeletal development abnormalities, 766 FGFR4, 765 fibrinogen, 817 fibroblast growth factor (FGF), 3, 4, 747–71 amphibians, 75 angiogenesis, 757–8, 758, 761 angiogenic, 755 apoptosis regulation, 765 birds, 76 cancer, 758, 759–60, 763–4 chromosomal location, 749 CNS, 757, 762 disease associations, 758, 759–60, 763–5 ECM actions, 747, 748, 750 binding, 753–4 embryogenesis, 755, 756 export mechanisms, 749–50 expression, 748, 754–5, 755 eye neovascular disease, 758, 763 family, 748–54 fibrosis, 758 fish, 75

xxxix functions, 755–8, 759–60, 761–3 gene-knockout studies, 755–6 genes, 748–9 hematopoiesis, 757, 762–3 HIV infection, 764–5 Tat protein, 764–5, 769–70 identification of novel, 769 intracellular trafficking, 750–4, 751, 752–3 invertebrates, 74–5 Kaposi’s sarcoma, 758, 759, 763, 764, 765 MMPs, 754, 761, 762, 764 neovascular disease, 758, 763 nervous system, 757, 762 neurodegenerative disease, 758 NLS, 749, 750, 751, Plate 31.1 nuclear trafficking, 751–2 organogenesis, 756 production, 749–50 protein sequence, 749 proteins endogenous, 751–2 exogenous, 752–3 regulation, 754–5 rheumatoid arthritis, 758, 759, 763 secretion mechanisms, 749–50 specificity, 768–9 structure, 748–9, 759–60, Plate 31.1 subcellular localization, 750–4 Tat protein, 764–5, 769–70 vasculogenesis, 756–7 VEGFs, 757, 761 wound healing, 757, 761–2 see also basic fibroblast growth factor (bFGF); individual named FGFs fibroblast growth factor-binding protein (FGF–BP), 764–5 fibroblast growth factor receptors (FGFR), 747, 748, 752 amphibians, 75 birds, 76 cancer, 766 dimers, 766, 767, Plate 31.2 disease associations, 766 expression, 765–6 fish, 75 inhibitory mechanisms, 768 regulation, 765–6 signaling, 766–8, Plate 31.2 skeletal development abnormalities, 766 specificity, 768–9 structure, 765, Plate 31.1 see also individual named FGFRs

xl fibroblasts CXC chemokines, 1062 G-CSF, 527 GM-CSF, 507 IL-4, 243 signaling, 236 IL-13, 416 IL-16, 467 IL-17, 488–9, 495 MIF expression, 1038 fibrodysplasia ossificans progressiva, 1182 fibronectin, 817, 1135, 1163 fibrosis activin, 1163 FGFs, 758 filamin, 1130 fish, 57 chemokines, 67–8 FGF, 75 IL-1, 61–2 IL-2, 63–4 IL-3, 65 IL-6, 66 IL-8, 67–8 IL-13, 71 interferons, 58, 60 PDGF, 76 TGFb, 73–4 TNF, 71–2 FKBP12, 1124 FLASH (FLICE-associated huge protein), 899 c-FLIP, 891, 893, 898–9, 901 v-FLIP, 898 flk-2, 989–90 flow cytometry, 1386, 1387, 1390, Plate 61.5 multicolor, 1387, Plate 61.5 flt-3/flt-2 receptor tyrosine kinase, 1264 Flt1 see vascular endothelial growth factor receptor 1 (VEGF-R1) Flt3, 989–1004 adjuvant properties, 1001–2 cancer, 997–9, 1000–1 clinical uses, 1002–4 colon cancer treatment, 1000 dendritic cells, 1001, 1002 regulation, 999–1000 expression, 993 gene, 992–3, 1220 gene therapy, 1003 hematopoietic cell development, 994–6 hematopoietic disorders, 996–7 immune response, 1001–2

INDEX

immunotherapy, 1003–4 infectious diseases, 1001 isoforms, 991–2 leukemia, 997–9 ligand cloning, 990–1 liver transplantation, 1002 radiation therapy, 1003 stem cells expansion, 1002–3 mobilization, 1003 structure, 992 tissue regeneration, 1003 tolerance induction, 1001–2 tyrosine kinase inhibition, 998, 1223 Flt3 receptor, 990, 992–3 expression, 993–4 Flt4 see vascular endothelial growth factor receptor 3 (VEGF-R3) c-Fms activating mutations, 938 dimerization, 937–8 Grb2, 941 internalization, 938 KI domain, 940 PI3K, 941 Src, 941 Stat1, 941 tyrosine phosphorylation, 938–42 Y807, 940 Y809, 939–40 Y973, 940–1 fms-like tyrosine kinase 3 see flt3 focal adhesion kinase, 129 follicle-stimulating hormone (FSH) regulator, 1155 follicular dendritic cells (FDC), 322 follistatin, 1153, 1155–7 activin, 1159 branching morphogenesis, 1162–3 production activation, 1167 variant forms, 1156 wound healing, 1164 follistatin-related protein (FSRP), 1155, 1156 function, 1157 wound healing, 1164 c-fos, 11 expression regulation by IL-2, 181–2 fowlpox virus (FPV), 59, 60, 61 genome, 71 fractalkine see CX3CL1 fractalkine receptor see CX3CR1 fractures angiogenesis, 1281 BMPs, 1183

FRIL, 991 fungal infection, M-CSF, 946, 947 G protein CSF-1, 933 dissociation, 11 G-protein-coupled receptor (GCPR) signaling, 970 G3139, 1359 Gab1, 798 gap junctions, 323 gastric carcinoma, 1069 gastrointestinal ischemia, 376–7 gastrointestinal mucosal atrophy, 377 gastrointestinal mucositis, 374–5 gastrointestinal tract antisense inhibitors, 1361 BTC activity, 973 EGF, 964 IL-1Ra, 683, 690 IL-18, 713 nucleases, 1361 TGFa, 966, 967 see also colon/colorectal cancer; Crohn’s disease; inflammatory bowel disease; ulcerative colitis GATA-1, 156–7 GATA-2, 157 GATA-3, 157, 229 asthma, 1324–5 IL-5 gene expression, 266, 267 IL-13 gene expression, 410 IL-13-induced activation, 418 TH2 cells, 236, 266 differentiation, 1324–5 cc (common cytokine receptor c chain), 173 expression, 175 IL-2 signal transduction, 176–7, 185–6 Jak3 association, 186 regulation, 175 negative by proteolysis, 181 regulatory elements, 171 SCID-X1, 186 signaling molecule association, 177–81 gene arrays, 1385 gene polymorphisms of human cytokines, 25–9 gene therapy, 1335–45 applications, 1341–5 approaches, 1336–41 cytokine function identification, 1344–5 gene transfer vectors, 1336–41 non-viral, 1340–1

INDEX

viral, 1337–40 genetic markers, 22 genetics of cytokines, 19–24, 25–52 gene polymorphisms, 20 genetic variation, 24 in vitro studies disease association, 32–52 expression, 30–1 genomic regions, 19 genomics for cytokines, 1389 giant cell tumor of bone, 879 giantism, 106 glial cell-derived neurotrophic factor (GDNF), 288, 1122 glioblastoma, 1220 glioma, 419 glomerulonephritis chronic hepatitis B, 1244 CX3CL1, 1104 CXC chemokines, 1068 IL-6, 292 MIF, 1042 Glu-110, 268, 269 glucocorticoids, 481 chemokine receptors, 1090 RANKL, 875 glucose homeostasis, abnormal, 106 gonadotrophin-releasing hormone (GnRH), 132 gonads, prolactin effects, 132 Goodpasture’s syndrome, 1191 gp130, 67, 288, Plate 12.4, Plate 12.6 cell growth induction, 290 IL-6R, Plate 22.5 IL-11 signaling, 369, 371–2 signal transduction pathways through, 289–91 gp130 receptor family, 1263 graft-versus-host disease (GVHD), 375, 1376 IL-1Ra, 683–4, 690, 691 IL-10, 611 IL-12, 396–7 IL-18, 719, 722 graft-versus-leukemia, 375 granulocyte chemotactic protein 2 see CXCL6 granulocyte colony stimulating factor (G-CSF), 525–38 biochemical properties, 526 biologic activities, 530 cancer therapy, 532, 1225 clinical uses, 532–3 deletion, 530–2 ex vivo stem cell expansion, 1271, 1272 gene

cloning, 526 expression, 526–8 promoter elements, 527 structure, 526–8 transcription, 527 IL-4, 242 IL-17-enhanced, 488, 489 ligand, 534 maternal/fetal interface, 527–8 myelodysplastic syndromes, 158 neutrophil stimulation of production, 1268 pro-inflammatory cytokine actions, 537–8 protein, 526 recombinant human, 532, 533 side-effects, 532 stem cell mobilization, 1269–70 structure, 526, Plate 22.1 therapeutic use, 1257 granulocyte colony stimulating factor (G-CSF):granulocyte colony stimulating factor receptor complex, 534, Plate 22.4 granulocyte colony stimulating factor (G-CSF) receptor, 525 acute myeloid leukemia, 534 alternative splicing of human transcript, 529–30 biochemical properties, 528 deletion, 530–2 differentiation mediation, 535 gene, 528–9 growth mediation, 535 isoforms, 529–30 JAK kinases, 535 ligand interaction, 534, Plate 22.4 pathology associated with altered expression, 533–4 PI3K, 537 protein structure, 528, Plate 22.2 signal transduction, 530, 534–7, Plate 22.5 constitutive in leukemogenesis, 538 SOCS, 536 Stat proteins, 535–6 transcription regulation, 530 tyrosine phosphorylation, 535, Plate 22.5 granulocyte–macrophage colony stimulating factor, human (hGMCSF), 156 granulocyte–macrophage colony stimulating factor, human (hGMCSF) receptor, 156 granulocyte–macrophage colony

xli stimulating factor (GM-CSF), 503–14 bc subunit deficient mice, 508 cancer therapy, 512, 1225, 1269 clinical applications, 510–12, 513, 514 deficiency in humans, 509–10 mouse models, 508 disease models, 509 DNA vaccination, 1344 eosinophilia, 270 ex vivo stem cell expansion, 1271, 1272 gene expression, 504–5 IL-3 gene relationship, 506 regulation, 504–5 hematopoiesis with FGF2, 763 IL-2 combination, 1226 IL-3 association, 201, 202, 208 IL-5 expression induction, 270 IL-6 synergy, 283 IL-17 induction, 486, 488 c-kit down-regulation, 208–9 myelodysplastic syndromes, 158 neutrophil stimulation of production, 1268 pharmacokinetics, 514 pharmacologic administration, 508–9 protein structure, 504 side effects, 514 stem cell factor synergy, 1012 stem cell mobilization, 1269–70 therapeutic use, 1257 TNF-a induction, 486 toxicity, 514 transgenic mice, 507 in vitro activity, 506–7 in vivo activity, 507–9 granulocyte–macrophage colony stimulating factor receptor (GM-CSF-R), 211, 505, 1262 genetics, 506 signal transduction, 505–6 Stat proteins, 506 subfamily, 8, 9, 13 granulocytes, 1257 GM-CSF, 507 IL-4 effects, 243 granulocytic precursor cells, G-CSF effect, 530 granulopoiesis, G-CSF/G-CSFR system, 531 Graves’ disease, 419 cytokine system, 1196

xlii Graves’ disease (continued) IL-16, 470 tissue damage, 1191 Grb-2, 212–13, 941 CSF-1, 935 gremlin, 1182 groa–c see CXCL growth factors, 3–4, 6, 7 hematopoietic, xxiii, 3 IL-6 function, 285–6 phylogeny, 73–6 growth hormone (GH), 103–9 disease states, 106 gene, 103–4 immune function, 107, 108 overexpression, 106 production, 105–6 control, 104 protein, 104–6 skin, 108–9 stress modulation, 107 therapeutic administration, 106 growth hormone (GH) receptors, 106–9 growth promoting activity (GPA), 67 GTPases, 657 guanylate-binding protein 1 (GBP-1), 93, 579, 761, 769 Guillain–Barré syndrome, 680 Hashimoto’s thyroiditis CD95/CD95L, 895 cytokine system, 1196 tissue damage, 1191 Helicobacter pylori, 495 helminth infections CC chemokines, 1087 IL-9, 354 IL-13, 417 hematological malignancy angiogenesis, 1281 erythropoietin, 158 IL-15, 449 hematopoiesis, 1255, 1256 activin, 1164–5 deterministic regulation, 1258, 1259–60 extrinsic regulation, 1260–2 FGFs, 757, 762–3 IL-3 effect, 207–9 IL-4 effect, 242 IL-6, 283–4 IL-12, 393 IL-13, 416 IL-17, 488–9 intrinsic regulation, 1260–2 lineage selection/commitment, 1260, 1261–2

INDEX

process, 1257–62 prolactin effects, 134, 135 stochastic regulation, 1258, 1259 stress, 208–9 hematopoietic cell development and Flt3, 994–6 hematopoietic cytokines, 1255–72 clinical use, 1267–72 neutrophil production stimulation, 1268 platelet production stimulation, 1268–9 red cell production stimulation, 1267 stem cell mobilization, 1269–70 hematopoietic growth factors, xxiii, 3 hematopoietic progenitor cells, 1265, 1266 adhesion molecules, 1265 GM-CSF, 506 IL-9, 351 receptor activity modulation, 1266 renewal, 1258–60, 1260–1 responses to cytokines, 1262–5 antagonists, 1266–7 inhibitors, 1266–7 modulation, 1265–7 suppression, 1266–7 VEGF, 1025–6 hematopoietic progenitor kinase 1 (HPK1), 1133 hematopoietic stem cells, 314, 1256 commitment, 317 differentiation, 317 ex vivo expansion, 1270–2 Flt3, 1003 G-CSF, 532–3 renewal, 1258–60, 1260–1 VEGF, 1025–6 hematopoietin family, 7 hematopoietin receptor superfamily, 1262–4 heparan sulphate, 753, 1265–6 heparan sulphate proteoglycans (HSPGs), 747, 748, 749, 750 ECM, 753–4 FGF trafficking, 752–3 FGFRs, 765, 766 dimers, 767 HB-EGF, 969 MMP reservoir, 754 VEGF binding, 1022 heparin, 1266 VEGF binding, 1022 heparin-binding EGF (HB-EGF), 959, 960, 969–72 activity, 962, 971–2

cancer, 971–2 gene, 971 precursor processing, 970–1 protein, 969–70 receptors, 971 signaling, 970 hepatitis IFN-a therapy, 1238–51 IL-15, 448 IL-18, 722 hepatitis B acute, 1240, 1241–2 advanced cirrhosis, 1244 chronic, 1240–1 anti-HBeAg-positive, 1243–4 antiviral therapy, 1242 HBeAg-positive, 1242–3 extrahepatic disease, 1244 fulminant, 1242 IFN treatment, 1237–8 type I, 557 IFN-a therapy, 1238–45 IFN-b therapy, 1245 IFN-c therapy, 1244–5 immunosuppressed patients, 1244 vaccine, 512 hepatitis C acute, 1246 antiviral treatment, 1245–50 chronic, 1246 cirrhosis, 1250 IFN treatment, 1238 type I, 557 IFN-a therapy combination with ribavirin, 1246–7 pegylated, 1247–9 IL-12 therapy, 398 liver transplantation, 1250 viral kinetics, 1249–50 hepatitis D, 1251 hepatocellular carcinoma, 795, 796 hepatocyte growth factor (HGF), 310, 783–99 active, 784 activin, 1159, 1163 angiogenesis, 791 autoimmune disease, 791 cancer, 789–91 co-receptors, 797 crystal structure, 786 disease states, 789–91 embryogenesis, 794–5 expression, 788–9 gene

xliii

INDEX

organization, 787 promoter, 787–8 human, 783–4 inflammatory bowel disease, 791 kringle domains, 786, 787, Plate 32.1 latent, 784 nervous system, 789 protein sequence, 785, Plate 32.1 signaling, 797–8 skeletal muscle generation, 788–9 structure, 784, Plate 32.1 TGFb synergy, 1132 tubulogenesis, 789, 798 ulcer-healing, 791 variant forms, 786 see also MET hepatocyte growth factor-like protein, 786 hepatocyte growth factor (HGF) receptor, 1263, 1264 hepatocytes apoptosis, 865 CD95/CD95L, 892 IL-4 effects, 244 TGFb-mediated apoptosis, 1138–9 hereditary hemorrhagic telangiectasia, 1122 herpes simplex virus (HSV) gene transfer vectors, 1340 IL-15, 451, 453 IL-18, 720 herpesvirus human, 441 saimiri see HVS see also human herpesvirus 8 (HHV8) HIF-1, 152, 1021 hip replacement, total, 368 histamine-producing cell stimulating factor (HCSF), 207 HIV-associated lymphoma, 1217–18 HIV-based vectors, 1338 HIV infection apoptosis, 893–4 CC chemokine antagonists, 1095 CCR5, 1087 CD4 T cells, 893–4 CD95 receptor, 893–4 CX3CL1, 1107 CX3CR1, 1110 CXCL12, 1063, 1066 CXCR4, 1066 FGF2, 764–5 GM-CSF, 511, 513 IL-2, 469 IL-7, 330

IL-12 therapy, 398 IL-15, 450–1 IL-16, 469–70 IL-18, 720 M-CSF, 946 progression rate, 22 Tat protein, 764–5, 769–70, 887, 894 HLA-A, 851 HLA-B, 851 HLA-DP, 851 HLA-DQ, 851 HLA-DR, 851 HLA-DR2, 850 HLA-DR3, 850 HLA-DR4, 135, 850 HMG box, 915–16 HMGB1, 916–17, 918 HMG (high mobility group) proteins, 914 HMGB1, 913–21 amino acid sequences, 914 antagonists, 918 autoimmune disease, 920 cellular localization, 914–15 cytoplasmic, 915 endotoxin stimulation, 916–17 extracellular roles, 919 HMG boxes, 916–17, 918 inflammatory responses, 917 lethal systemic inflammation mediation, 916–18 MAPK, 920 NFjB, 920 nuclear, 914–15 post-translational modification, 916 RAGE interaction, 918–20 release, 919 rheumatoid arthritis, 920 sepsis, 918 sickness behavior, 917–18 structure, 915–16 t-PA, 918 tissue injury, 917–18 HMGB-1 receptors, 918–20 HMGB-2, 915 HOG-1 stress gene, 660 homeostasis maintenance, xxiii hormones polypeptide, 6, 7 sex, 125, 682, 1190 steroid, 119–20 see also glucocorticoids host defense IL-1Ra, 678, 696–7 IL-18, 720 innate, xxiii

host response, 1040 hsp60, 817 hsp65, 1192 hsp70, 817 HSV-1, 451 HTLV-1 IL-2, 1216–17 IL-9, 350, 353 IL-15, 449, 451 leukemia, 1216–17 human chromosome 9, 550 human chromosome 19, 551 human GH-variant (hGHV), 103 human herpesvirus 8 (HHV8) CC chemokines, 1093 CX3CR1, 1111 CXC chemokines, 1066 multiple myeloma, 1217 see also Kaposi’s sarcoma human herpesvirus infection, 441 human prolactin-binding protein, 121 human prolactin receptor gene, 121–2 human T cell lymphotropic virus see HTLV-1 humoral immunity, IFN-c, 584–5 HVS, 486 CXC chemokines, 1066 genome, 476 IL-26, 636 ORF-13, 477 HVS-13, 475, 476, 479, 486 HVS-14, 476 hypercalcemia of malignancy, 880 hypereosinophilic syndrome, 1376 hypergammaglobulinemia, 23 hyperprolactinemia, 120–1, 125 stress-induced, 133–4 hypersensitivity contact CXC chemokines, 1067 IL-1Ra, 681 delayed-type autoimmune disease, 1343 IL-16, 470 hypoestoxide, 494 hypoxia-induced factor-1 see HIF-1 hypoxia response element (HRE), 1020–1 hypoxic conditions, IL-8, 1057 ICE, 648, 649, 650 IL-18, 711, 712 inhibition, 712, 900 TNFR1, 845 IFN-a, 7, 549–50, 1233–51 allograft rejection, 1298–9

xliv IFN-a, 7 (continued) anti-inflammatory properties, 1235 antiangiogenic activity, 1285, 1286, 1287 cancer gene therapy, 1342 hepatitis C therapy, 1246–7 hepatitis D, 1251 host defense, 1235–7 IL-10 regulation, 608 IL-11 down-regulation, 366 IL-18R induction, 715 immunotherapy in cancer, 1225 pegylated, 1247–9, 1250–1 side effects of therapy, 1250–1 structure, 552, Plate 23.1 therapeutic use, 1225, 1238–51 viral hepatitis therapy, 1238–9, 1240–4, 1245–50 IFN-a/b, 549 antagonism with IFN-, 12 family, 7 proteins, 4 IFN-a/b receptors, 8, 9, 10 IFN-b, 549–50 anti-inflammatory therapy in autoimmunity, 1200–1 antiangiogenic activity, 1286 hepatitis B therapy, 1245 multiple sclerosis therapy, 12 structure, 552, Plate 23.1 IFN-d, 552 IFN-, xxiv, 4, 567–90 allografts acceptance, 1303–5 rejection, 1299–300, 1307 antagonism with IFN-a/b, 12 with IL-4, 12 antiangiogenesis, 1286, 1287 antigen presentation, 582–3 antiproliferative activities, 581, 586 antisense oligonucleotides, 1362 antiviral activity, 580–1 apoptosis, 586 asthma, 1318, 1322–3, 1324 B cells, 585 biologic activities, 580–6 biosynthesis, 569–70 birds, 60 cytokine stimulation, 13 erythropoiesis inhibition, 152 FGF2 expression, 755 gene, 567–8, 569 promoter elements, 568 hepatitis B treatment, 1244–5 humoral immunity, 584–5

INDEX

Ig heavy chain switching, 585 IL-10 regulation, 608 IL-12-induced production, 388, 389, 390, 396 IL-12 receptor regulation, 389 IL-18-induced, 570, 717 innate immunity, 581–2 LPS induction, 24 macrophage activation, 581–2 MHC class I, 586 MHC class II, 24 multicolor flow cytometry, 1387, Plate 61.5 nickel dermatitis, 497 NK cells, 569–70 production stimulation, 13 protein, 568, 569, 569 regulation, 567–8 signaling protein disruption, 586–7 Stat1-dependent genes, 579–80 structure, 569 synergism with TNF, 12 T cells, 326, 568, 569, 570, 583–4, 1323 TH1 cells, 583–4 TH2 cell inhibition, 232 TNF receptor expression modulation, 14 tumor immunity, 585–6, 1224 vaccine adjuvant use, 61 IFN--inducible protein 10 see CXCL10 IFN- receptor, 8, 9, 10, 570–2 JAK kinases, 573–7 signal transduction, 569, 573–9 alternative pathways, 579 dysfunction, 586–7, 588, 589–90 Jak–Stat pathway, 574–9 Stat inhibitors, 577–8 ubiquitin–proteasome pathway, 578 SOCS, 576–7, 578 Stat proteins, 573–8, 579 subunits, 570–2, 574 expression/regulation, 578–9 IFN--regulated genes, 579–80 IFN-inducible T-cell a chemoattractant see CXCL11 IFNj, 550 IFN-Ra, 553, 554 IFN-Rb, 553–4 IFN regulatory factor (IRF), 1234 IFN stimulatable genes (ISGs), 1234 IFN-stimulatable response element (ISRE), 1234 IFN-stimulated gene factor 3 (ISGF3), 1234

IFN-, 7, 551 structure, 552, Plate 23.1 IFN-, 7, 549–50 IFNGR subunits, 570–2, 574, 578–9 deficiency in humans, 587, 588, 589–90 deficient mouse models, 587 tumor regulation, 586 Ig heavy chain switching, IFN-, 585 Ig repeats, 815 IgA eosinophil degranulation, 271 IL-5 enhancement of production, 273 IL-6 increase in production, 282–3 IgA nephropathy, 451 IgE asthma, 1315, 1319 IL-4 isotype switching, 239–40 synthesis IL-4, 282, 414 IL-6, 282 IL-13, 414 IGF-1 GH secretion regulation, 104–5 production, 106 IGF-1 receptors, 106–9 immune function, 107, 108 IgG, 239, 292 IgM, 238, 239 IB, 1044 IB kinase (IKK) complex, 10, 11 IL-10, 616 VEGF, 1027 IL-1, xxv, 4 amphibians, 62 anti-TNFa therapy, 1199 antiangiogenesis, 1287 birds, 62–3 blocking, 661–3 cachexia, 1223 chromosomal localization, 736–7, 738 cytokine production stimulation, 13 erythropoiesis inhibition, 152 family, 643–63 biochemical actions, 658 cells producing, 647 cytoplasmic signaling cascades, 657–9 F5–F10, 735–44 members, 645–51 signal transduction, 657–61 structures, 646–7 fish, 61–2 gene, 736–7, 738

INDEX

human disease, 644 IL-2 receptor expression modulation, 14 IL-10 regulation, 608 inflammation, 643 NFB inhibition, 1202 nomenclature, 5 ovulation, 678 pyrogen assay, 1377 rheumatoid arthritis, 1195–6 TLR-like signals, 815 TNF receptor downregulation, 14 see also IL-18 IL-1 receptor, 8, 9 amplification, 658 binding, 660 birds, 63 blockade, 661–3 cellular specificity, 658 deficiency, 644 fish, 62 genes coding for, 653–4 R1–9, 653–4, 1365 rheumatoid arthritis, 1196 signal transduction, 10–11, 655, 735, 817–18, 1196 Toll homology, 814, Plate 33.3 type I, 654–6, 735 cytoplasmic domain, 659 gene regulation, 656–7 human studies, 661–3 soluble, 661–3 surface expression, 656–7 type II, 653, 656, 676 decoy, 735 see also IL-1F; IL-1Ra IL-1 receptor-activating kinases see IRAKs IL-1 receptor antagonist see IL-1Ra IL-1a, 644, 645–6, 650–1 antisense oligonucleotides, 1364 autoantibodies, 651 autocrine growth factor, 650 deficiency, 644 deficient mice, 651–2 human systemic response, 662–3 membrane, 650–1 precursor, 650 receptor binding, 655, 656 transcriptional regulation, 647, 648 IL-1a receptor, 650, 651 IL-1b, 644, 645–6 activin, 1166–7 birds, 62, 63 converting enzyme, 649, 650 deficiency, 644 deficient mice, 651, 652

FGF2 expression, 755 fish, 61–2 function, 1345 IL-1a production control, 652 polymorphisms, 1219 production, 647 receptor binding, 655, 656 rheumatoid arthritis, 1344 secretion, 649–50 structure, 738, Plate 30.5 transcriptional regulation, 647–8 IL-1b-converting enzyme see ICE IL-1F, 5 IL-1F5, 645–6, 736, 737 expression, 739–40, 743 properties, 741 protein sequence/structure, 738–9 receptor binding, 743 regulation, 740 structure, 738, Plate 30.5 IL-1F6, 736, 737 expression, 740 properties, 741 protein sequence/structure, 738–9 regulation, 740 IL-1F7, 646, 736, 737 antitumor activity, 742 expression, 740, 743 gene transfer, 744 properties, 741 protein sequence/structure, 738–9 receptor binding, 743 regulation, 740–2 IL-1F8, 736, 737 expression, 742 properties, 741 protein sequence/structure, 738–9 regulation, 742 IL-1F9, 736, 737 expression, 742, 743 properties, 741 protein sequence/structure, 738–9 receptor binding, 743 regulation, 742 IL-1F10, 736, 737 expression, 742–3 properties, 741 protein sequence/structure, 738–9 receptor binding, 743 regulation, 743 IL-1R-AcP, 654, 655, 657, 658 IL-1R interaction, 676 IL-1R-related proteins (IL-1Rrp), 736 IL-1R1, 653, 1365 IL-1R2, 653 IL-1R3, 653 IL-1R4, 653–4

xlv IL-1R6, 654 IL-1R8, 654 IL-1R9, 654 IL-1Ra, 414, 643, 645–6, 669–98, 1196, 1200 animal models of disease, 686–90 arthritis, 679–80, 687–8, 691–2 gene therapy, 693–4, 696, 698 autoimmune disease, 677, 679–80, 687–8 blood, 677 bone, 682, 689–90 cancer, 683 cardiovascular disease, 681–2, 689 gene therapy, 695 CNS, 678 gene therapy, 694–5 combination therapy, 697–8 crystal structure, 675–6 delivery, 686, 697 diabetes mellitus, 683, 690 gene therapy, 695 in disease, 679–84 gastrointestinal tract, 683, 690 gene allelic polymorphisms, 684–6 structure, 670–1 transcription regulation, 671–2 gene therapy, 692–3 animal models of disease, 693–6 human trials, 696, 698 host defense, 678, 696–7 human trials, 690–2 infection, 677, 679, 687, 690–1 inflammation, 678 inflammatory bowel disease, 683 intracellular, 670, 673–4, 675, 677–8 host defense, 697 kidney disease, 682, 689 gene therapy, 695–6 lung disease, 681, 689 gene therapy, 696 mechanism of action, 675, 676 nervous system, 680, 688 normal physiologic function, 677–8, 696–7 organ transplantation, 683–4, 690, 691 osteoporosis, 682, 689–90 ovaries, 678 phosphorothioate oligonucleotide-induced, 1357 production, 674 protein isoforms, 672–4 receptor binding, 655, 656, 675 recombinant, 684 reproductive system, 682

xlvi IL-1Ra (continued) secretory, 670, 672 sepsis, 687, 690–1 skin, 680–1, 688–9 soluble, 675, 677, 697 therapeutic uses, 686 tissue distribution, 676 tissue localization, 674–5 IL-1RI birds, 63 deficient mice, 652–3 fish, 62 glycosylation, 655 IL-1RI-associated kinase, 659 IL-1RII, 62 IL-2, xxv, 4, 167–88 actions, 170–2 allografts acceptance, 1303–5 rejection, 1297–8, 1305 amphibians, 64 anti-CD3 stimulation, 169, 170 asthma, 1317, 1323–4 B cell lymphoma cell lines, 170 B cells, 171, 185 birds, 64–5 CD4+ T cells, 169 cDNA cloning, 168 chromosomal localization, 168 CTTL-2 cell line bioassay, 1380 cytokine stimulation, 13 dendritic cells, 170 DNA vaccination, 1344 fish, 63–4 function, 168–9 gene chromatin remodeling, 170 expression, 169–70 genomic structure, 168 GM-CSF combination, 1226 HIV infection, 469 HTLV-1, 1216–17 IL-6 effects, 283 IL-10 regulation, 608 immune system, 170 immunoglobulin secretion, 239 immunotherapy cancer, 1224–5 targeting, 186–7 LAK cell induction, 325 leukemia, 171, 1216–17 lymphoma, 171 NK cells, 171–2 nomenclature, 5 production stimulation, 13 reptiles, 64 signal transduction, 176–7, 440

INDEX

signaling pathways, 181–3 structure, 168–9, Plate 8.1 synergism with IL-5, 12 T cells, 184, 185–6, 1304 in vivo effects, 445 IL-2-activated genes, 181–3 IL-2/IL-4 family, 7 IL-2 receptor, 167, 1262–3 chromosomal localization, 173 classes, 168–9, 170–1, 172–3 cloning, 173 expression, 174–5 modulation, 14  subunit, 1263 IL-2 signal transduction, 176 immune system, 184–6 immunotherapy targeting, 186–7 LAK cells, 172 regulation, 174–5 signaling, 10, 177–81 structure, 437 subfamily, 8, 9 IL-2Ra, 172–3 brain, 436 expression, 174–5 immune system, 184–5 inhibitors in cancer therapy, 1222 regulation, 174–5 regulatory regions, 171, 174–5 signaling molecule association, 177–81 soluble, 183–6 structure, 437 IL-2Rb, 172–3 brain, 436 expression, 175 IL-2 signal transduction, 176 regulation, 171, 175 regulatory regions, 171 IL-2R, 172–3 gene, 185 see also c (common cytokine receptor  chain) IL-3, 201–17 administration, 207 antagonists, 216 apoptosis suppression, 215 asthma, 1314 B cells, 203, 204, 205–6 birds, 65–6 bone marrow stimulation, 215 clearance, 202, 207 clinical significance, 215–17 eosinophilia, 270 ex vivo stem cell expansion, 1271, 1272 FGF2 combination, 763

fish, 65 gene and GM-CSF gene relationship, 506 glycoforms, 202 hemopoiesis effect, 207 hemopoietic stem cell actions, 203 IL-5 expression induction, 270 IL-6 synergism, 283 IL-10 regulation, 608 immune system, 207–8 infection management, 216 JAK kinases, 212, 214 c-jun, 215 c-kit down-regulation, 208–9 leukemia, 216–17 lung carcinoma, 216 lymphoid cells, 204 MAP kinase activation, 213 mature cells of hematopoietic origin, 204 c-myc, 215 neurons in brain, 205 PI3K activity, 212, 213, 214–15 platelet stimulation, 215 progenitor cell actions, 203 Ras activation, 212–13 receptor binding, 211–12 serum, 207 signal transduction, 210–15 sources, 206 Stats, 214 steady-state lymphohemopoiesis, 207–9 stem cell factor synergy, 1012 stress, 208–9 structure, 201–3 synergism with other cytokines, 203 synthesized, 202–3 T cells, 203, 204–5, 206, 216 activation, 206–7, 208 tyrosine phosphorylation, 212 IL-3 receptor, 209–15, 1262 b-chain, 209, 210 IL-4, 227–51 allograft rejection, 1297–8, 1300, 1305 gene therapy, 1343 animal models, 245–9 antagonism with IFN-, 12 anti-tumor effects, 247–50 antiangiogenesis, 1287 antigen receptor triggering, 239 antisense oligonucleotides, 1364 asthma, 1314, 1316, 1317, 1319–20, 1323, 1325 B cells, 230 biologic effects, 237–40

INDEX

biologic properties, 237 cellular sources, 231–3 circulating IgE level regulation, 239 cytokine stimulation, 13 gene Crohn’s disease, 230 NRE regions, 229 PRE-I region, 227–8 regulatory sequences negative, 229–30 positive, 227–9 structure, 227–30 gene therapy, 248–9, 250 hematopoiesis effects, 242 IgE synthesis, 282, 414 IL-5 coexpression, 264–5 IL-10 regulation, 608 IL-12 production stimulation, 246 IL-12R, 389, 390 IL-13 relationship, 410, 411 inflammatory conditions, 497 inhibition by IFN-g, 585 isotype switching, 239–40 JAK kinases, 234, 1316 mediator production, 242–3 mRNA expression, 230 myelomonocytic cell effects, 242–3 neoplasia experimental model, 247–8 gene therapy, 248–9 neutralization, 1302, 1303 NK cell effects, 241–2 PI3K, 234, 235 promoter, 228, 229 protein, 230–1 recombinant cancer models, 247–8 clinical trials, 249–50 signaling, 411 in B cells, 235 cascade, 234–6 in T cells, 235–6 Stat3, 412 Stat6, 234–5, 236, 240, 1316 binding site, 228–9 structure, 227–31 T cells, 229, 230, 231–3, 412 biologic effects, 240–2 mature, 240–1 TH1 cells, 235–6, 241 TH2 cells, 235–6, 241, 412 TH2 differentiation, 1325 thymocyte effects, 240 TNF activity suppression, 841 toxin therapy, 250 tumor immunity, 248–9

IL-4 receptor/receptor complex, 233–4, 247, 411, 412 cancer cells, 250 signaling pathway, 234 soluble, 1327 types I and II, 234 IL-4Ra antisense oligonucleotides, 1365 birds, 71 gene, 454 variants and allergic disease, 234 IL-5, 263–74 antisense oligonucleotides, 1364 asthma, 1314, 1315, 1316, 1317, 1319–20 inhibition, 1327 autoimmune disease, 272–3 B cells, 272–3 basophils, 272 biological activities, 263–4 bone marrow, 269, 270 bone metabolism, 273 eosinophil activation, 271 eosinophilia, 269–70 gene CLE0 sequence, 265–6 coexpression, 264–5 expression, 264–7 GATA consensus site, 267 IL-4/IL-5/IL-13 locus, 267 NFAT site, 266–7 posttrascriptional regulation, 267 promoter region, 265–7 regulatory elements, 266, 267 response elements, 266 structure, 264, Plate 11.3 IgA production enhancement, 273 IL-7-stimulated release, 316 IL-9 synergism, 351–2 mast cell secretion, 233 nervous system, 273 protein structure, 267–9, Plate 11.3 receptor interaction, 268–9 sources, 274 synergism with IL-1, 12 T cells, 263, 274 IL-5 receptor, 268, 1262 IL-6, 5, 281–94 ACTH secretion, 285 activin, 1166 acute phase response, 284–5 antagonist, 1200 anti-TNFa therapy, 1199 antisense oligonucleotides, 1364–5 B cell malignancies, 1222

xlvii B cells, 273, 282 B9 cell proliferation, 1379–80 biological activity, 282–6 birds, 66–7 cachexia, 1223 cancer, 1220, 1222–3 cardiac myxoma, 291, 292 Castleman’s disease, 292, 1222–3 deficiency, 284 disease, 291–4 Fanconi anemia, 284 febrile response, 285 fish, 66 gene deregulated expression, 294 promoter, 286–7, Plate 12.1 promoter polymorphism, 22 structure, 286–7, Plate 12.1 growth arrest induction, 290 growth factor function, 285–6 hematopoiesis, 283–4 IgA production, 282–3 IL-1 stimulation of glioblastoma/astrocytoma cells, 285 IL-2 receptor expression modulation, 14 IL-3 synergism, 283 IL-4-dependent synthesis, 282 IL-10 regulation, 608 IL-11 relationship, 368, 369 IL-17-induced, 488 immune response, 282–3 inflammation, 292 inflammatory proliferative disease, 292–3 inhibitors in cancer therapy, 1222–3 invertebrates, 66 multiple myeloma, 879, 1217, 1218, 1220, 1222–3 myeloma cells, 293 NFB, 287 inhibition, 1202 osteoclast development, 285 phosphorothioate oligonucleotide-induced, 1357 plasma cell neoplasia, 293–4 production, 286 stimulation, 13 protein structure, 286–7 receptor binding, 287, Plate 12.3 responsive element, 289 rheumatoid arthritis, 292–3, 1195 Stat proteins, 289 T cell activation, 283 tumor microenvironment, 1220

xlviii IL-6 receptor, 67, 287–8, 1263, Plate 12.2, Plate 12.3 diversity generation, 288, Plate 12.5 inhibitors in cancer therapy, 1222–3 JAK–STAT signal transduction pathway, 289–91 signaling, 10 IL-10R signaling interaction, 615 inhibitors, 291 subfamily, 8, 9 IL-7, 305–33 allograft rejection, 1305 B cells, 314–18 developmental gene upregulation, 317 bone marrow, 322 transplantation, 327, 1226 cancer, 322, 323–8 therapy, 324–8 cellular immune response, 331 clinical importance, 315 colitis, 322 colorectal cancer, 322, 326 CTL induction, 326 developing tissues, 322–3 expression, 309 gene, 308, Plate 13.1 cloning, 305–6, 308 growth factor for DTEC, 321 iIEL expression, 320–1 IL-5 release stimulation, 316 IL-10 regulation, 608 immune response to infection, 328–31 immune system, 321–2, 328–31 immunodeficiency, 327 immunotherapy, 324, 328, 330 JAK tyrosine kinase, 312 LAK cell induction, 325–6 PI3K, 312 proteolysis protection, 1266 purification, 305 SCID-X1, 311 serum level elevation, 330 skin immune reactions, 321 Stat proteins, 312, 313, Plate 13.2 structure, 306–7 d T cell homing, 321 T cells, 318–21, 326 homeostasis, 305, 331–2 TCR, 319 thymocytes, 332 tissue-specific immune responses, 321–3 tumor cell effects, 324 IL-7-associated cytokines, 308, 310

INDEX

IL-7 receptor, 310–18, Plate 31.2 B cells, 314 signaling, 311, 312, 313, 327 defective, 186 T cells, 311, 314, Plate 13.3 TCR, 313 IL-7Ra, 310, 311, Plate 31.2 anti-apoptotic effects, 313 cancer, 323–4 cell viability maintenance, 313 developing tissues, 322–3 gene rearrangement signals, 313 lymphoma, 324 RAG expression, 320 Src kinase enzymes, 311–12, Plate 13.2 IL-8, 1049, 1050, 1054, 1055 angiogenesis, 1057 anti-TNFa therapy, 1199 antisense oligonucleotides, 1366 AP-1, 1057 ARDS, 1067 asthma, 1068 biologic properties, 1060–2 birds, 68, 69, 70 cancer, 1069 cancer cells, 1057 cytomegalovirus, 1062 dendritic cell production, 1091 fish, 67–8 glomerulonephritis, 1068 hypoxic conditions, 1057 IL-1-induced, 659 IL-17-induced, 488, 495 infection, 1065, 1066 inflammatory responses, 495 MIF induced, 1043 modifications in mice, 1063–4 NFB, 1057 inhibition, 1202 production of human, 1056–7 psoriasis, 1067 pulmonary fibrosis, 1067–8 receptor system, 1058–9 reperfusion injury, 1067 rheumatoid arthritis, 1068 structure, 1054 T cells, 1061–2 IL-9, 347–58 asthma, 354–5, 1314, 1317, 1320–1 B cells, 349 cerebral palsy, 355 gene, 352–4 cloning, 347–8 expression, 353–4 HTLV-1, 350, 353 IL-5 synergism, 351–2

IL-16 production stimulation, 467 IRS-2, 357 leukemia, 350, 351, 353 lymphoma, 350, 351 mast cells, 347 parasitic infections, 354 protein, 348 responsive cells, 348–52 T cells, 349–50, 353 thymus, 350 IL-9 receptor, 355–8 characterization, 355–6 JAK1, 356, 357 JAK3, 356 M-Ras, 357 NF-B, 357 signal attenuation, 357–8 signal transduction, 356–7 SOCS proteins, 358 Stat proteins, 356–7 IL-10, 603–19 actions, 605 allograft rejection, 1300, 1305, 1306–7 gene therapy, 1343 angiogenesis, 610–11 anti-inflammatory therapy in autoimmunity, 1200 antiangiogenesis, 1287 antisense oligonucleotides, 1362, 1365 asthma, 1317, 1321–2 autoimmune disease, 605, 609 gene therapy, 1342, 1343 B cell malignancies, 1217–19 cancer, 1220 cellular, 606–7 cellular functions, 618 chemokine receptors, 1090 cytokine inhibition, 13, 417 diabetes type 1, 610 disease outcome, 23 regulation, 617 risk, 21 expression, 607–8 family, 7, 627–36 receptor redundancy, 636 subfamily of homologues, 628–9 gene, 23–4 structure, 605–6 human disease, 608 IFN- inhibition, 570 IL-12 inhibitor, 386 IL-18 synergy, 717 immune system, 605 infectious disease, 20– 21

INDEX

inflammation, 608–9 inflammatory bowel disease, 609 LPS induction, 24 pathophysiologic functions, 619 promoter, 608 protein structure, 605–6 receptor interaction sites, 606 recombinant, 21 regulation, 608 rheumatoid arthritis, 1195–6 SNPs, 23–4 structure, 606 systemic lupus erythematosus, 22–3 T cells, 607 TNF activity suppression, 841 transcriptional activation, 616 transcriptional regulation, 614–15 transgenic animals, 21 transplant rejection, 611 tumor progression, 1220 viral, 606–7, 611, 629 IL-10 receptor, 8, 9, 636 disease states, 611–12 downstream genes, 617 expression, 612 inflammatory responses, 612 interaction sites, 606 JAK-Stat activation, 614–15 signaling, 604, 612, 613–15, 629–30 signaling pathway interactions, 615–16 SOCS, 616–17 Stat proteins, 614–15 structure, 612–13 IL-10M1, 607 IL-11, 363–78 activity, 363–4 anti-inflammatory therapy in autoimmunity, 1200 cancer therapy, 1225–6 cellular sources, 366 disease states, 368–9 down-regulation by IFN-a, 366 expression regulation, 368–9 gene chromosome location, 364 downstream gene activation, 372 expression, 364–5 promoters, 365–6 structure, 364 suppressors, 365–6 IL-6 relationship, 368, 369 immunoreactive, 365 ligand, 370–1 MGF combination, 316

protein, 366 receptors, 369–71 recombinant human, 363, 366–8 gastrointestinal mucositis, 374–5 immunomodulation, 375–8 inflammatory bowel disease, 377–8 intestinal ischemia, 376–7 megakaryocytes, 371 pharmacology, 373–5 short bowel syndrome, 377 systemic inflammatory conditions, 376 rheumatoid arthritis, 1195–6 tissue expression, 366 in vitro activity, 366–8 IL-11 receptor, 1263 distribution, 371 expression, 371 function, 372–3 signaling, 369, 371–2 structure, 369–70 IL-11Ra, 71 IL-12, 383–99 allograft rejection, 1298–300 angiogenesis, 393 antiangiogenic activity, 1285, 1287 asthma, 1324, 1325 B cells, 392–3 Bcl-2, 392 biologic activities, 391–3 bone marrow transplantation, 396–7 cancer, 585 gene therapy, 1342 therapy, 395, 397–8, 585 cellular immunity, 392 cellular sources, 385–6, 387 clinical trials, 397–8 cytokine stimulation, 13 deficiency in MSMD disorder, 590 gene, 383–4 gene therapy, 395 GVHD, 396–7 hematopoiesis, 393 IFN- production induction, 388, 389, 390, 396, 570 IL-10 regulation, 608 IL-15 antitumor combination, 452 IL-18 synergy, 717, 723 IL-18R induction, 715 infectious disease, 395–6 inflammation, 392 inhibitors, 386 LAK activity, 392

xlix NK cells, 392 organ transplantation, 396, 397 production stimulation, 13 IL-4, 246 recombinant human, 397–8 regulation, 385–6 T cells, 392 TH1 differentiation, 1325 TH1/TH2 response pattern, 391–2 tumor rejection, 585 IL-12 receptor, 388–91 expression, 389 regulation, 390 IL-4 role, 389, 390 immune defects, 389–90 interactions with IL-12 family members, 390–1 signaling, 389 Stat proteins, 389 IL-12RA, 385, 387–8 biologic functions, 394 IL-12R interaction, 390 IL-13, 409–19 asthma, 417–18, 1314, 1316, 1317, 1319–20, 1322, 1323 autoimmune disease, 418–19 B cells, 413–14 biologic properties, 410 cancer, 419 cytokine inhibition, 13, 417 fish, 71 gene, 409–10 polymorphisms, 418 hematopoiesis, 416 IgE synthesis, 414 IL-1R antagonist, 414 IL-4 relationship, 410, 411 IL-5 coexpression, 264–5 IL-10 regulation, 608 IL-18 stimulation of production, 413 suppression of production, 720 immune response-induced pathology, 417–19 immune response regulation, 416–17 immune system, 416–19 inflammation, 416–17 mannose receptors, 415 NK cells, 413 production, 412–13 promoter, 22 protective immunity, 416 protein, 409–10 signal transduction, 411–12 skin, 418 Stat proteins, 410, 412, 1322

l IL-13 (continued) T cells, 412–13 in vitro biologic activities, 413 IL-13 receptor, 247 asthma therapy, 1327 complexes, 410–11 IL-13Ra, 71 IL-15, 431–53 allograft rejection, 1297–8, 1305 angiogenesis, 446 autoimmune disease, 447–9 B cells, 443 biologic activities, 439–46 birds, 70 cancer, 443–4, 449, 451, 1221–2, Plate 53.4 antitumor therapy, 452 CD8+ T cells, 442 CD34+ stem cells, 441 chemokines, 442, 444 DNA vaccination, 1344 expression regulation, 436–7 gene, 432–3 genomic organization, 432–3 IL-10 regulation, 608 IL-12 antitumor combination, 452 immunotherapy, 451–3 infection, 441, 452–3 inflammation, 447–9 isoforms, 436–7 knockout animals, 446–7 leukemia, 1221–2 lymphocyte development regulation, 439–41 NK cells, 440, 441–2, 451, 1214–15, Plate 53.4 prostate cancer, 443–4 IL-15 receptor, 436 distribution, 438 MAPK pathways, 438 PI3K, 438 SHP-2, 438 signal transduction, 178, 438, 440 defective, 186 skeletal muscle, 445 sources, 433, 434–5, 436 Stat proteins, 438 structure, 432–3, 437–8 T cells, 442–3, 448, 1214–15, Plate 53.4 TCRs, 440 transgenic animals, 446–7 in vivo effects, 445–6 IL-15Ra, 173, 436, 437–8 IL-16, 465–71 bioactivity, 466, 468–9 birds, 71

INDEX

CD4 interaction, 468 cell sources, 467 function, 466–7, 471 gene post-translational modification, 466, Plate 19.2 structure, 465–6, Plate 19.1 HIV infection, 469–70 inflammation, 470 neuronal (NIL-16), 471 properties, 471 sequence, 466–7 signaling, 468 structure, 466–7 synthesis, 465–6 T cells, 466, 467, 468–9 TCR, 469 IL-16Ra and b chains, 655 IL-17, 412, 475–97 airway diseases, 495–6 autoimmune disorders, 491–5 biologic activities, 486, 487, 488–9 cancer, 490–1 cartilage effect, 493 cytokine induction, 486, 487–8 fibroblasts, 488–9 function, 478 gastric disorders, 495 gene, 476 hematopoiesis, 488–9 human, 477, 478–9 IL-8 stimulation, 488, 495 IL-17B–F, 482–6 immunopathologic conditions, 489–97 inflammatory skin disorders, 496–7 MAP kinases, 481 mouse, 476–7 NF-B, 479, 481 nickel dermatitis, 497 rheumatoid arthritis, 483–4, 491–4 signaling, 479, 481–2 structure, 478 synergy with other cytokines, 486, 488 T cells, 478 ab T cells, 477 TNF-a synergy, 486, 488, 492 transplantation, 489–90 IL-17 receptor, 486 birds, 71 cloning, 479 sequence, 479, 480 signaling, 479, 481–2 IL-17B, 482 IL-17B receptor, 482

IL-17C, 482 IL-17E, 483 IL-17F, 483–6 sequence, 484, 485 structure, 484 IL-18, 233, 644, 645–6, 709–24, 735–6 antiangiogenesis, 721–2, 1287 antitumor activity, 721–2 asthma, 1318, 1323–4 bioassay, 1381 biologic activity, 710 birds, 70–1 bone, 713 cancer antitumor activity, 721–2 gene therapy, 1342 tumor growth promotion, 722 cartilage, 713 cellular expression, 712–13 CNS, 713 cytokine stimulation, 13 cytolytic activity effect, 718 deficiency, 644 diagnostic use, 724 Fas/Fas ligand signaling, 719, 722 Fas ligand expression, 719 gastrointestinal tract, 713 gene, 710–11 mapping, 710 regulatory regions, 710–11 Stat proteins, 711 hormonal regulation, 711 host defense, 720 human, 71 human disease states, 723–4 IFN- induction, 570, 717 IL-10 synergy, 717 IL-12 synergy, 717, 723 IL-13 production stimulation, 413 suppression, 720 infection, 720–1 inflammatory response, 722, 723 knockout mouse phenotypes, 719–20 microvesicle rapid release, 712 pathological role, 722–4 physiological role, 719–24 processing, 711–12 receptor binding, 655 receptor-mediated release, 712 skin, 712–13 T cells, 712 TH2 cytokine induction, 718 TLR-like signals, 815 transcriptional regulation, 648–9 tumor growth promotion, 722

INDEX

viral infection, 720–1 in vitro activity, 717–19 IL-18 binding protein (IL-18BP), 653, 657, 715–16, 736 IL-18 receptor, 8, 9, 714–15 composition, 714 deficiency, 644 expression, 714–15 homology, 714 IRAKs, 659–60 localization, 714 NK cells, 717 regulation, 715 signal transduction, 655, 716–17 T cells, 715, 717 IL-19, 627, 628–31 expression, 630 gene, 628, 630 IL-20, 627–8, 629, 630, 631–2 biologic functions, 631–2 clinical relevance, 632 protein, 631 skin, 632 IL-20 receptor, 631, 636 IL-21, 431–2, 453–6 NK cells, 456 protein, 453 IL-21 receptor, 453–4 birds, 71 genomic structure, 455 signaling, 454–5 IL-22, 627–8, 629, 630, 632–4 binding protein, 634 biologic effects, 633 gene, 632–3 IL-22 receptor, 633, 634, 636 birds, 71 signal transduction, 633, 634 IL-23, 383, 384–5, 387 biologic functions, 393–4 IL-12R interaction, 390 TH1 deficiency, 391 IL-24, 627–8, 629, 630, 634–5 IL-25, 13, 412, 483 IL-26, 627–8, 629, 630, 635–6 IL-27, 385, 388 IL-12R interaction, 390–1 IL-28A, 551 IL-28B, 551 IL-29, 551 IL-TIF see IL-22 IL1RN gene, 670–2 allelic polymorphisms, 684–6 disease association, 685–6 IL10-1082 SNP, 23–4 immune response, 57 Flt3, 1001–2

IL-7, 331 lymphotoxin, 829–30, 831–2 NF-B, 814 RANKL, 875 VEGF, 1026–7 immune stimulation, 1356–7 immune system, 19, 20, 810–11, Plate 33.1 activin, 1161–8 CD95/CD95L, 889–92 CX3CL1, 1106 genetic variation, 20 IL-2, 170 IL-2R, 184–6 IL-3, 207–8 IL-6, 282–3 IL-7, 305–6, 321–2, 328–31 IL-10, 605 IL-11, 371 IL-12R, 389–90 IL-13, 416–19 IL-15, 451–3 IL-24, 635 lymphotoxin, 831–2 MIF, 1040–1 prolactin effects, 131, 134 RANKL, 875 regulation, xxiii immunity, adaptive, 809–10, Plate 33.1 immunity, innate, 809–10, Plate 33.1 defense systems, 831–2 IFN-, 581–2 lymphotoxin, 831–2 immunoassays, 1377, 1378, 1382–4 sandwich-type, 1384 two-site principle, 1382, Plate 61.2 immunodeficiency IL-7, 327 see also HIV infection immunogenetics, 19 immunoglobulin superfamily, 715 see also individual named Igs immunohistochemistry, 1386–7, Plate 61.4 immunometric assay (IRMA), 1382 immunomodulation IFN type I, 556 rhIL-11, 375–8 immunostaining, 1386–7, Plate 61.4 immunotherapy cancer, 324, 328 Flt3, 1003–4 IL-2/IL-2R system, 186–7 IL-7, 324, 328, 330 IL-15, 451–3 in situ hybridization (ISH), 1387, 1388 infection/infectious disease

li cancer association, 1214–15 CD95L, 892 CXC chemokines, 1065–6 Flt3, 1001 fungal, 946, 947 G-CSF, 531, 532 GM-CSF, 509, 511, 513 IL-1Ra, 679, 687, 690–1 induction, 677 IL-1RI, 652–3 IL-4 animal models, 246–7 IL-7 immune response, 328–31 IL-9, 354 IL-12 therapy, 395–6 IL-15, 441, 452–3 IL-18, 720–1 lymphotoxin, 832 M-CSF, 946, 947 MIF, 1040, Plate 45.3 parasitic IL-9, 354 IL-18, 720 pneumonia, 496 TLR signaling, 817 TNF effect on susceptibility, 850–1 TRAIL/Apo2L therapeutic use, 866 vaccines, 509, 512 see also HIV infection; mycobacterial infection; viral infection inflammation activin, 1165, 1166–7 clinical syndromes, 1167–8 antisense inhibitors, 1360, 1361–2 atherosclerosis, 681 B cells, 292 cancer association, 1214–15, 1221–2 CC chemokines, 1087 CSF-1, 942–3, 945 CX3CL1, 1104 gene therapy, 1343 HMGB1, 916–18 IL-1, 643 IL-1a, 652 IL-1b, 651, 652 IL-1Ra, 678 induction, 677 IL-1RI, 652–3, 661 IL-6, 292 IL-10, 608–9 IL-12, 392 IL-13, 416–17, 418 IL-15, 447–9 IL-16, 470 M-CSF, 946 systemic, 376

lii inflammation (continued) TNF therapy, 853 vagus nerve stimulation, 853 inflammatory bowel disease activin, 1167–8 HGF, 791 IL-1Ra, 683 IL-10, 609 rhIL-11, 377–8 IL-15, 448–9 IL-16, 470 MIF, 1042 see also Crohn’s disease; ulcerative colitis inflammatory demyelinating polyneuropathy, 724 inflammatory lung disease, 418 GM-CSF production, 510 inflammatory proliferative disease, chronic, 292–3 inflammatory response angiogenesis, 1281 IL-8, 495 IL-10R, 612 IL-18, 722, 723 infliximab, 852, 1197, 1198, 1199, 1201 influenza vaccine, 512 virus, 651 inhibin, 1153, 1156 activin signaling antagonism, 1157 cancer, 1155, 1161 genes, 1154, 1155 inhibin A, 1122, 1155 inhibin B, 1155 inhibin-binding protein, 1156, 1157 insulin, 6 sensitivity, 285 insulin-like growth factors, 103 see also IGF-1 insulin receptor substrates, 129 integrins, 761 endostatin interaction, 1284 interferon/IL-10 gene cluster, 61 interferon-induced genes, 553–5 interferon-inducible protein 10 (IP-10), 1286, 1287 interferon stimulated gene factor 3 (ISGF3) see ISGF3 complex interferon stimulated response element see ISRE interferons, 3, 4 amphibians, 58 antiangiogenic activity, 1286 antiviral mechanisms, 1235–6 birds, 59–61

INDEX

fish, 58, 60 hepatitis B, 1237–8 hepatitis C, 1238 host defense, 1235–7 phylogeny, 58–61 proteins, 1233–4 receptor chains, 61 reptiles, 58–9 signaling, 1234 viral antagonists, 1237–8 type I, 58–60, 61, 549–58 antiproliferative functions, 555–6 antiviral mechanisms, 553–4 apoptosis regulation, 556 CSF-1, 937 genes, 549–50 immunomodulation, 556 inducers, 552 NK cells, 556 receptors, 553 signal transduction, 553, Plate 23.2 Stat proteins, 554, Plate 23.2 structure, 552, Plate 23.1 subtypes, 549–50 T cells, 557 therapeutic applications, 557–9 type II, 60–1, 567 see also named IFNs interleukins antiangiogenesis, 1286–7 phylogeny, 61–71 terminology, xxv see also named ILs intestinal epithelial cell 6 (IEL-6), 367 intestinal intraepithelial lymphocytes (iIEL), 320–1 intracellular adhesion molecule(s) (ICAMs) IFN- receptor, 575 IL-17 enhanced surface expression, 486, 495 intracellular adhesion molecule 1 (ICAM-1), 243 IL-1, 643 inhibitor, 1355, 1358–9 intracytoplasmic cytokine (ICC) assays, 1390 invertebrates, 57 FGF, 74–5 IL-6, 66 TGFb, 73 TNF, 71 IRAKs, 659–60 IL-1R signaling, 735 IL-18R, 659

IL-18R signaling, 716 IRF-1 transcriptional factor, 579 induction, 12 inhibition, 131 IRF-3 transcriptional factor, 552, 1235–6 IRF-7 transcriptional factor, 552 Irinotecan see CPT-11 IRS-1, 236 IRS-2, 236 IL-9, 357 IL-13 signaling, 411 ISGF3 complex, 88, 554, Plate 23.2 ISIS 2302, 1355, 1358–9, 1363 ISIS 2503, 1359 ISIS 3521, 1359 ISIS 5132, 1359 ISIS 104838, 1363 ISRE, 554, Plate 23.2 JAB, IL-2 signaling, 183 Jab1, 1043 JAK kinases, 10, 85–7 activation, 10, 234 G-CSF signaling, 525, 535 IFN- signaling, 573–7 IFN signaling, 1234, 1237 IL-2R signaling molecules, 177 IL-3, 212, 214 IL-4, 1316 IL-11 signaling, 371 JAK1, 10, 85 activation, 86 G-CSFR signaling, 535 IL-2R signaling molecules, 177 IL-6, 289 IL-7, 312 IL-9 receptor, 356, 357 IL-10R signaling, 614 IL-13 signaling, 411 IL-15, 438 Stat6 activation, 235 JAK2, 10, 85, 115 activation, 86, 126, 130 erythropoietin receptor, 154–5 G-CSFR signaling, 535 GM-CSF receptor signaling, 506 IL-2 signaling, 181 IL-2R signaling molecules, 177 IL-6, 289 IL-11, 372 IL-12 receptor, 389 IL-15, 438 Stat5 signaling, 127–8 Stat6 activation, 235 substrates, 126–7 tyrosine phosphorylation, 130

INDEX

JAK3, 10, 85, 86–7 c association, 186 IL-2 signaling, 181, 182 IL-2R signaling molecules, 177–8 IL-4 signaling, 236 IL-7, 312 IL-9 receptor, 356 IL-13 signaling, 412 IL-15, 438 immunotherapy target, 187 tyrosine phosphorylation, 236 JAK–STAT signal transduction pathway, 10, 11, 127–8, 235 CSF-1, 936 IFN-, 574–80 through IL-6R, 289–91 Janus kinases see JAK kinases Jehovah’s Witnesses, 1267 JNK kinases, 129, 213–14, 481 activin, 1161 CD95 signaling, 899 IL-1, 660 IL-17, 481 TGFb-induced expression, 1136, 1137 TGFb signaling, 1133, 1135 TNF production, 840 c-jun, 11 expression regulation by IL-2, 181–2 IL-3, 215 TGFb-induced expression, 1136 jun activation-domain binding protein (Jab1), 1043 Jun N-terminal kinase (JNK) see JNK kinases Kaposi’s sarcoma antiangiogenesis, 1281 CC chemokines, 1093 CXC chemokines, 1066 FGF2, 750, 765 FGFs, 758, 759, 763, 764 GM-CSF, 511 IFN-a immunotherapy, 1225 IFN type I, 557, 559 IL-6, 292 see also human herpesvirus 8 (HHV8) Kaposi’s sarcoma cells, 750 keratinocyte growth factor (KGF), 322, 632 keratinocytes activin, 1163 apoptosis, 444, 445, 447 epiregulin, 975 IL-1 family, 647

IL-1Ra, 677 IL-4, 497 IL-8, 1062 IL-10, 607 IL-15, 433, 434, 444–5, 447 IL-17, 497 IL-18, 712–13 IL-20, 632 MIF expression, 1038, 1042 kidney erythropoietin, 151 HGF in development, 789 MIF, 1042 prolactin receptors, 133 kidney disease chronic renal failure, 157 CX3CL1, 1104 CXC chemokines, 1068 EGF precursor, 962–3 IgA nephropathy, 451 IL-1Ra, 682, 689 gene therapy, 695–6 IL-18, 722 KILLER, 863 c-kit, 991, 992 down-regulation, 208–9 structure, 992 LAK cells IL-2, 172 IL-2-induced, 325 IL-4, 242 IL-7 cancer therapy, 325–6 IL-12, 392 IL-15-induced, 450, 452 latency-associated peptide (LAP), 1120, 1121, Plate 49.1 latent TGFb-binding protein (LTBP), 1120 lectins, mannose-binding, 991 Leishmania infection, IL-7, 329, 330 lentiviruses, 1338 leucine-rich repeats (LRRs), 814, 815, Plate 33.4 leukemia B-cell, 1217–18 chronic lymphocytic, 324 FGF2, 765 Flt3, 997–9 G-CSF, 533, 534 G-CSFR, 534 constitutive signaling, 538 GM-CSF, 511–12, 513, 1269 HTLV-1, 1216–17 IFN-a immunotherapy, 1225 IFN type I, 557, 558, 559 IL-1R, 662

liii IL-1Ra, 683 IL-2, 171, 1216–17 IL-3, 216–17 IL-7, 324 IL-9, 350, 351, 353 IL-10, 1217–18 IL-15, 449, 1221–2, Plate 53.4 IL-17, 491 NFB, 1215 prolactin, 135 stem cell mobilization, 1270 leukemia inhibitory factor (LIF), IL-17-induced, 488 leukemia inhibitory factor (LIF) receptor, 1263 leukemic cells, differentiation induction, 284 leukemogenesis, 1222, Plate 53.4 leukocyte functional antigen 1 (LFA-1), 243, 1270 leukocytes anti-TNFa therapy, 1199 CC chemokines, 1084, 1088 CX3CL1, 1104, 1106 CX3CR1, 1107–8 function modulation, 3 LIGHT, 825 limb morphogenesis, 1182 limitin, 550 lipid metabolism, IL-15, 445 lipopolysaccharide (LPS), 811 activin release activation, 1167 chemokine receptors, 1090 HMGB1 release, 916–17 IL-1b-deficient mice, 651 IL-1Ra in blood, 677 IL-1RI, 653 IL-18 induction, 711, 712 interleukin stimulation, 242, 243 macrophage activation, 367 monocyte activation, 415 sensor, 811–12 signaling pathway, 812 TLR4 activation, 818 5-lipoxygenase, 1060 15-lipoxygenase, 414–15 liver CD95/CD95L, 892 erythropoietin, 151 injury and IL-18-deficient mice, 719 prolactin receptor up-regulation, 125 liver transplantation Flt3, 1002 hepatitis C, 1250 long signal peptide (LSP), 436, 437 see also IL-15

liv lovastatin, 1094 LTa, 825 cancer, 1219 expression, 827–8, 830 lymph nodes, 829–30 lymphoid tissue biogenesis, 828–9 spleen, 829–30 structure, 826–7, Plate 34.3 LTb, 825 expression, 827–8, 830 lymph nodes, 829–30 lymphoid tissue biogenesis, 828–9 spleen, 829–30 structure, 826–7 LTb receptor, 828 lymph nodes, 829–30 signaling, 827, 832 spleen, 829–30 lung antigen presenting cells, 1324 dendritic cells, 1324 HGF in development, 789 TGFa expression, 966 lung cancer IL-3, 216 IL-18, 722 resistance, 851 lung disease allergic, 417–18 CXC chemokines, 1067–8 IL-1Ra, 681, 689 gene therapy, 696 inflammatory, 418 GM-CSF production, 510 Lyme arthritis, 478 Lyme disease, 494 lymph nodes, lymphotoxin, 829–30 lymphocyte-activating factor (LAF), xxiv, 4 lymphocyte-derived chemotactic factor (LCF), xxiii lymphocytes, xxiii activation-induced cell death, 719 activin, 1165 homeostasis, 331–2, 442, 446 IL-15 regulation of development, 439–41 intrathymic positive selection, 314 TGFb-mediated apoptosis, 1139–40 traffic regulation by CC chemokines, 1091–2, Plate 47.2 see also B cells; CD4+ T cells; CD8+ T cells; cytotoxic T cells (CTL); T cells; TH1 cells; TH2 cells lymphoid cells, IL-3, 204 lymphoid follicle formation, 831

INDEX

lymphoid microarchitecture formation, 830–1 lymphoid tissue biogenesis, 828–9 lymphokine-activated killer cells see LAK cells lymphokines, xxiii, 20 discovery, xxiii nomenclature, 5 research, 3, 4 lymphoma, 449 B cell, 1217–18 cutaneous T-cell, 323, 449 G3139, 1359 IFN-a immunotherapy, 1225 IFN type I, 557, 558 IL-1Ra, 683 IL-2, 171 IL-7Ra, 324 IL-9, 350, 351 IL-10, 1217–18 IL-13, 419 IL-15, 449, 1222, Plate 53.4 IL-16, 469 NFB, 1215 prolactin, 135 stem cell factor, 1014 T-cell, 323–4 lymphopoiesis, IL-7 mediated, 305 lymphoproliferative disease, CD95, 889, 890 lymphotoxin (LT), xxiv, 825–32, Plate 34.1 B cells, 830 cancer, 1219 expression regulation, 827–8 functions, 828–32 immune response, 829–30, 831–2 immune system, 831–2 infection, 832 lymph nodes, 829–30 lymphoid follicle formation, 831 lymphoid microarchitecture formation, 830–1 peripheral lymphoid tissue biogenesis, 828–9 signal transduction, 827, 829–31, Plate 34.4 spleen, 829–30 T cells, 830 tissue distribution, 827–8 TNFR, 825, 826–7, Plate 34.1 see also LTa; LTb Lyn expression, 155, 537 M-Ras, 357 macrophage-activating factor (MAF), xxiv

birds, 60 IFN-, 581 macrophage colony-stimulating factor (M-CSF), xxiii, 927–47, 1263 cancer, 947 cells, 945–6 cell surface, 928 cellular sources, 929 clinical trials, 947 gene regulation, 928–9 structure, 928 HIV infection, 946 IL-4, 242 IL-6 synergy, 283 infection, 946, 947 inflammation, 946 microglia, 945 osteoclasts, 944–5 protein structure, 929 soluble, 928 macrophage colony-stimulating factor (M-CSF) receptor, 7 macrophage-derived chemokine see CCL22 macrophage inflammatory protein 1 (MIP-1), 1366 macrophage inflammatory protein 2 (MIP-2), 1362–3 macrophage inflammatory protein 3a (MIP-3a), 492 macrophage inflammatory protein 3b (MIP-3b), 1306 macrophage inflammatory proteins, 1084 macrophage migration inhibitory factors (MIF), xxiv, 4, 1037–44, 1045 activity, 1038–9, 1042–3 ARDS, 1041–2 asthma, 1041 cancer, 1043, 1215–16, Plate 45.4 cytokine expression, 1039–43 disease states, 1039–43 gene structure, 1038, Plate 45.1 host response, 1040 immune system, 1040–1 infection, 1040, Plate 45.3 p53 inhibition, 1043 PAG interaction, 1043–4 pathologic process association, 1045 properties, 1045 protein preformed stores, 1038–9 production, 1038 structure, 1038–9, Plate 45.2

INDEX

receptors, 1043–4 regulation, 1038, 1039–43 rheumatoid arthritis, 1041 sepsis, 1040–1 signaling, 1044 tumor growth promotion, 1043, Plate 45.4 macrophage-stimulating protein (MSP), 786–7 macrophages, 1257 activin, 1166 CSF-1, 932, 943 CX3CL1, 1106 G-CSF, 527 GH effects, 108 GM-CSF, 507 HMGB1, 918 IFN-, 581–2 IL-1 family, 647, 649 IL-1Ra, 677, 679 IL-2R complex, 1225 IL-4 effects, 242–3 IL-6 effects, 286, 290 IL-9, 351 IL-10, 607–8 IL-11, 367 IL-13, 415 IL-15, 433, 434, 443 IL-18, 711 TH2 cytokine induction, 718 LPS activated, 367 MIF expression, 1038 macroprolactins, 117 c-maf, 229 MAL, 814, 816 TLR signaling, 817 malignancy hypercalcemia, 880 IL-7, 323–4 see also cancer; tumor(s) mammals, 57 mammary gland amphiregulin, 969 BTC activity, 973–4 EGF, 964 prolactin effects, 131–2 prolactin receptor upregulation, 125 RANKL in development, 876 TGFa, 966, 967 mannose receptors, 415 marimastat, 1288 mast cell growth-enhancing activity (MEA), 347 mast cells IL-3 production, 206, 207, 208 IL-4, 242

production, 233 signaling, 236 IL-5 production, 233 IL-9, 347, 348–9, 354, 355 IL-13, 416 IL-15, 438, 444 mastocytes, CX3CR1 expression, 1107, 1108 mastocytosis, IL-3 induction, 207 matrix metalloproteinase (MMP) antiangiogenesis, 1285, 1288 endostatin interaction, 1284 FGF2, 754, 761, 762 FGFs, 761, 764 HSPGs, 754 IL-1b cleaving, 649 IL-10, 610–11 IL-18, 712 inhibitors, 842 rheumatoid arthritis, 492, 1202 TNF post-translational regulation, 841 MD-2 protein, 815 MDA-7 see IL-24 megakaryocyte growth and development factor (MGDF), 1268–9 megakaryocytes IL-6, 284 IL-11, 366–7, 371 thrombopoietin, 1268 MEK kinase 1 (MEKK1), 616–17, 845 melanocytes, 109 melanoma CXC chemokines, 1069 CXCL1, 1062 cytokine gene transfer, 1341 G3139, 1359 GM-CSF, 1269 IFN-a immunotherapy, 1225 IFN type I, 557, 559 IL-7 immunotherapy, 328 IL-12 therapy, 397, 398 IL-18, 713, 722 melanoma-stimulatory activity see CXCL1 membrane-anchored metalloprotease-disintegrin glycoprotein (ADAM), 970–1 Mendelian susceptibility to mycobacterial disease (MSMD), 587, 588, 589–90 meningitis, IL-10 levels, 23 menstrual cycle angiogenesis, 1281 IL-1Ra, 678 IL-15 effects, 433

lv mesangial cells, IL-13, 415 mesenchymal cells, 1179 mesothelial cells, G-CSF, 527 MET, 784, 791–8 cancer, 795, 796, 797 prognostic use, 795, 797 co-receptors, 797 disease states, 795, 796, 797 docking sites, 798 embryogenesis, 794–5 endocytosis with E-cadherin, 797 exons, 793–4 expression, 794–5 gene organization, 792–4 promoter structure/function, 794 related receptors, 792 signaling, 797–8 skeletal muscle generation, 788–9 c-met proto-oncogene, 791–2 expression, 794–5 metastases, 1220–1, 1376 bone, 879–80 methotrexate, 1197, 1198, 1199, 1201 2-methoxyestradiol (2-ME), 1287 2O-(2 methoxyethyl) chimeras, 1359–61 mevastatin, 1094 MG132 proteasome inhibitor, 578 MHC, 1237 autoimmunity, 1189–90, 1192 class I, 19, 550 genes, 57 IFN-g, 582–3, 586 IFNs, 1237 class II, 19 genes, 57 IFN-g, 583 IFNs, 1237 immune function, 24 TNF, 24 cytokine effects, 24 role, 1189–90 vertebrate genes, 57 microbial molecules, 810–11 microglia CX3CL1, 1107 CX3CR1, 1108–9 M-CSF, 945 microplate-based assays, 1385 microtubule (MT), 1130 mini-chromosome maintenance 5 (MCM5), 575 minor histocompatibility antigen, 1303 vMIPII, 1111

lvi mitogen-activated protein kinase (MAPK) activation, 481 activin, 1159 CD95 signaling, 899 CX3CR1 signaling, 1109–10, Plate 48.4 cytoplasmic cross-talk with Smads, 1134–5 family, 129 FGF2, 761 FGFR signaling, 767 G-CSFR-induced activation, 536, 537 HMGB1, 920 IL-1R binding, 660 IL-3-induced activation, 213 IL-10, 616 IL-15R signaling, 438 IL-17, 481 IL-18R signaling, 716 nuclear cross-talk with Smads, 1133–4 phosphorylation, 1126 Ras, 931–2 rheumatoid arthritis, 1202–3 Shc-Ras-Raf pathway, 179 signaling pathway, 155–6 TGFb, 1136 signaling, 1132–5 TNF production, 840 TNFa, 1202–3 see also p38 MAP kinase mitogen-activated protein kinaseactivating protein-2, 661 ML-1, 484–6 MLV vectors, 1337–8 molecular mimicry, 1192 monensin, 1387, Plate 61.5 monocyte chemotactic protein 1 (MCP-1), 580, 1083, 1084, 1088, 1089 antagonists, 1095 anti-TNFa therapy, 1199 antisense oligonucleotides, 1366 cancer, 1092, 1093–4 incidence, 1222 dendritic cell production, 1091 inhibition, 1094 monocyte chemotactic protein 2 (MCP-2), 1089 monocyte chemotactic protein 3 (MCP-3), 1089, 1092 monocyte chemotactic protein 4 (MCP-4), 1089 monocytes, 4, 1257 activin, 1166

INDEX

CSF-1, 943 CX3CR1, 1107, 1108 G-CSF, 527 GM-CSF, 507 IL-1 family, 647, 649 IL-1Ra, 677 IL-2, 172 IL-4, 242–3 IL-4-induced inhibition of cytokines, 236 IL-9, 351 IL-10, 607–8 IL-13, 414–15 IL-19, 631 inflammatory pathway, 615 LPS-activated, 415 MIF expression, 1038 migration, xxiv monokine induced by IFNg (MIG) see CXCL9 monokines, xxiii, xxiv, 4, 20 leukocyte function modulation, 3 monopoiesis, 928 MRA see anti-IL-6 receptor antibody mRNA measurement, 1385 mucin production, IL-9, 352 mucositis gastrointestinal, 374–5 GM-CSF, 512 multiple myeloma apoptosis, 449 IL-6, 879, 1217, 1218, 1220, 1222–3 osteoprotegerin, 879 PTHrP, 879 RANKL, 879 thalidomide, 1223, 1281 multiple sclerosis activation-induced cell death, 894 apoptosis, 895 CD95/CD95L, 894–5 CX3CL1, 1107 IFN-b therapy, 1200–1 IFN type I, 557, 559 IL-1Ra, 680 IL-15, 448 IL-17, 495 IL1RN gene association, 684 sex bias, 1190 T cells, 894 tissue damage, 1191 Mx gene, 58 proteins, 553, 1235, 1236–7 Mx GTPase, 1236–7 myasthenia gravis IL-18, 722 sex bias, 1190

c-myb, 181–2 c-myc expression regulation by IL-2, 181–2 IL-3, 215 mycobacterial infection IFN- signaling defects, 587, 588, 589–90 IL-7, 328, 329 IL-15, 452–3 IL-16, 470 IL-18, 720 Stat1 mutation, 590 mycosis fungoides, IL-15, 449 MyD88, 659–60 IL-18R, 659–60, 716, 717 TLR proteins, 814, 815–16 signaling, 817 myelin basic protein (MBP), 1192 myelodysplasia/myelodysplastic syndromes erythropoietin, 158 Flt3, 997 GM-CSF, 511, 513 myeloid cells Flt3, 995–6 G-CSF, 530 GM-CSF receptors, 505 IL-4 signaling, 236 VEGF-induced differentiation, 1026–7 myeloma cells IL-6, 293 IL-15, 449 myelomonocytic cells, 242–3 myelomonocytic growth factor (MGF), 65 myelopoiesis G-CSFR deletion, 531 Stat3 isoform expression, 538 myelosuppression chemotherapy-induced, 532 G-CSF, 532 GM-CSF, 509, 510 rhIL-11 pharmacology, 373, 374 myocardial infarction, 681–2 myositis ossificans progressiva, 1182 myostatin, 1157, 1162 MZ (marginal zone) cells, 317 N-SMase, 845 Na+/H+ antiport activity, CSF-1, 933–4 Nb2 lymphoma cell line gene regulation, 131 prolactin activity, 125, 126, 129 Nck insulin receptor substrate, 129

INDEX

neonates paraventricular leukomalacia, 724 premature and GM-CSF, 511, 513 sepsis G-CSF, 533 GM-CSF, 511 neoplasia, IL-4 experimental model, 247–8 neovascular disease, FGFs, 758, 763 nephrotic syndrome, minimalchange, 451 nerve growth factor (NGF), 3, 484 nervous system erythropoietin, 159 FGFs, 757, 762 HGF expression, 789 IL-1Ra, 680, 688 IL-3, 205 IL-5, 273 IL-7, 323 IL-15, 436 IL-18, 713 TGFa expression, 966 neural cells, erythropoietin function, 159 neuregulin, 959 neurodegenerative disease, FGFs, 758 neurokinin A, 496 neurons, CX3CR1, 1108–9 neuropilin-1 (NP-1), 1020, 1022, 1022, Plate 44.1 signal transduction, 1024 neutral sphingomyelinase domain (NSD), 845 neutropenia chemotherapy-induced, 510, 511, 532 G-CSF, 532 sepsis complication and GM-CSF, 510–11 stem cell factor clinical use, 1012 neutrophil activating protein 2 (NAP-2) see CXCL7 neutrophils G-CSF, 525, 530, 531–2, 533 G-CSFR deletion, 531 GM-CSF, 506 IL-1Ra, 679 IL-2, 172 IL-4 administration, 247 IL-6, 286 IL-8, 1061 IL-13, 416 IL-15, 444 IL-17, 496 stem cell factor, 1012 stimulation of production, 1268

Newcastle disease virus, 59–60 NFB activation, 10, 11 anti-cytokine cancer therapy, 1223 binding protein induction, 287 cancer, 1215, Plate 53.2 therapy, 1223 CD95 signaling, 899 CD95L, 887 glucocorticoid regulation, 481 HMGB1, 920 IL-6, 287 IL-8, 1057 IL-9R, 357 IL-10, 616 IL-11, 367 IL-17, 479, 481 multiple sclerosis, 495 IL-18R signaling, 716, 717 immune response, 814 LT-TNF receptor signaling, 827, Plate 34.4 reactive oxygen intermediates, 1066–7 rheumatoid arthritis, 1201–2, 1203 TLR4, 815 TNF, 839, 840 TNF-a production, 1201–2 TNF-induced, 846 TNFR1, 845 VEGF, 1027 NFB-inducing kinase (NIK), 827, 831 NFB/Rel factors, 1138–9, 1140 NIL-16, 471 nitric oxide ICE inhibition, 712 IFN-, 582 IL-18 inhibition, 712 multiple sclerosis, 495 osteoarthritis, 493 TNF-induced, 848 VEGF-R2 stimulated, 1024 nitric oxide synthase, constitutive, 1024 nitric oxide synthase, inducible, 582 IFNs, 1237 IL-1, 643 NK cells, 4 CX3CR1, 1107, 1108, 1110 cytolytic activity effects of IL-18, 718 Flt3, 996 GH effects, 107, 108 IFN-, 569–70 IFN type I, 556 IL-1 family, 647 IL-2, 171–2

lvii IL-2R complex, 1225 IL-4, 241–2 administration, 247 IL-10, 607 IL-12, 392 IL-13, 413 IL-15, 440, 441, 451, 1214–15, Plate 53.4 regulation by, 441–2 IL-18R, 715, 717 IL-21, 456 TRAIL/Apo2L, 863, 864 tumor resistance, 1224 NK receptors (NKR), 1224 NK-T cells, 232–3 GM-CSF, 507 IL-18, 718 noggin, 1182, Plate 51.1 non-steroidal anti-inflammatory drugs (NSAIDs), 1215 novel erythropoiesis-stimulating protein (NESP), 157 NRDc, 971 nuclear factor of activated T cells (NFAT), 839, 840, 886 nuclear localization sequences (NLS) of FGF, 749, 750, 751, Plate 31.1 obesity, 285 ODF expression, 493 2,5 oligoadenylate synthetase (OAS), 1235, 1236 family, 580–1 system, 553 oligonucleotides, 1349, 1350 antisense, 1351–2 capped, 1353 chimeric, 1351 medicinal chemistry, 1353 phosphorothioate, 1353–9 RNase H activation, 1350–2 oncostatin M, 182–3, 1263 OPG production, 494 orf virus (OV), 606 organ transplantation, 1297–307 allograft acceptance, 1301–5, 1343 Flt3, 1002 gene transfer, 1343 IL-1Ra, 683–4, 690, 691 IL-10, 611 IL-12, 396, 397 IL-15, 451 IL-17, 489–90 kidney, 490 tolerance induction, 1001–2, 1301–5

lviii organ transplantation (continued) see also allograft rejection; liver transplantation organogenesis, FGFs, 756 osmoregulation, prolactin effects, 133, 135–6 osteoarthritis IL-1Ra, 679, 688, 698 gene therapy, 694 IL-11, 368 IL-17, 493 osteoblasts, 1179 IL-11, 367–8 ODF expression, 493 osteocalcin, 1179 osteoclasts bone resorption in rheumatoid arthritis, 494 development, 285 IL-1Ra, 682 IL-6, 285 IL-11, 367–8 IL-15, 445 M-CSF, 944–5 RANK, 876 RANK/RANKL, 875–6, Plate 37.1 osteogenic protein 1 (OP-1), 1181, 1183 osteogenin see BMP3 osteoporosis IL-1Ra, 682, 689–90 osteoprotegerin, 878–9 postmenopausal, 285 RANKL/RANK, 878–9 osteoprotegerin, 863, 871, 872, 873 chromosome location, 877 M-CSF, 945 metastatic bone disease, 880 multiple myeloma, 879 osteoporosis, 878–9 RANKL activity regulation, 877 association, 876 rheumatoid arthritis, 879 structure, 877 osteosarcoma, BMP4, 1182 ovarian cancer IL-12 therapy, 398 IL-17, 490 stem cell factor, 1014 ovarian TNF receptor (OTR), 72 ovaries IL-1Ra, 678 prolactin effects, 132 ovulation, 678 oxygen sensing mechanisms, 152

INDEX

P-selectin, 415 P2X-7 receptor, 649–50 p19, 384–5, 387, 393–4 p28, 385, 388, 394–5 p35, 384, 385 p38 MAP kinase, 213–14 activation, 660 FGFR signaling, 767 IFN type I, 554, Plate 23.2 IL-1, 660–1 IL-10, 616 inhibition, 660–1 TNF-a expression, 1203 TNF production, 840 p38Hog, 129 p40, 385, 387–8, 394 p53 activin, 1160 cancer, 1215–16 CD95, 889 IL-4 expression, 229–30 inhibition by MIF, 1043 p97/Gab2, 178 p150SHIP, 936 p200, 556 p202, 556 PAG (proliferation association gene), 1043–4 Paget’s disease, 878 pain, neuropathic, 680 PAK1 (p21-activated kinase 1), 1133 pancreas BTC activity, 973 MIF expression, 1038 pancreatic carcinoma, 1069 parasitic infections IL-9, 354 IL-18, 720 parathyroid hormone, 945 parathyroid hormone-related peptide (PTHrP) breast cancer, 880 multiple myeloma, 879 paraventricular leukomalacia, 724 PARP (poly(ADP-ribose) polymerase), 1140 Pax5, 316 peripheral blood mononuclear cells (PBMC) assay, 1385–6, Plate 61.3 peripheral blood progenitor cells, 1012, 1269–70 peripheral blood stem cells, 510, 513 peripheral tissues CX3CL1, 1104, 1106 CX3CR1 expression, 1107–8 phosphatases CSF-1, 935–6

IFN- signaling, 576 phosphatidyl-inositol 3-kinase (PI3K), 10, 129, 156 CSF-1, 934–5 CX3CR1 signaling, 1110, Plate 48.4 c-Fms, 941 G-CSFR signaling, 537 IL-2 signaling, 179 IL-3, 212, 213, 214–15 IL-4, 234, 235 IL-7, 312 IL-10R signaling pathway, 615 IL-15R, 438 phospholipase, 932–3 phospholipase A, 643 phospholipase A2, 1044 phospholipase C, phosphatidylinositol-specific (PI-PLC), 11 phospholipase C-gamma (PLC-), 156 VEGF-R2 stimulated, 1024 phosphorothioate oligonucleotides, 1353–9 absorption, 1354 aerosol administration, 1355 cellular uptake, 1354 clearance, 1355 clinical activities, 1358–9 clotting inhibition, 1356, 1357–8 complement activation, 1356, 1357 distribution, 1354–5 human safety, 1357–8 immune stimulation, 1356–7 localization, 1355 pharmacokinetics, 1354–5 platelets, 1358 protein interactions, 1353–4 topical administration, 1355 toxicological properties, 1356–7 phylogeny of cytokines, 57–76 colony-stimulating factors, 65–6 growth factors, 73–6 interferons, 58–61 interleukins, 61–71 TNFs, 71–2 PIAS, 93–4, 291, Plate 12.8 IFN- signaling, 577–8 IL-6 signaling, 291 PIAS1, 93, 94 PIAS3, 93–4 pigment epithelium-derived factor (PEDF), 1282 pit-1 gene, 104, 116 pituitary anterior, 119 IL-18, 713

INDEX

placenta, prolactin effects, 132 placental lactogen, 123 plasma cell neoplasia, 293–4 plasminogen, 1286 kringles, 1284 plasminogen activator inhibitor 1 (PAI-1), 761 platelet activating factor (PAF), TNF-induced, 848 platelet basic protein (PBP), 1054–5 platelet-derived growth factor (PDGF), 3, 4, 76 platelet-derived growth factor (PDGF) receptor, 7 platelet factor-4 see CXCL4 platelets phosphorothioate oligonucleotides, 1358 production stimulation, 1268–9 transfusion, 374 PlGF, 1020, 1025 pneumonia, bacterial, 496 polycystic kidney disease, congenital, 962–3 polymorphonuclear leucocytes (PMNs), 450–1 poxvirus protein, 716 PP2A, 936 PPRIV, 171, 174–5 pre-pro B-cell factor (PPBSF), 308, 310 cofactor, 310 pregnancy angiogenesis, 1281 IL-1Ra, 678, 682 IL-15 effects, 433 pro-IL-16, 470–1, Plate 19.3 pro-IL-18, 711–12 pro-TNF, 838 progenitor cells, 315 prolactin, 115–37 activities, 116 autoimmune disease, 135 backup mechanisms for functions, 124 behavioral effects, 133 biological effects, 131–5 breast cancer, 136 calcium metabolism, 134 cellular sources, 119 disease states, 120–1, 135–6 expression, 118, 119 gene activation, 122 chromosome location, 116 regulation, 116–17, 131 structure, 116–17

glycosylation, 118 gonad effects, 132 hematopoiesis, 134, 135 homologies, 118 immune system effects, 131, 134 inhibitors, 119 mammary gland effects, 125, 131–2 MAPK activation, 129 metabolism, 134 modulators, 119–20 normal levels, 135 osmoregulation, 133, 135–6 overexpression, 125 pathophysiological role, 116 phosphorylation, 118–19 PI3K activation, 129 placental effects, 132 posttranslation modifications, 118–19 proangiogenic effects, 132 prostate cancer, 136–7 prostate effects, 125, 132–3 protein, 117 proteolysis, 118 psychological effects, 133 regulation, 119–20 reproductive effects, 131–2, 135 skin effects, 134–5 SOCS upregulation, 129–30 Src tyrosine kinase activation, 128–9 Stat activation, 127–8, 130–2 stimulators, 119, 120 stress, 133–4 synthesis, 116 uterus effects, 132 in vitro activity, 131 in vivo activity, 131–5 prolactin receptor, 106, 107, 121–5 binding, 123 cell lines expressing, 125 cytokine receptor homology (CRH) region, 122 cytoplasmic domain, 123 dimerization, 125–6 down-regulation, 125 expression, 123–5 extracellular domain, 122–3 gene human, 121–2 rodent, 122 inactive dimers, 126 isoforms, 125 Jak2 activation, 126, 130 substrates, 126–7

lix kidney, 133 ligand specificity, 123 mammary gland effects, 131–2 protein, 122 signaling, 122 Jak2-Stat5 pathway, 127–8 signal transduction, 125–31 splice variants, 130 splice variants, 122, 130 Stat5 activation, 128 transmembrane domain, 123 up-regulation, 125 proliferation assays, 1380 proline-directed kinases, 1126 prop-1 gene, 104 prostaglandin E2 inhibition, 494 osteoarthritis, 493 prostate prolactin effects, 132–3 prolactin receptor upregulation, 125 TGFb-mediated apoptosis in epithelium, 1137–8 prostate cancer IL-15, 443–4 prolactin, 136–7 TGFb, 1137 prosthetic joints hip replacement, 368 IL-1Ra gene therapy, 695 protease-activated receptors (PARs), 712 protein inhibitors of activated stats see PIAS protein kinase A (PKA) IL-17 expression regulation, 482 MIF signaling, 1044 protein kinase C (PKC) activators, 659 CD95 up-regulation, 889 CSF-1, 933 IL-1 signaling, 657, 659 IL-11 signaling, 372 IL-13, 414 inhibitors, 841, 1359 protein kinase R (PKR), 554, 580, 581, 1235, 1236 protein metabolism, IL-15, 445 proteinase 3, 712 proteoglycan macrophage colonystimulating factor (PG-M-CSF), 928 proteoglycans, HGF–MET signaling, 797 proteomics for cytokines, 1389–90 proto-oncogenes, 1219–20

lx provirus insertion in murine leukemia 1 (PIM-1), 580 Pseudomonas exotoxin A (PE), 250 psoriasis amphiregulin, 969 anti-TNFa therapy, 1199–200 CX3CL1, 1106 CXC chemokines, 1067 ICAM-1 inhibitor, 1355 IL-1Ra, 680–1 IL-6, 292 IL-11, 369 IL-15, 447 IL-17, 496–7 IL-20, 632 ISIS 2302, 1358 pulmonary alveolar proteinosis (PAP), 503, 509, 512, 513 pulmonary fibrosis, idiopathic, 1067–8 Pyk2, 312–13 pyrogen assay for IL-1, 1377 radioimmunoassay (RIA), 1382, 1383 radiotherapy Flt3, 997, 1003 rhIL-11, 373, 374–5 mucositis, 512 Raf-1, 129 RAG expression, 313 B cell differentiation, 315 IL-7Ra, 320 RAGE, 918–20 RANK, 871, 872, 873 bone, 876 disorders, 877–8 CSF-1, 943, 944 dendritic cells, 876–7 T cells, 876–7 tissue distribution, 876 RANKL, 481, 494, 871–80 biological activity, 873, 875 chromosome location, 873 CSF-1, 943–4, 945 expression, 873, 875, 876 gene promoter, 875 structure, 873 giant cell tumor of bone, 879 immune system, 875 metastatic bone disease, 879–80 multiple myeloma, 879 osteoprotegerin regulation of activity, 877 rheumatoid arthritis, 879 structure, 873, 874 RANKL/RANK

INDEX

osteoclastogenesis, 875–6, Plate 37.1 osteoporosis, 878–9 pathway, 873, 875 RANTES, 22 Ras activation and CSF-1, 930–1 activator, 129 G-CSFR activation, 536 IL-3-mediated activation, 212–13 MAPK, 931–2 TGFb signaling, 1132, 1134, 1135 M-Ras, 357 reactive oxygen intermediates, 1066–7 Rebetron, 1247 receptor activator of the NFkB ligand see RANKL receptor activator of the NFkB receptor see RANK receptor for advanced glycation endproducts see RAGE receptor tyrosine kinases (RTKs), 1219–20 recombinase system, 811 red cell production stimulation, 1267 regulator of G protein signaling (RGS), 1059 Rel family activation, 10 renal cell cancer IFN-a immunotherapy, 1225 IL-7, 323, 326 IL-11, 369 IL-12 therapy, 397, 398 IL-13, 419 renal failure, chronic, 157 renal transplantation, 490 reperfusion injury, CXC chemokines, 1066–7 reproduction angiogenesis, 1280–1 IL-1Ra, 678, 682 inhibins, 1155 prolactin effects, 131–2, 135 reptiles IL-2, 64 interferons, 58–9 respiratory syncytial virus, 1111 restin, 1286 retinal pigment epithelium, 323 retroviruses, gene transfer vectors, 1337–8 reverse transcriptase–polymerase chain reaction (RT-PCR), 1387–8 rhabdomyosarcoma cell lines, IL-15, 433, 434 rheumatoid arthritis

angiogenesis, 1282 anti-IL-1 therapy, 1200 anti-IL-6 receptor antibody, 294 therapy, 1200 anti-TNFa therapy, 1197, 1198, 1199, 1201, 1202, Plate 52.6 cartilage loss, 1364 CX3CL1, 1106 CXC chemokines, 1068–9 cytokine system, 1193, 1194, 1195–6 cascade, 1195 FGFs, 758, 759, 763 HMGB1, 920 IL-1b, 1344 IL-1R, 662 IL-1Ra, 679–80, 687–8, 691–2 combination therapy, 698 gene therapy, 693–4, 696, 698 IL-6 abnormal production, 292–3 IL-11, 368 IL-15, 447–8, 451 IL-16, 470 IL-17, 491–4 IL-17F, 483–4 MAPK, 1202–3 MIF, 1041 MMPs, 1202 NFB, 1201–2, 1203 osteoprotegerin, 879 prolactin levels, 135 RANKL, 879 sex bias, 1190 T cells, 492 tissue damage, 1191 TNF, 851, 852 TNF-a expression blocking, 1201–3, Plate 52.6 VEGF, 1282 ribavirin, 1246–7 RIP-associated Ich-1/CED-3 homologous protein with death domain (RAIDD), 844–5 rituximab, 1226 RNase H activation, 1350–2 substrate, 1359 RNase protection assay (RPA), 1387, 1388 RNase L, 554, 555, 1235, 1236 RON receptor, 792 RTK oncogenes, 1223 S1, 936 S6 kinase, 933 Salmonella, 66

INDEX

SARA (Smad anchor for receptor activation), 1126–7, 1130 sarcoidosis IL-15, 448 IL-16, 470 SEA receptor, 792 self-antigens, 1190–1, 1192 sepsis HMGB1, 918 IL-1Ra, 687, 690–1 MIF, 1040–1 TNF, 848–9 septic shock, 848–9 severe combined immunodeficiency, X-linked (SCID-X1), 185–6 correction, 186 IL-7, 311 severe combined immunodeficiency (SCID), 315 sex hormones, autoimmune disease, 1190 Sézary syndrome, 449 Shc, CSF-1, 935 Shc-Ras-Raf-MAP kinase pathway, 179 short bowel syndrome, rhIL-11, 377 short signal peptide, 436, Plate 18.1 SHP-1, 156, 935–6 G-CSFR signaling, 536 IFN- signaling, 576 SHP-2, 129, 156 G-CSFR signaling, 536 IFN- signaling, 576 IL-2 signaling, 178, 290, Plate 12.7 IL-6 signaling, 289 IL-9 signaling, 357 IL-15 signaling, 438 sickness behavior, HMGB1, 917–18 signal transducers and activators of transcription see Stat proteins signal transducin factor (STF)-IL-4, 234 signaling/signal transduction, 10–11, 85–94 activin, 1156, 1157 BMP, 1124, 1125 BMP receptors, 11, 1182, Plate 51.1 CD95 inhibitor, 893 CD95 receptor, 896–901, Plate 38.2 CD95L, 888 IL-18, 719, 722 chemokine receptors, 1058–9 CSF-1, 941–2 CX3CR1, 1109–10, 1112, Plate 48.4 cytokine receptors class I, 10 erythropoietin receptor, 154–7 G-CSFR, 530, 534–7, Plate 22.5

GCPR, 970 GM-CSF receptor, 505–6 HB-EGF, 970 HGF, 797–8 HMGB1 receptors, 920 IFN type I, 553, Plate 23.2 IL-1 family, 657–61 IL-1R, 10–11, 655, 735, 817–18, 1196 IL-2, 176–7, 185–6, 440 pathways, 181–3 IL-2 receptor, 10, 177–81 IL-3-mediated, 210–15 IL-4, 234–6, 411 IL-6R, 10 inhibitors, 291 IL-7, 311 defective, 186 IL-7R, 312, 313, 327 IL-9R, 356–7 IL-10R, 612, 613–15 signaling pathway interactions, 615–16 IL-11R, 369, 371–2 IL-12R, 389 IL-13, 411–12 IL-15, 178 defective, 186 IL-15R, 438, 440 IL-16, 468 IL-18R, 655, 716–17 IL-20R, 631 IL-21R, 454–5 IL-22R, 633, 634 interferons, 1234 viral antagonists, 1237–8 lipopolysaccharide pathway, 812 lymphotoxin, 827, 829–31, Plate 34.4 MAPK pathway, 155–6 TGFb, 1132–5 MET, 797–8 MIF, 1044 prolactin pathway, 127–8, 130–1 prolactin receptors, 122, 125–31 TGFa, 965 TGFb, 1124–31, 1132–6, Plate 49.3, Plate 49.5 TGFb receptors, 11, 1122–3, 1124, 1125, Plate 49.3 TLR, 817–18 TNFR, 10, 842, 843–6 TNFR2, 846 TRAIL/Apo2L, 865, Plate 36.4 VEGF, 1022–4, Plate 44.2, Plate 44.3 see also JAK–STAT signal transduction pathway

lxi single nucleotide polymorphisms see SNPs Sjögren’s syndrome, 24 cytokine system, 1196 IL-1Ra, 685 sex bias, 1190 skeletal development abnormalities, 766 skeletal metastases, 879–80 skeletal muscle generation, 788–9 IL-15, 445 Ski, 1129 skin amphiregulin, 969 GH role, 108–9 IL-1a, 650 IL-1Ra, 677, 680–1, 688–9 IL-7, 321 IL-11, 369 IL-13, 418 IL-15, 447 IL-17, 496–7 IL-18, 712–13 IL-20, 632 prolactin role, 134–5 TGFa, 966, 967 skin cancer, 1222 see also melanoma skin disease CX3CL1, 1104, 1106 CXC chemokines, 1067 IL1RN gene association, 684 T-cell accumulation, 1067 see also psoriasis Smad-binding element, 1127 Smad-interacting proteins, nontranscription factor, 1130 Smad-interacting transcription factors, 1128–9 Smad nuclear interacting protein (SNIP1), 1129 Smad signal transducers, 11, 1125–31, Plate 49.4 activin, 1158–9, 1163, 1164 adaptors, 1131 BMPs, 1182 co-repressors, 1129 cytoplasmic cross-talk with MAPK, 1134–5 functions, 1126 inhibitory, 1130 negative regulators of signaling, 1129–30 nuclear cross-talk with MAPK, 1133–4 phosphorylation, 1126–7

lxii Smad signal transducers (continued) TGFb-mediated apoptosis in prostate, 1137 transcriptional activity, 1128–9 smooth muscle cells activin, 1163 GM-CSF, 507 IL-13, 416 Smurf2, 1129, 1130 SnoN, 1129 SNPs, IL-10, 23–4 SOCS, 90–3, 94 cytokine therapy outcome, 1266–7 G-CSFR signaling, 536 GM-CSF receptor signaling, 506 hematopoietic progenitor cells, 1266–7 IFN- signaling, 576–7, 578 IL-2 signaling, 183 IL-6 signaling, 291 IL-10R signaling, 616–17 IL9 signaling, 358 prolactin effects, 129–30 SOCS box, 90 SOCS1, 90, 91–2 CSF-1, 936 G-CSFR signaling, 536 IFN- receptor signaling, 576–7 IL-6 signaling inhibition, 291 IL-10R signaling, 616 SOCS2, 90, 92 G-CSFR signaling, 536 IL-9 signaling, 358 SOCS3, 90, 92–3, 580 G-CSFR signaling, 536 IFN- receptor signaling, 576–7 IL-3-induced up-regulation, 204 IL-6 signaling inhibition, 291 IL-9 signaling inhibition, 358 somatogenic receptor see growth hormone (GH) receptors somatomedin C see IGF-1 somatotropin see growth hormone (GH) sorting nexin 6 (SNX6), 1131 SP1, 11 SPARC, 1155 spleen hemopoiesis, 207 IL-4 administration, 247 lymphotoxin, 829–30 spondylarthropathy, anti-TNFa therapy, 1199 squalamine, 1285, 1287–8 squamous cell carcinoma, 713 Src family kinases c-Fms, 941

INDEX

IL-2 signaling, 179–80 Src homology 2 protein tyrosine phosphatase 1 and 2 see SHP-1 and -2 Src tyrosine kinase, 128–9 FGFR signaling, 767 SSI protein, 183 SSP–IL-15, 436, Plate 18.1 STAM (signal transducing adaptor molecule), 180–1 Stat proteins, 10, 87–90 CD95, 889 CSF-1, 936 erythropoietin receptor, 155 G-CSFR signaling, 535–6 GM-CSF receptor signaling, 506 IFN, 1234, 1237 type I, 553, Plate 23.2 IFN- signaling, 573–8 IL-2R signaling molecules, 177–8 IL-3 signaling, 214 IL-4, 1316 IL-6, 289 IL-7, 312, Plate 13.2 IL-9R, 356–7 IL-10 family, 631, 633, 636 IL-10R signaling, 614–15 IL-12R, 389 IL-13, 410, 412, 1322 IL-15R, 438 IL-18 gene, 711 IL-20R signaling, 631 IL-22R, 633 inhibitors, 577–8 prolactin signaling pathway, 127–8, 130–1 VEGF-R2, 1024 Stat1, 87 activation, 88, 551 erythropoietin, 155 IL-3, 214 IL-9R, 356 prolactin, 128, 131 c-Fms, 941 G-CSFR signaling, 535, 536 GM-CSF receptor signaling, 506 IFN- receptor signaling, 573–5, 579 IFN type I, 554, Plate 23.2 IL-10R, 614–15 IL-22R, 633 mutation in mycobacterial disease, 590 Stat1-dependent genes, 579–80 Stat1-independent genes, 580 Stat2 activation, 88–9, 551

G-CSFR signaling, 535–6 IFN type I, 554, Plate 23.2 Stat3, 87, 551 activation, 89 erythropoietin, 155 IL-3, 204, 214 IL-4, 412 IL-9R, 356 IL-12R, 389 IL-13, 412 prolactin, 128 SHP-2, 290, Plate 12.7 CD95, 889 GM-CSF receptor signaling, 506 IL-6, 289, 290 IL-10R, 614–15 IL-15R, 438 IL-18 gene, 711 IL-20R signaling, 631 IL-22R, 633 isoform expression during G-CSFmediated myelopoiesis, 538 VEGF-R2, 1024 Stat4, 87 activation, 89, 389 Stat5 activation, 128, 551 IL-3, 214 IL-9R, 356–7 erythroid differentiation, 1265 erythropoietin receptor, 155 G-CSFR signaling, 535, 536 GM-CSF receptor signaling, 506 IL-2R signaling molecules, 177–8 IL-7, 312, 313 IL-10R, 614–15 IL-15R, 438 IL-18 gene, 711 IL-22R, 633 prolactin effect mediation, 127 signaling pathway, 127–8 serine phosphorylation, 128 translocation, 128 tyrosine phosphorylation, 128 VEGF-R2, 1024 Stat5a, 87, 115–16 activation, 89 erythropoietin receptor, 155 IRF-1 promoter inhibition, 131 prolactin mediation, 131–2 Stat5b, 87 activation, 89 erythropoietin receptor, 155 IRF-1 promoter inhibition, 131 Stat6, 87 activation, 89–90, 234

INDEX

IL-13, 410, 412 IL-4, 234–5, 236, 240, 1316 binding site, 228–9 IL-13, 1322 stem cell(s) Flt3, 1002–3 IL-3 administration, 209 mobilization, 1003, 1269–70 peripheral blood, 510 GM-CSF mobilization, 513 stem cell factor (SCF), xxiii, 1011–15 biology, 1012–13 birds, 65–6 cancer, 1014–15 clinical indications, 1014 clinical use, 1014–15 ex vivo stem cell expansion, 1271–2 IL-9, 348, 349 IL-17, 489 recombinant human, 1014 survival factor role, 1264 synergy, 1012 stem cell factor (SCF) receptor, 1263–4 STEMGEN, 1014 steroid hormones, 119–20 steroid receptors, 125 Still’s disease, 724 STRAP (serine threonine kinase receptor-associated protein), 1131 stress ACTH release, 1037 hematopoiesis, 208–9 IL-3, 208–9 prolactin, 133–4 stress-activated protein kinase (SAPK), 213–14 IL-1, 660 stroke, erythropoietin use, 159 stromal cell-derived factor see CXCL12 SU5416, 1285, 1289 SU6668, 1285, 1289 substance P, 496 sulfhydryl donors, 1286 suppressors of cytokine signaling see SOCS Sweet’s syndrome, 532 Syk protein tyrosine kinase G-CSFR signaling, 537 IL-2 signaling, 180 synergism, 12–13 systemic lupus erythematosus (SLE), 22–3 cytokine system, 1196–7 HGF, 791

IL-1Ra, 685, 688 IL-10 secretion, 23 IL-15, 448 IL-17, 494–5 sex bias, 1190 susceptibility, 1190 tissue damage, 1191 TNF, 851–2 systemic sclerosis IL-1Ra, 681 IL-17, 497 T-bet transcription factor, 584 T cell(s) allograft acceptance, 1301–5 antigen presentation, 1189–90 apoptosis, 447 asthma, 1324–6 autoimmune disease, 1191 autoimmunity, 1192 bystander activation, 1192 CD95, 889, 891, 900–1 CD95 receptor, 893–4 CD95L, 887 chemokines, 1091–2, Plate 47.2 CX3CR1, 1107, 1108 dendritic cell primed, 1342 differentiation, 314, 1319, 1325 ELISPOT frequency analysis, 1389, Plate 61.6, Plate 61.7 eosinophilia, 269 fate, 314 Flt3, 996 GH effects, 107, 108 homeostasis, 331–2, 442, 446 IFN-, 326, 568, 569, 570, 583–4, 1323 IFN type I, 556 IL-2, 184, 185–6, 1304 IL-2 gene regulation, 183 IL-3, 203, 204–5, 206 activation, 206–7, 208 IL-4, 229, 230, 231–3, 412 biologic effects, 240–2 signaling, 235–6 IL-5 production, 263, 274 IL-6-induced activation, 283 IL-7, 318–21, 326 regulation of homeostasis, 305 IL-7R, 311, 314, Plate 13.3 IL-8, 1061–2 IL-9, 349–50, 353 IL-10, 607 IL-12, 392 IL-13, 412–13 IL-15, 442–3, 448, 1214–15, Plate 53.4

lxiii IL-16, 466, 467, 468–9 IL-17, 478 IL-18, 712 IL-18R, 715, 717 immune response, 187 lymphotoxin, 830 mature and IL-4 effects, 240–1 multiple sclerosis, 894 RANK, 876–7 regulation of growth/function, 4 renal allograft rejection, 490 rheumatoid arthritis, 492 thymic development, 318–19 see also CD4+ T cells; CD8+ T cells; cytotoxic T cells (CTL); TH1 cells; TH2 cells T-cell lymphoma, 323–4 T cell receptor (TCR) CD95, 889, 890–1 IFN-, 570 iIEL, 320 IL-7, 319 IL-15, 440 IL-16, 469 IL-18 induction of IFN-, 717 invariant complex, 320 signaling complex, 312 T cell receptor  (TCR), 313 ab T cells, 477 d T cells, 319, 321 IL-7 infection response, 328 IL-15, 443 TACE, 841, 965, 1104 TAG ruthenium ion containing reporter molecule, 1385 TAK binding protein 1 (TAB1), 1133 TAKI, 1133 TAP1 (transporter protein 1), 1218 TAPs (transporters associated with antigen processing), 583, 586 Tat protein, 769–70 CD95L expression, 887, 894 HIV infection, 764–5, 769–70, 887 TbR-1 associated protein 1 (TRAP-1), 1131 TbR-I receptor chain, 11 TbR-II receptor chain, 11 TEC tyrosine kinase, 129, 537 testes, prolactin effects, 132 testosterone, 125 tetanus toxoid, 60 TFR expression, 158 TGFa, 5, 959, 964–7 activity, 966–7 cancer, 966 discovery, 964 gene, 965–6

lxiv TGFa (continued) precursor processing, 960, 965 protein, 964–5, 966 receptor, 966 signaling, 965 soluble, 965 transgenes, 967 TGFb, xxiii, 4, 5, 1119–40 activin synergism, 1163 allograft rejection, 1300, 1306 amphibians, 74 antiapoptotic effects, 1136 apoptosis mediation, 1135, Plate 49.6 regulation, 1136–40, Plate 49.6 biologic actions, 1165 birds, 74 cancer, 1220 cytokine inhibition, 13 FGF2 expression, 755 fish, 73–4 genes, 1121 IFN- inhibition, 570 IL-10 regulation, 608, 1220 invertebrates, 73 MAPK signaling pathway, 1132–5, 1136 cytoplasmic cross-talk with Smad, 1134–5 nuclear cross-talk with Smad, 1133–4 regulation, 1133 molecular structure, 1120–1, Plate 49.1 prostate cancer, 1137 signaling, 1126–7 activin cross-regulation, 1159 Smad-independent, 1135–6 transcriptional regulation mediating, 1135, Plate 49.5 Smad cytoplasmic cross-talk with MAPK, 1134–5 nuclear cross-talk with MAPK, 1133–4 Smad signaling pathway, 1124–31, Plate 49.3, Plate 49.5 ubiquitin–proteosome pathway, 1129–30 tumor progression, 1220 see also activin; inhibin; Smad signal transducers TGFb-activated kinase (TAL1), 1133 TGFb receptors, 8, 9, 1121–5, Plate 49.2 activation, 1124

INDEX

endoglin, 1121, 1122, 1282 gene deletions, 1158–9 modulators, 1124–5 regulation, 1124–5 signaling, 11, 1122–3, Plate 49.3 type I and II, 8, 9, 1121, 1122–3, Plate 49.2 type III, 1121–2, Plate 49.2 TGFb1, 1120 allograft rejection, 1307 gene, 1121 TGFb–LAP activation, 1120, Plate 49.1 TH1 cells allograft acceptance, 1301–5 asthma, 1324–6 CC chemokines, 1087, 1091–2, Plate 47.2 CD95L, 887 chemokines, 1091–2, Plate 47.2 differentiation, 1319, 1324–6 IFN-, 583–4, 1323 IL-4, 231–2, 235–6, 241 IL-10, 607, 611 IL-12, 391–2, 393 IL-12R, 390 IL-17, 478 SLE, 495 IL-18-deficient mice, 719 IL-18R, 715, 717 immune response, 1376 infection, 246 polarized responses, 13 TH2 cells allograft acceptance, 1301–5 asthma, 1313, 1314–15, 1324–6 CC chemokines, 1087, Plate 47.2 CD95L, 887 chemokines, 1091–2, Plate 47.2 differentiation, 229, 1319, 1324–6 humoral response, 248 IFN-, 583–4, 1323 IL-3, 204, 206, 216 IL-4, 229, 231–2, 235–6, 241, 412 IL-9, 350 IL-10, 607, 611 IL-12, 391–2, 393 IL-12R, 389, 390 IL-17, 478 SLE, 495 IL-18R, 715, 717 immune response, 1376 infection, 246 inhibition by IFN-, 232 polarized responses, 13 TH2 cytokines, IL-18-induced, 718 TH3 cells, 1376 thalidomide, 756, 1223

antiangiogenesis, 1285, 1288 multiple myeloma, 1281 thrombocytopenia IL-11, 368 rhIL-11 pharmacology, 373–4 stem cell factor clinical use, 1012 thrombocytopenic purpura, 1191 thrombopoiesis, rhIL-11 pharmacology, 373 thrombopoietin, 1268–9 thrombospondin 1 (TSP-1), 1121 Thy-1 antigen, 204, 238 thymic stromal lymphopoietin (TSLP), 305, 307, 310 B lymphopoiesis, 316 high-affinity binding, Plate 31.2 receptor, 310, Plate 31.2 thymocytes apoptosis, 891 CD95, 891 Flt3, 996 IL-4 effects, 240 IL-7, 332 phenotype of immature, 319 thymosin, 1245 thymus atrophy, 332 GH receptors, 107 IL-7 effect, 332 IL-9, 350 thymus and activation-regulated chemokine (TARC) see CCL17 thyroid disease, autoimmune, 1190 thyroid peroxidase (TPO), 1192 TIR domain, 8, 9 TIRAP, 814, 816 TLR signaling, 817 tissue injury, 917–18 regeneration, 1003 repair and angiogenesis, 1281 transplantation IL-12, 396 IL-15, 451 tissue inhibitors of metalloproteinase (TIMP) antitumor activity, 1288 FGFs, 761 IL-10, 610 rheumatoid arthritis, 492 tissue plasminogen activator (t-PA), 1286 HGF cleaving, 784 HMGB1, 918 TLR, 8, 9, 647, 809–19 chromosomal distribution, 813, Plate 33.2

INDEX

cytoplasmic transducers, 815–16 endogenous ligands, 817 function, 816–18 genes, 813, Plate 33.2 Ig repeats, 815 IL-1R, 654, 655 IL-18R signaling, 716–17 LLRs, 814, 815, Plate 33.4 locus polymorphism, 819 MyD88, 814, 815–16 orphan receptors, 812–13 promoter function, 818–19 signal transduction, 817–18 specificity, 816–18 structure, 814–16, 817 TIR domain, 814–15, 816, Plate 33.4 tissue distribution, 818–19 TLR1, 814, 815 structure, 817 TLR2, 812, 813, 814 structure, 817 TLR3, 813 TLR4, 812, 813, 814, 815 endogenous ligands, 817 IL-1R, 818 LPS activation, 818 promoter function, 818–19 TLR6, 814, 815 TNF, 4, 837–53 anti-inflammatory pathway, 853 ASD-mediated pathway, 845–6 assay, 1379 autoimmune disease susceptibility, 851–2 biologic effects, 846–50 biosynthesis, 838–41 birds, 72 cancer, 849–50 susceptibility, 851 cardiovascular system, 848 celiac disease, 851 CNS effects, 846–8 cytokine stimulation, 13 cytotoxicity, 850 diabetes mellitus type 1, 852 disease risk, 21 susceptibility, 850–3 erythropoiesis inhibition, 152 fish, 71–2 gene polymorphisms, 1219 genetics, 21–2 homology domain, 826 IL-2 receptor expression modulation, 14 infectious disease, 20, 21, 832

susceptibility, 850–1 invertebrates, 71 knockout mice, 21 ligand family, 826 MHC class II, 24 NFB induction, 846 NSD domain, 845 phylogeny, 71–2 polymorphisms, 850 post-translational regulation, 841 production regulation, 840–1 stimuli, 838, 839 promoter, 839 regulation, 838–41 rheumatoid arthritis, 851, 852 septic shock, 848–9 SLE susceptibility, 851–2 structure, 837–8 synergism with IFN-g, 12 therapeutic inhibition, 841 toxicity prevention, 852 TRAIL/Apo2L, 861–2 transcriptional regulation, 839–40 translational regulation, 840 TNF-a allograft rejection, 1298–9, 1306–7 antisense oligonucleotides, 1362–3 apoptosis, 1215, Plate 53.2 cachexia, 1223 cancer, 1215, 1219, 1222, Plate 53.2 CSF-1, 937 cytokine stimulation, 13 expression blocking, 1201–3 FGF2 expression, 755 fish, 71–2 IL-1 family, 644 IL-10 regulation, 608 IL-11, 365 IL-15-induced, 447 IL-17, 482 synergy, 486, 488, 492 MAPK, 1202–3 nickel dermatitis, 497 phosphorothioate oligonucleotides-induced, 1357 rheumatoid arthritis, 1195, 1202–3, Plate 52.6 skin cancer, 1222 see also anti-TNFa therapy TNF-a-converting enzyme (TACE), 841, 965, 1104 TNF-b see LTa; LTb; lymphotoxin (LT) TNF-binding protein, 842–3 TNF receptor-associated factor (TRAF), 287 TNF receptors (TNFR), 8, 9, 842–3

lxv death domains, 10, 827 downregulation by IL-1, 14 expression, 828 modulation, 14 fish, 72 lymphotoxins, 825, 826–7, Plate 34.1 signaling, 10, 842, 843–6 soluble, 842–3, 849, 1196 TNF-related apoptosis-inducing ligand (TRAIL), 862, 877 TNFR-associated death domain protein (TRADD), 827, 843, 844–5 TNFR1, 826–7, 842–3, Plate 34.3, Plate 34.4 antisense oligonucleotides, 1363 binding, 838 caspase recruitment domains (CARD), 844 death domain, 843, 844–5 expression, 828 signaling, 843–6 TNF functions, 842 TNFR2, 826, 842–3 activation, 838 antisense oligonucleotides, 1363 binding, 838 expression, 828 signaling, 842, 846 TNFSF6 see CD95L tolerance induction, 1301–5 Flt3, 1001–2 Toll/IL-1 receptor (TIR), 814–15, 816, Plate 33.4 Toll-like receptors see TLR TRAF-2, 482 TRAF-6, 481–2, 659–60 IL-1R signaling, 735 IL-18R, 659–60 signaling, 716 TRAF family, 827 TRAIL, 862, 877 TRAIL/Apo2L, 861–6 apoptosis, 864, 865–6 cancer, 865 expression upregulation, 863–4 functional forms, 864–5 physiological functions, 863–4 receptors, 863 recombinant human, 864 sequences, 862 signaling, 865, Plate 36.4 structure, 864, Plate 36.2 therapeutic uses, 866 transcriptional regulatory element, 863 TRAIL receptor-R1, 863, 865

lxvi TRAIL receptor-R1 (continued) death domains, 863 signaling, 865, Plate 36.4 TRAIL receptor-R2, 863, 865 death domains, 863 signaling, 865, Plate 36.4 TRANCE see RANKL transferrin receptor (TFR) expression, 158 transplantation see organ transplantation transporter protein 1 (TAP1), 1218 transporters associated with antigen processing (TAPs), 583, 586 TRAP-1 (TbR-1 associated protein 1), 1131 trastuzumab, 1220 TRFK-5, 1327 TRICK2, 863 TrkA, 484 tuberculin antigen, xxiv tuberculosis anti-TNFa therapy, 1201 see also mycobacterial infection tubulogenesis, 789, 798 tumor(s) growth, 1220 promotion and MIF, 1043, Plate 45.4 microenvironment, 1220 progression, 1216–20 rejection, 1224 see also cancer; cancer cells tumor-associated dendritic cells (TADC), 1220 tumor-associated macrophages (TAM), 1220 tumor counterattack theory, 892, 1093, Plate 38.1 tumor infiltrating lymphocytes (TILs), 893, 1220 Tyk2, 85, 86 activation, 87 G-CSFR signaling, 535 IL-2R signaling molecules, 177 IL-6, 289 IL-10R signaling, 614 IL-12 receptor, 389 IL-13 signaling, 411 tyrosine kinase inhibitors, 1223 tyrosine kinase receptors, 792, 793, 1263–4 inhibition, 769 tyrosine phosphorylation CSF-1, 934–7 c-Fms, 938–41 inhibition by FGFRs, 768

INDEX

ubiquination, CSF-1, 935 ubiquitin–proteasome pathway, 578 ulcerative colitis activin, 1167–8 IL-1Ra, 683 IL-10, 609 IL-15, 448 IL1RN gene association, 684 umbilical cord blood, 1013 urokinase, 1286 urokinase-type plasminogen activator (u-PA) HGF cleaving, 784 MET interaction, 797 urokinase-type plasminogen activator receptor (u-PAR), 797 US28, 1111 uterus angiogenesis, 1281 IL-15 effects, 433 prolactin effects, 132 vaccines cancer, 1224, 1226 DNA, 1344 gene transfer, 1344 GM-CSF, 509, 512, 513 IFN- as adjuvant, 61 tumor cell, 512 vagus nerve stimulation, 853 vascular cell adhesion molecule 1 (VCAM-1), 243, 244, 272 IL-1, 643 IL-13, 415 vascular endothelial cells, 1024 vascular endothelial growth factor(s) (VEGF), 4, 1017–28 angiogenesis, 1017, 1019, 1024–5 anti-TNFa therapy, 1199 antiangiogenesis strategies, 1285, 1289–90 antisense oligonucleotides, 1367–8 cancer, 1026–7 FGF2 synergy, 764, 765 FGFs, 757, 761 hematopoietic progenitor cells, 1025–6 IL-10, 610–11 immune response, 1026–7 myeloid cell differentiation, 1026–7 NFB, 1027 PlGF potentiation, 1025 promoter region, 1020–1 rheumatoid arthritis, 1282 signal transduction, 1022–4

structure, 1018–21, Plate 44.1 tumor growth, 1220 VEGF-A–E, 1018–20, Plate 44.1 vascular endothelial growth factor receptor (VEGF-R), 1021–2, Plate 44.1 hematopoietic progenitor cells, 1026 inhibitors, 1289 vascular endothelial growth factor receptor 1 (VEGF-R1), 1018, 1020, 1021, 1022, 1282 hematopoietic progenitor cells, 1026 signal transduction, 1022–3, Plate 44.2 vascular endothelial growth factor receptor 2 (VEGF-R2), 244, 757, 1018, 1020, 1021, 1022, 1022 hematopoietic progenitor cells, 1026 signal transduction, 1023–4, Plate 44.3 vascular endothelial growth factor receptor 3 (VEGF-R3), 1019, 1021, 1022 hematopoietic progenitor cells, 1026 signal transduction, 1023–4 vascular endothelium, 527 vascular injury, 972 vascular permeability factor (VPF), 451, 1017 Vav proto-oncogene, 937 VEGF-A, 1018, 1020, Plate 44.1 VEGF-B, 1018–19, 1020, Plate 44.1 VEGF-C, 1019–20, Plate 44.1 VEGF-D, 1019–20 VEGF-E, 1020 vertebrates, 57 very late activation antigen 4 (VLA-4), 272, 1269–70 very late activation antigen 5 (VLA-5), 1269–70 Vig-1 gene, 58 viral infection CC chemokines, 1093 CD95L, 892 Dengue virus, 551 EMCV, 551 IL-12 therapy, 398 IL-15, 450–1 sindbus virus, 551 TNF family, 832 TRAIL/Apo2L therapeutic use, 866 VSV, 551 vitamin D, 875

lxvii

INDEX

vitravene, 1358 von Hippel–Lindau tumor suppressor (VHL), 1021 wound healing activin, 1163–4, 1167–8

angiogenesis, 1281 FGFs, 757, 761–2 GH therapy, 109 GM-CSF, 509, 512, 513 HB-EGF, 971 HGF, 791

MIF, 1042 TGFa, 967 XC chemokine receptor, 8, 9 XIAP (X chromosome-linked inhibitor of apoptosis protein), 1131–2, 1133, 1135

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1 The cytokines: an overview Jan Vilˇcek Department of Microbiology, New York University Medical Center, New York, NY, USA

Explanation separates us from astonishment, which is the only gateway to the incomprehensible. Eugène Ionesco

GENERAL FEATURES OF CYTOKINES Origins of cytokine research The field of cytokine research as it exists today has evolved from four originally independent sources. The first and probably most significant source is immunology and, more specifically, the field of lymphokine research. The origins of lymphokine research can be traced to the mid-1960s when it was demonstrated that lymphocyte-derived secreted protein mediators regulate the growth and function of a variety of white blood cells. Soon it would become apparent that monocytes too are the source of important proteins (‘monokines’) that can modulate leukocyte function. The second source of cytokine research derives from the study of the interferons. Originally described in the 1950s as selective antiviral agents, interferons gradually became recognized as proteins exerting a The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

broad range of actions on cell growth and differentiation, both within and without the immune system. As a result, the dividing line between lymphokines/ monokines and the interferons began to dwindle and today it is clear that interferons in fact are cytokines. The third source of cytokine research is the field of hematopoietic growth factors – colony-stimulating factors. In addition to promoting the growth and differentiation of hematopoietic stem cells, colonystimulating factors have been shown to regulate some functions of fully differentiated hematopoietic cells, thus blurring the dividing line between these agents and lymphokines/monokines. The fourth source of cytokine research derives from the study of growth factors acting on nonhematopoietic cells. One might be reluctant to count ‘classical’ growth factors – e.g. PDGF, EGF, FGF or NGF – among the cytokines. Nevertheless, it is clear that, in addition to promoting cell growth, many of these agents exert other, what might be referred to as ‘cytokine-like’ actions. Moreover, at least one of the Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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THE CYTOKINES : AN OVERVIEW

offsprings of growth factor research, i.e. transforming growth factor-b (TGF-b), is now considered a bona fide cytokine.

A brief outline of the history of cytokine research The beginnings of lymphokine research are usually traced to the demonstration that migration of normal macrophages is inhibited by material released from sensitized lymphocytes upon exposure to antigen (David, 1966; Bloom and Bennett, 1966). The putative factor responsible for this action was termed macrophage migration inhibitory factor (MIF). The description of MIF activity was followed by the discovery of ‘lymphotoxin’ activity, i.e. selective cytotoxicity for some target cells, in supernatants of activated lymphocyte cultures (Williams and Granger, 1968; Ruddle and Waksman, 1968). Dumonde et al. (1969) coined the term ‘lymphokine’ to designate ‘cell-free soluble factors (responsible for cell-mediated immunological reactions), which are generated during interaction of sensitized lymphocytes with specific antigen’. Among the lymphokines a central role in the regulation of T cell growth and function is played by interleukin-2 (IL-2). It was known since the early 1970s that lymphocytes can produce one or more factor(s) mitogenic for other lymphocytes (reviewed by Oppenheim et al., 1979). Morgan et al. (1976) reported that supernatants of mitogen-activated human mononuclear cells could support the continuous growth of human bone marrow-derived T cells. The responsible mitogenic factor is IL-2, then designated T cell growth factor (TCGF) and also known under a variety of other names that by now are largely forgotten (Aarden et al., 1979). The first monocyte/macrophage-derived cytokine described was tumor necrosis factor (TNF) – originally identified as a cytotoxic protein present in the serum of animals sensitized with Bacillus CalmetteGuérin and challenged with LPS (Carswell et al., 1975). In addition to its direct cytotoxicity for some tumor cells in vitro, TNF was identified as the mediator of LPS-induced hemorrhagic necrosis of Meth A sarcoma in mice. Among the first monocyte-derived cytokines described is also lymphocyte activation factor (LAF), now known as interleukin-1 (IL-1). LAF activity,

defined as a mitogenic signal for thymocytes, was originally detected in supernatants of adherent cells isolated from human peripheral blood (Gery et al., 1971). Other investigators described activities that are now known to be mediated by IL-1 under a variety of other names, e.g. mitogenic protein, leukocytic pyrogen, endogenous pyrogen, B cell-activating factor, leukocyte endogenous mediator, etc. (reviewed by Oppenheim et al., 1979; Aarden et al., 1979). While the early studies of lymphokines and monokines were largely the domain of immunologists who sought a better understanding of delayed-type hypersensitivity and other cell-mediated immune reactions, interferons were the brainchildren of virologists. Interferon was first described by Isaacs and Lindenmann (1957) as a factor produced by a variety of virus-infected cells capable of inducing cellular resistance to infection with homologous or heterologous viruses. That interferons would affect immune reactions was initially not even suspected. However, several years later, Wheelock (1965) described a functionally related virus-inhibitory protein (today known as IFN-c) produced by mitogen-activated T lymphocytes. It is now known that T cell and NK cell-derived IFN-c is structurally completely distinct from the large family of IFN-a/b proteins, which are produced by a variety of cell types, including dendritic cells, monocytes, NK cells and B cells in addition to sundry nonhematopoietic cells (De Maeyer and De MaeyerGuignard, 1988; Vilˇcek and Sen, 1996; Siegal et al., 1999; Biron and Sen, 2001). Colony-stimulating factors (CSF) are proteins whose major function is to support the proliferation and differentiation of hematopoietic cells. Their name reflects the early observation that CSFs promote the formation of granulocyte or monocyte colonies in semisolid medium (Bradley and Metcalf, 1966; Sachs, 1987). Years of effort by many groups of investigators have led to the isolation and characterization of several distinct proteins, to be described in greater detail elsewhere in this volume (see Chapters 7, 21, 22, 40). Many proteins that can stimulate the growth of non-hematopoietic cells have been identified. Best known among these are epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF) – to name just a few. Although these and other growth factors are generally not

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GENERAL FEATURES OF CYTOKINES

included among the cytokines, some cytokine-like actions of classical growth factors on immunocytes and other cells have been described. de Larco and Todaro (1978) described a growth factor, originally termed sarcoma growth factor, whose most interesting property was that it promoted the growth of normal rat fibroblasts in soft agar. Since then, two families of ‘transforming growth factors’ have been identified – TGF-a and TGF-b. These are distinct peptides with very different spectrums of biological activities. TGF-a is closely related to EGF whereas the family of TGF-b proteins plays important roles not only in cell growth control and neoplasia but also in inflammation and immunoregulation. Among the important actions of TGF-b proteins are the recruitment and activation of mononuclear cells, promotion of wound healing, fibrosis and angiogenesis, and a potent immunosuppressive action on numerous functions of T lymphocytes (Roberts and Sporn, 1990; Flanders and Roberts, 2001). Based on what is known about the actions of TGF-b proteins today, these polypeptides undoubtedly qualify for inclusion among the cytokines.

Cytokine nomenclature Inasmuch as the cytokine field evolved from several separate sources, a unifying concept of what cytokines are has been slow to emerge. The term ‘lymphokine’ – originally denoting the product of sensitized lymphocytes exposed to specific antigen (Dumonde et al., 1969) – has often been used less discriminately for secreted proteins from a variety of cell sources, affecting the growth or functions of many types of cells. To dispel the wrong notion that such proteins could be produced by lymphocytes alone, Cohen et al. (1974) proposed the term ‘cytokines’, which has become the generally accepted name for this group of proteins. Subsequently, a group of participants at the Second International Lymphokine Workshop held in 1979 proposed the term ‘interleukin’ in order to develop ‘a system of nomenclature . . . based on [the proteins’] ability to act as communication signals between different populations of leukocytes’ (Aarden et al., 1979). As a first step, the group introduced the names ‘IL-1’ and ‘IL-2’ for two important cytokines, which up until then had been described under a variety of different names. At the

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time of writing, the interleukin series has reached IL-25 (Fort et al., 2001) and provisionally IL-26 (Knappe et al., 2000). Although the name ‘interleukin’ implies that these agents function as communication signals among leukocytes, Aarden et al. (1979) suggested that the term not be reserved for factors that can act only on leukocytes. Indeed, a number of the proteins that have been labeled as interleukins not only are produced by a variety of nonhematopoietic cells, but also affect the functions of many diverse somatic cells (e.g. IL-1 or IL-6). Whereas many cytokines are now termed interleukins, others remain to be known by their older names (e.g. IFN-a/b, IFN-c, TNF, lymphotoxins (LT-a and LT-b), TGF-b, leukocyte inhibitory factor (LIF), most colony-stimulating factors and many others). Although these older names are easier to remember, they suggest only one (usually the earliest recognized) function of these pleiotropic agents. Recently, a new nomenclature was proposed for IL-1 family genes and proteins (Sims et al., 2001). Newly described members of the IL-1 family (other than the ‘classical’ four members listed in Table 1.3) would be designated IL-1F plus a consecutive number (IL1F5–IL1F10 have been assigned so far). New numerical designations have been approved for the four subfamilies of chemokines and chemokine receptors (see Table 1.3) consisting of the letters CXC, C, CX3C and CC plus a consecutive number (International Union of Immunological Societies, 2001).

Cytokines, hormones and growth factors Having outlined their history and nomenclature, it is appropriate to try to define what cytokines are. We propose the following general definition: ‘Cytokines are regulatory proteins secreted by white blood cells and a variety of other cells in the body; the pleiotropic actions of cytokines include numerous effects on cells of the immune system and modulation of inflammatory responses’. Since no short definition can encompass all essential properties, cytokines are best defined by a set of characteristic features, as listed in Table 1.1. Many characteristic features of cytokines outlined in Table 1.1 are shared by two other groups of protein

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THE CYTOKINES : AN OVERVIEW

TABLE 1.1 Characteristic features of cytokines Most cytokines are simple polypeptides or glycoproteins 30 kDa in size (many cytokines form homodimers or homotrimers and one cytokine (IL-12) is a heterodimer). Constitutive production of cytokines is usually low or absent; production is regulated by various inducing stimuli at the level of transcription or translation. Cytokine production is transient and the action radius is usually short (typical action is autocrine or paracrine, not endocrine). Cytokines produce their actions by binding to specific high affinity cell surface receptors (kDa in the range of 109–1012 M). Most cytokine actions can be attributed to an altered pattern of gene expression in the target cells. Phenotypically, cytokine actions lead to an increase (or decrease) in the rate of cell proliferation, change in cell differentiation state and/or a change in the expression of some differentiated functions. Although the range of actions displayed by individual cytokines can be broad and diverse, at least some action(s) of each cytokine is (are) targeted at hematopoietic cells.

mediators – growth factors and hormones. The relationship between cytokines and growth factors was already mentioned earlier in this chapter. Although the dividing line is rather tenuous, one difference is

that the production of growth factors (e.g. PDGF, EGF or TGF-a) tends to be constitutive and not as tightly regulated as that of cytokines. Another difference is that, unlike cytokine actions, the major actions of growth factors are targeted at nonhematopoietic cells. It is also not easy to distinguish clearly between cytokines and classical polypeptide hormones (Table 1.2). One of the major distinguishing features is that classical hormones are produced by specialized cells, e.g. insulin is produced by beta cells of the pancreas, growth hormone by the anterior pituitary and parathormone by the parathyroid. In contrast, cytokines tend to be produced by less specialized cells, and more often than not, several unrelated cell types can produce the same cytokine (e.g. IL-1 is produced by monocytes-macrophages, mesangial cells, NK cells, B cells, T cells, neutrophils, endothelial cells, smooth muscle cells, fibroblasts, astrocytes and microglial cells). However, there are exceptions, e.g. IL-2, IL-3, IL-4, IL-5, lymphotoxins and IFN-c are produced exclusively or mainly by lymphoid cells. Perhaps the most characteristic features of cytokines, that distinguish them from hormones, are the redundancy and pleiotropy of cytokine actions, i.e. the fact that structurally dissimilar cytokines (e.g. TNF and IL-1) can be remarkably similar in their actions (Le and Vilˇcek, 1987), and that individual cytokines tend to exert a multitude of actions on different cells and tissues.

TABLE 1.2 Distinguishing features between polypeptide hormones and cytokines Hormones Characteristic features

Cytokines Characteristic features

Exceptions

Secreted by one type of specialized cells

Made by more than one type of cells

Many (e.g. IL-2, IL-3, IL-4, IL-5, LT-a, LT-b) are made only by lymphoid cells

Each hormone is unique in its actions

Structurally dissimilar cytokines have an overlapping spectrum of actions (‘redundancy’)

Restricted target cell specificity and a limited spectrum of actions Act at a distant site (endocrine mode of action)

Exceptions

Insulin

Multiple target cells and multiple actions (‘pleiotropy’) Usually have short action radius (autocrine or paracrine mode of action) BASIC CYTOKINE BIOLOGY

Many (e.g. TNF, IL-1 or IL-6 in septic shock)

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CYTOKINE RECEPTORS AND SIGNAL TRANSDUCTION

Despite some differences, it is apparent that cytokines, growth factors and polypeptide hormones all function as extracellular signaling molecules featuring fundamentally similar mechanisms of action. This conclusion is supported by the finding that receptors for several cytokines and hormones (i.e. IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, G-CSF, erythropoietin, prolactin and growth hormone) show several common structural features (Bazan, 1989; D’Andrea et al., 1989; Gearing et al., 1989; Kishimoto et al., 1994; Ihle, 1995; Taga and Kishimoto, 1995; Hirano, 1998). Earlier, Roberts et al. (1988) described regions of structural homology in the receptors for PDGF and M-CSF. (Unlike other cytokine receptors, the M-CSF receptor comprises a functional intrinsic tyrosine kinase.) In addition, similar molecular pathways transmit signals from the growth factor, polypeptide hormone or cytokine receptors to the nucleus, and several components in the signal transduction pathways are shared by cytokines, growth factors and polypetide hormones.

CYTOKINE RECEPTORS AND SIGNAL TRANSDUCTION

TABLE 1.3 Structural features of some cytokines permit their grouping into families Family

Representative members

IL-2/IL-4

IL-2 IL-4 IL-5 GM-CSF

IL-6/IL-12

IL-6 IL-12a

Interferons-a/b

IFN-a (many subtypes) IFN-b IFN-x IFN-s

Tumor necrosis factors TNF-a LT-a (TNF-b) LT-b Fas ligand CD40 ligand TRAIL BAFF APRIL RANK LIGHT IL-10

IL-10 IL-19 IL-20 IL-22 IL-24 (MDA-7)

IL-17

IL-17 IL-25

Interleukin-1

IL-1a IL-1b IL-1 receptor antagonist IL-18

TGF-b

TGF-b Bone morphogenetic proteins Inhibins Activins

Chemokines

CXC subfamily (CXCL1–16) CC subfamily (CCL1–28) C subfamily (CL1/Lymphotactin) CX3C subfamily (CX3CL1/Fractalkin)

Structural features of cytokines Structural analysis has made it possible to group many cytokines within ‘families’ (Table 1.3). Some of these families include proteins whose primary sequences show a high degree of homology to one another, e.g. all members of the IFN-a/b family (further subdivided into subfamilies IFN-a, IFN-b, IFN-x, and IFN-s) show at least 30% homology in their amino acid sequences (De Maeyer and De Maeyer-Guignard, 1988). In other instances, the structural relationship is more distant and is based mainly on a common proposed tertiary structure and spatial organization. An example of the latter type of structural relationship is the IL-2/IL-4 family (Bazan, 1992; Boulay and Paul, 1992). Members of this family contain four a-helical regions in a spatially similar arrangement. Other cytokines that bind to the hematopoietin family of cytokine receptors (see below) might also belong to this family. Most recently characterized is the IL-10 family, which includes five novel cytokines: IL-19, IL-20, IL-22, IL-24 (MDA-7) and AK155 (provisionally termed IL-26) (Kotenko, 2002).

a

IL-12 is a heterodimer in which one subunit is structurallyrelated to IL-6, and the other subunit shows partial homology to the extracellular domain of the IL-6 receptor (a chain).

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THE CYTOKINES : AN OVERVIEW

Cytokine receptor families Progress in the characterization of cytokine receptors and the cloning of genes encoding cytokine receptors has led to the recognition that many cytokine receptors can be grouped into families based on common structural features (Table 1.4). The most extensive family is represented by the class I cytokine receptors that encompass receptors for many important cytokines, including IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, IL-13, the hematopoietic growth factor G-CSF, GM-CSF and erythropoietin, as well as growth hormone and prolactin (Bazan, 1989; Kishimoto et al., 1994; Taga and Kishimoto, 1995). Most of these receptors form heterodimers, some are homodimers (e.g. G-CSF and erythropoietin receptors) and some (e.g. IL-2, IL-15 and CNTF receptors) are heterotrimers (Hirano, 1998). Interestingly, many of these receptors form subfamilies in which one of the chains forming the heterodimeric or heterotrimeric receptor is common to all members (Taga and Kishimoto, 1995). The latter receptors characteristically contain one or two unique subunits that act as specific binding components for a single cytokine, linked to a signal transducing chain that is shared with other members of the same subfamily. Three such receptor subfamilies, the IL-6, GM-CSF and IL-2 group of receptors, utilize signaling components common to each of these subfamilies (the gp130 chain, b chain and c chain, respectively). Some members of the class I cytokine receptor family also share sequences in the intracellular domains, termed Box 1 and Box 2 regions. These sequences, found in the receptors for IL-2, IL-3, IL-4, IL-6, IL-7, erythropoietin, G-CSF and in gp130 (Ihle, 1995; Theze et al., 1996), are important for the activation of signaling pathways. Structural features form the basis for three other cytokine receptor families. One of these is the interferon family receptors, also called class II cytokine receptors because they have features related to the large family of class I cytokine receptors (Bazan, 1990). This family includes the heterodimeric receptors for IFN-a/b and IFN-c (Bach et al., 1996; Domanski and Colamonici, 1996; Pestka et al., 1997; Stark et al., 1998) and the receptor for IL-10 (Ho et al., 1993) and other IL-10 family ligands (Kotenko, 2002). Another large (and still growing) family are the TNF receptors (Smith et al., 1994; Bodmer et al., 2002). This

family comprises two separate TNF receptors (p55 or TNF-RI and p75 or TNF-RII), each of which binds both TNF-a and LT-a. All members of the TNF receptor family are single-chain receptors (most are thought to form homotrimers) that become activated when cross-linked by their trimeric ligands. Another family is formed by the members of the IL-1 receptor proteins, which now includes the IL-18 receptor (Sims et al., 1993; Greenfeder et al., 1995; O’Neill, 2000; O’Neill and Dower, 2001). The functional IL-1 receptor is a heterodimer of the type I IL-1 receptor (IL-1RI) and the ‘IL-1 receptor accessory protein’. The IL-18 receptor too is a heterodimer comprising the IL-18RI and IL-18R accessory protein. Most of these receptor chains are structurally related to one another not only in their extracellular domains, but they also show sequence similarity in their cytosolic regions. This newly described receptor family includes the Drosophila melanogaster protein Toll, the IL-18 receptor (IL-18R), and the mammalian Tolllike receptors TLR-2 and TLR-4 (the last two receptors bind molecules from Gram-positive and Gramnegative bacteria, respectively). The conserved sequence in the cytosolic region of these receptors has been termed the Toll-IL-1 receptor (TIR) domain. A number of distinctive features is shared by receptors for the TGF-b family proteins. A unique feature of these receptors is that they contain intracellular serine/threonine kinase domains (Wrana and Attisano, 2000; Massagué, 2000). Structural features are the basis for the division of TGF-b receptors into type I and type II receptors. TGF-b signaling requires both a type II (the ligand binding component) and a type I (the signaling component) receptor. Finally, there is the family of chemokine receptors, structurally quite dissimilar from the other cytokine receptors (Murphy, 1996; Horuk, 2001). Chemokine receptors are seventransmembrane-domain, G protein-coupled receptors, related to rhodopsin-like receptors that mediate neurotransmission, light perception and responses to other sensory stimuli (Wu et al., 1993). A recent review lists 18 chemokine receptors that have been molecularly cloned and identified (Horuk, 2001). On the basis of their ligand specificity they are subdivided into four subclasses (Table 1.4): CXC (six known members), CC (ten members), XC (one member) and CX3C (one member).

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CYTOKINE RECEPTORS AND SIGNAL TRANSDUCTION

TABLE 1.4 Structural features of some cytokine receptors permit their grouping into families Receptor family

Common features

Receptor subfamilies

Representative ligands

Class I cytokine receptors (‘hematopoietin’ family receptors)

Conserved cysteines, WSXWS motif in extracellular domains; some of these receptors also share conserved sequences in intra-cellular domains (‘Box I’ and ‘Box II’)

IL-6 (sharing gp130)

IL-6 IL-11 CNTFa LIFa Oncostatin Ma Cardiotrophina GM-CSF IL-3 IL-5 IL-2b IL-4 IL-7 IL-9 IL-15b IL-13 IL-14 IL-12 G-CSF Erythropoietin Growth hormone Prolactin

GM-CSF (sharing b chain)

IL-2 (sharing c chain)

IL-13 (sharing a chain) None

Class II cytokine receptors (interferon/ IL-10 family receptors)

Conserved cysteines in extracellular domains

None

IFN-a/b family IFN-c IL-10 IL-20 IL-22

TNF receptor family

Partial homology in extracellular domains; conserved ‘death domains’ in intracellular portions of TNF-RI, Fas, TRAIL and NGF receptors

None

TNF-a LT-a (TNF-b) LT-a/LT-b heterotrimer NGF Fas ligand CD40 ligand TRAIL BAFF

IL-1 receptor family

Immunoglobulin superfamily structure in extracellular domains; cytosolic region related to Toll-like receptors (TIR domain)

None

IL-1a IL-1b IL-1 receptor antagonist IL-18

TGF-b receptors

Cysteine-rich extra-cellular domains, kinase domains, GS domains (type I receptors), serine/ threonine-rich tail (type II receptors)

None

TGF-b Bone morphogenetic proteins Activins Inhibins

Chemokine receptors

Seven transmembrane domains

CXC chemokine receptors CC chemokine receptors C chemokine receptors CX3C chemokine receptors

CXC chemokines CC chemokines C chemokines CX3C chemokines

a

CNTF, LIF, oncostatin M and cardiotrophin 1 receptors share another common chain, the LIF receptor chain.

b

IL-2 and IL-15 receptors also have a common b chain. BASIC CYTOKINE BIOLOGY

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THE CYTOKINES : AN OVERVIEW

Cytokine signal transduction Very significant progress has been made in recent years in our understanding of the mechanisms and pathways whereby signals are generated and transmitted from the cell surface cytokine receptors to the inside of cells. Only a very general and superficial overview of this information can be conveyed here. It is not surprising that the patterns of signal transduction are largely determined by the structural characteristics of the cytokine receptors. Thus each of the cytokine receptor families listed in Table 1.4 exhibits a distinct, characteristic pattern of signal transduction. The revolution in the understanding of cytokine signaling began with the identification of JAK tyrosine kinases and Stat proteins as essential elements in interferon actions (Pellegrini and Schindler, 1993; Darnell et al., 1994). The JAK-Stat signal transduction pathway represents an elegant, efficient, rapid and simple mechanism, utilized by both the IFN-a/b and the IFN-c receptors. All essential components preexist in unstimulated cells. Although the IFN receptors lack intrinsic kinase activity, their intracellular domains bind cytoplasmic JAK tyrosine kinases. (JAK1 and Tyk2 associates with the two chains of IFNa/b receptor, JAK1 and JAK2 with the IFN-c receptor chains.) Binding of the IFNs to their receptors results in activation of the receptor-associated JAK kinases. The most important consequence of JAK kinase activation is the tyrosine phosphorylation and resulting activation of the SH-2 domain-containing Stat proteins. (IFN-a/b activates Stat1 and Stat2, IFN-c only Stat1.) Activated Stat proteins then form homo- or heterocomplexes that migrate to the nucleus, bind to recognition sequences in the promoter regions of IFN-inducible genes and thereby activate their transcription. The Stat protein complexes formed upon IFN-a/b receptor activation are partly different from the Stat protein complexes formed on stimulation with IFN-c, which explains why the actions triggered by the two IFN receptors are both overlapping and unique (Sato et al., 2001). It appears that most (though not all) major actions generated by the IFNs can be traced back to the activation of the JAK-Stat pathway. In contrast, signaling by the class I cytokine receptors relies on elements of the JAK-Stat pathway, as well as on additional signaling mechanisms. A case in point is signaling by the

IL-2 receptor (Lin and Leonard, 1997). IL-2 binds to a receptor composed of three chains of which two, the b and c chain, generate signals. In a manner similar to the IFN receptors, two JAK kinases, JAK1 and JAK3, associate with the signaling chains of the IL-2 receptor and produce activation of two Stat proteins (Stat3 and Stat5). However, additional signaling pathways are activated by the IL-2 receptor, including the tyrosine kinases Syk and p56Ick. IL-2 receptor activation also leads to the activation of the PI3 kinase pathway. Finally, there is evidence that IL-2 receptor activation also leads to the activation of the Ras-dependent MAP kinase pathway. The EPO receptor, the IL-6 receptor and other members of the class I cytokine receptor family also utilize signaling mechanisms that include the JAK-Stat pathway, the Ras-Raf-MAP kinase pathway, PI3 kinase and possibly other elements (Ihle, 1995; Taga and Kishimoto, 1995; Hirano, 1998). Signaling by members of the TNF receptor family proceeds through pathways that are distinct from those utilized by the class I and II cytokine receptors. There is no evidence for the involvement of components of the JAK-Stat pathway. Instead, the primary mechanism for signal transduction is the recruitment/activation of a variety of other cytoplasmic proteins to specific regions in the intracellular domains of these receptors (Hsu et al., 1996; Wallach et al., 1999; Nagata, 1999). The signaling proteins include those that specifically bind to the ‘death domains’ within the cytosolic regions of some of these receptors (e.g. TRADD, FADD/MORT1 and RIP), as well as others that bind to different domains (e.g. the TRAFs). The death domain binding proteins initiate events that lead to apoptosis, but some can also mediate NF-jB activation involving activation of the IjB kinase (IKK) complex. The main feature of signaling by the IL-1 receptor family (which includes the IL-1, IL-18 and Toll-like receptors) is that these cytokines lead to the activation of the Rel family (NF-jB) proteins (O’Neill, 2000; O’Neill and Dower, 2001). Although it is well known that IL-1 exhibits many activities in common with TNF (Le and Vilˇcek, 1987; Neta et al., 1992), with both TNF and IL-1 producing NF-jB activation, the initial intracellular cascades activated by the TNF family receptors are quite different from those operating in IL-1 family receptor signaling. The IL-1 signaling cascade involves activation of the adapter protein

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CYTOKINE NETWORKS

Myd88, kinases IRAK1/2, and the adapter TRAF6. Further downstream, IL-1 and TNF signaling pathways converge on the kinases NIK or MEKK1, which then (in some cases along with TAB-1 and TAK-1) lead to activation of the IKK complex and the resulting IkB degradation and NF-jB activation. Other signaling pathways activated by ligand binding to the IL-1 and IL-18 receptors in mammals lead to the activation of the p38 MAP kinase. A unique pattern of signaling is used by the TGF-b receptor family. The signaling cascades have been most thoroughly investigated with TGF-b1, the prototypical member of this extensive family. TGF-b binding induces complex formation of the two receptor chains, TbR-I and TbR-II (the complex is thought to be a tetramer consisting of two chains each of TbR-I and TbR-II). A family of intracellular signal transducers termed Smads, is responsible for most of the actions of TGF-b family ligands (Massagué, 2000; Wrana and Attisano, 2000). Originally identified in drosophila and termed Mad, the family of Smad genes and proteins has grown to at least nine members, of which seven have been identified in vertebrates. Phosphorylation of TbR-I by the intrinsic kinase of TbR-II leads to the recruitment of Smad2 and Smad3 and their phosphorylation by the activated TbR-I. Phosphorylated Smad2 and Smad3 then heterodimerize with Smad4, and the resulting complexes move to the nucleus, where they combine with other transcription factors and co-activators before they activate transcription of a variety of target genes. Other Smads are specific for the BMP receptor signaling pathway (Reddi, 2001). In some of its general features TGF-b signaling resembles the events occurring during the activation of the JAK/STAT pathway by interferons and many other cytokines. One difference is that TGF-b receptors contain serine/threonine kinase domains, whereas class I and II cytokine receptors use extraneous JAK tyrosine kinases. More importantly, unlike the STATs, the Smads do not act as transcriptional activators on their own, but must combine with coactivators (e.g. CBP/P300) or with members of other transcription factor families in order to regulate the transcription of specific target genes (Zhang and Derynck, 1999). Best known is the propensity of the Smad3/4 heterodimer to form complexes with members of the AP1 family of transcription factors, such as

c-jun, c-fos and SP1. The fact that Smads are promiscuous in the choice of transcription factors with which they partner, undoubtedly contributes to the unusual pleiotropy of actions of TGF-b family members, which is remarkable even when compared with other multifunctional cytokines. Ligation of chemokine receptors induces a conformational change that affects the dissociation of the associated G proteins into their a and bc subunits. It is believed that the dissociated G protein subunits can act as second messengers, leading to the activation of enzymes (Horuk, 2001). Among the target enzymes is a phosphatidylinositol-specific phospholipase C (PIPLC), leading to the generation of IP3 and diacylglycerol, and resulting increased Ca2 influx with activation of protein kinases. As a result, leukocyte chemotaxis is activated, along with many other leukocyte functions including an increased respiratory burst, phagocytosis and degranulation.

CYTOKINE NETWORKS Synergistic and antagonistic interactions Most of the recent studies of cytokine actions are being carried out with homogeneous cytokine preparations produced by recombinant DNA techniques. These studies have led to the assembly of an enormous body of information on the spectrum of actions displayed by individual cytokines. Most of this information has been derived from the analysis of recombinant cytokine actions in various in vitro systems. Although this information is important, it may not provide a realistic picture of the functions of cytokines in the intact organism. One reason is the already mentioned pleiotropy and redundancy in cytokine actions. Another reason is that actions of cytokines can be profoundly influenced by the milieu in which they act and especially by the presence or absence of other biologically active agents (i.e. other cytokines, as well as hormones, growth factors, prostaglandins, microbial components, etc.). Cytokine action is contextual (Sporn and Roberts, 1988). Under natural conditions a cell rarely, if ever, encounters only one cytokine at a time. Rather, a cell is likely to be exposed to a cocktail of several cytokines and other biologi-

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THE CYTOKINES : AN OVERVIEW

cally active agents, with the resulting biological action reflecting various synergistic and antagonistic interactions of the agents present. A certain pattern of characteristic features of cytokine actions has emerged that, somewhat tongue-in-cheek, might be referred to as the ‘molecular philosophy of cytokine actions’ (Table 1.5). While it is impossible to include in this chapter a comprehensive survey of the myriad of synergistic or antagonistic interaction that have been reported, some typical examples will be mentioned. A large number of publications described synergistic actions of TNF and IFN-c. For example, IFN-c was found to potentiate the cytotoxic action of TNF on tumor cells (Lee et al., 1984; Fransen et al., 1986). Other examples of synergistic actions of TNF and IFN-c include enhancement of CSF-1 and G-CSF production by monocytes or lymphocytes (Lu et al., 1988), induction of differentiation of human myeloid cell lines (Trinchieri et al., 1986), antiviral activity (Wong and Goeddel, 1986), and the induction of nitric oxide production in murine macrophages (Ding et al., 1988). It is significant that while TNF and IFN-c act on similar target cells and they partly overlap in their ability to activate genes in the target cells (Beresini et al., 1988; Lee et al., 1990), they exert their actions through intracellular pathways that are quite distinct. Interestingly, TNF and IFN-c share the capacity to induce the transcription factor IRF-1, which is involved in the regulation of expression of IFN-b, the iNOS gene and some other IFN-induced genes (Fujita et al., 1989; Kamijo et al., 1994; Sato et al., 2001). However, the pathways whereby TNF and IFN-c induce IRF-1 synthesis are distinct and therefore synergistic (Pine, 1997).

Not only can a mixture of two cytokines produce an action that represents more than the sum of the separate actions of the individual cytokines (classical definition of a synergistic effect), but a cytokine mixture can result in actions that are qualitatively different from those seen with the individual cytokines. For example, in the HT29 colon carcinoma cell line, TNF-a alone or IFN-c alone even at high concentrations does not exert a marked effect on cell viability. However, when the two cytokines are applied together, there is a rapid and marked cytotoxic action resulting from the induction of apoptosis (Feinman et al., 1987; Abreu-Martin et al., 1995). Another example of such synergy is the induction of IgM secretion by IL-2 and IL-5 (Matsui et al., 1989). Although not as frequently documented, there are also many examples of antagonistic interactions among cytokines. Since in many types of cells IFNs tend to be growth-inhibitory whereas some other cytokines are growth-stimulatory, the presence of IFN (either IFN-a/b or IFN-c) together with a growthstimulatory cytokine (or growth factor) will result in a mutually antagonistic relationship (De Maeyer and De Maeyer-Guignard, 1988). Other examples of an antagonistic interaction include the actions of IL-4 and IFN-c on the synthesis of immunoglobulin subclasses in B cells (Snapper et al., 1988), or the inhibitory action of IFN-a/b on IFN-c-induced enhancement of class II HLA antigen expression (Ling et al., 1985; Kamijo et al., 1993). Antagonism between the actions of IFN-a/b and IFN-c may be partly responsible for the therapeutic efficacy of IFN-b in multiple sclerosis (Karp et al., 2001). Analysis of the synergistic or antagonistic interactions of cytokines helps to appreciate the complexi-

TABLE 1.5 Molecular philosophy of cytokine actions Pleiotropy

Cytokines often have multiple target cells and multiple actions

Redundancy

Different cytokines may have similar actions

Synergism/Antagonism

Exposure of cells to two or more cytokines at a time may lead to qualitatively different responses

Cytokine cascade

A cytokine may increase (or decrease) the production of another cytokine

Receptor transmodulation

A cytokine may increase (or decrease) the expression of receptors for another cytokine or growth factor

Receptor trans-signaling

A cytokine may increase (or decrease) signaling by receptors for other cytokines or growth factors BASIC CYTOKINE BIOLOGY

CYTOKINE NETWORKS

ties of cytokine actions in the intact organism. Nevertheless, it seems that the experimental systems employed still greatly underestimate the variables influencing the actions of cytokines in their natural setting.

Stimulatory and inhibitory actions of cytokines on cytokine production Another characteristic feature of cytokines is their ability to stimulate or inhibit the production of other cytokines. As a result, many cytokine actions are indirect, i.e. due to an increase or decrease in the level of production of other cytokines, which then results in an altered biological response. Among the earliest discovered examples of such an indirect action was the demonstration that the mitogenic action of IL-1 in murine thymocytes involves the stimulation of IL-2 production, and that IL-2 is the actual effector molecule responsible for the stimulation of thymocyte proliferation (Smith et al., 1980). The stimulatory effect of IL-1 on IL-2 production and the role of this interaction in T cell proliferation has become a paradigm for the actions of many other cytokines. In addition to IL-2, IL-1 was found to stimulate the production of IL-6 (Content et al., 1985), GM-CSF (Zucali et al., 1986), and chemokines (Matsushima etal., 1988) in various types of cells. All of these cytokines are also induced by TNF – in accord with the many other similarities seen between the actions of IL1 and TNF (Le and Vilcek, 1987; Neta et al., 1992). In monocytes both TNF and IL-1 are also autostimulatory and, in addition, they stimulate each other’s production (Neta et al., 1992). Other known examples of stimulatory interactions involve the ability of IL-2 and IFN-c to augment IL-1, TNF-a, IL-6 and LT-a production (Svedersky etal., 1985; Collart etal., 1986; Kamijo et al., 1993), and the stimulation of IFN-c production by IL-2 (Torres et al., 1982). IL-12 (originally termed natural killer cell stimulatory factor) induces IFN-c production in T and NK cells and appears to be a major regulator of IFN-c production in the intact organism (Trinchieri, 1995). Similarly, IL-18 (originally identified as IFN-c inducing factor) is an inducer of IFN-c in NK cells and T cells that in the intact organism controls LPS-induced IFN-c production (Ghayur et al., 1997). Conversely, IFN-c, together with TNF-a, stimulate IL12 production in macrophages, and in mice infected

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with Mycobacterium bovis IL-12 production requires both IFN-c and TNF (Flesch et al., 1995). IL-25, one of the most recently described cytokines, was shown to stimulate IL-4, IL-5 and IL-13 production in T lymphocytes (Fort et al., 2001). Although not as numerous as the reports of stimulatory interactions, there is increasing evidence of inhibitory actions of cytokines on cytokine production. IL-10 is a cytokine whose major biological function appears to be inhibition of cytokine production by TH1 cells and by monocytes/macrophages (Fiorentino et al., 1991). Many of the immunosuppressive and anti-inflammatory actions of TGF-b also appear to be due to its ability to suppress cytokine production in T cells and mononuclear phagocytes (Roberts and Sporn, 1990; Flanders and Roberts, 2001). Another example is a strong inhibitory activity of IL-13 on inflammatory cytokine production (IL-6, IL-1b, TNF-a, IL-8) in LPS-stimulated monocytes (Minty et al., 1993). A prime example of stimulatory and inhibitory interactions involving multiple sets of cytokines is the development of polarized T helper cell 1 (TH1) and T helper cell 2 (TH2) responses (O’Garra and Arai, 2000). TH1 cells secrete IL-2, IFN-c and LT-a and are efficient in activating cellular immune responses that promote the elimination of intracellular pathogens. TH2 cells secrete IL-4, IL-5, IL-10 and IL-13, which promote humoral immune responses, especially IgE production leading to allergy. One major player in these processes is IL-12, which promotes IFN-c production and TH1 development via signaling pathways that lead to activation of Stat4. Another key cytokine is IL-4, leading to Stat6 activation, which can drive a naive Thp cell toward the TH2 differentiation pathway. It has been proposed that a balance between the TH1-specific T-bet and the TH2-specific GATA3 transcription factors can explain how IL-12 and IL-4 can reciprocally drive the initial TH1 and TH2 polarization, respectively (Rengarajan et al., 2000). Molecular details of the ensuing processes are the subject of intense studies.

Transmodulation of cytokine receptors Another mechanism important in the network of cytokine actions is the modulation of the level of cytokine receptor expression. One of the earliest studied models involves induction of the high-affinity

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IL-2 receptor on T cells by IL-1 (Kaye et al., 1984). Appearance of high-affinity receptors is the consequence of the induced expression of the IL-2 receptor complex, mainly due to the regulation of its a chain (Smith, 1988; Hatakeyama et al., 1989). Other cytokines, including TNF and IL-6 (Noma et al., 1987; Lowenthal et al., 1989), can also affect IL-2 receptor expression, though perhaps not as efficiently as IL-1. Other examples of the modulation of cytokine receptors include the stimulatory action of the interferons, especially IFN-c, on the expression of TNF receptors on many different cell lines (Aggarwal et al., 1985). This action might contribute to the widely documented synergism between IFN-c and TNF. Conversely, TNF was also shown to up-regulate IFN-c binding (Raitano and Korc, 1990). In some instances, receptor transmodulation by cytokines results in a reduced level of receptor expression. One example is the down-regulation of TNF receptors by IL-1 (Holtmann and Wallach, 1987). There are also examples of down-regulation of receptor function that do not result from the decreased expression of cell surface receptors, but fall within the general category of ‘receptor trans-signaling’ (Castellino and Chao, 1996). Thus, TNF inhibits insulin signaling by decreasing tyrosine phosphorylation of the insulin receptor and its substrate, IRS-1 (Hotamisligil et al., 1994).

Cytokine chaos? The important roles of cytokines in the regulation of immune and inflammatory responses are by now clearly recognized. Availability of purified, potent cytokine preparations and the use of transgenic mouse models have helped to define the major actions of cytokines and their in vivo functions. However, the extensive redundancy and pleiotropy in cytokine actions makes it difficult to predict how unique and essential individual cytokine actions are in the intact organism. The quest to learn how cytokines function in their natural environment of the intact host has also been greatly aided by the development of the technique of targeted gene disruption. Nevertheless, much remains to be learned about the complex interactions that ultimately determine the outcome of cytokine actions in the complex environment of the intact organism. In an article titled ‘Cytokines, Chaos and Complex-

ity’, Callard et al. (1999) argue that the network of interacting cytokines and cytokine receptors is of such staggering complexity that ‘the long-term behavior of the system is essentially unpredictable’. They further point out that the network of cytokine interactions, which includes positive and negative feedback, is nonlinear because the output is not proportionally related to the input. They relate the cytokine system to Poincaré’s discovery some 100 years ago that the motion of the planets is unpredictable because it takes the movement of only three celestial bodies, whose interactions are governed by Newton’s laws, to defy a complete understanding of their behavior. Mathematicians refer to such behavior as ‘chaotic’. Yet, Callard et al. (1999) correctly point out that despite its enormous complexity the cytokine system is deterministic because the outcome of most cytokine interactions in nature is orderly and compatible with homeostasis. So if there is chaos, it exists only in our interpretations and understanding of the systems and not in the cytokine networks per se ! One of the main points made by Callard et al. (1999) is that even though cytokine networks are nonlinear and therefore ‘chaotic’ (according to the mathematical definition), mathematical models of how cytokine networks work could help unravel some of the complexities involved. My own prejudice is that mathematical modeling may be helpful, but that a better understanding of the complexity of cytokine actions will ultimately emerge mainly as a result of the painstakingly slow, detailed analysis of the molecular mechanisms involved. Examples of recent progress in the understanding of processes that until not long ago appeared ‘nonlinear’ and ‘chaotic’ include the intricate regulatory cascade of IFN-a/b synthesis (Levy et al., 2002; Taniguchi and Takaoka, 2002) or the initial steps in the cascade leading to TH1/TH2 polarization (Rengarajan et al., 2000; Grogan et al., 2001). Whether my prediction is correct should be fairly clear by the time revisions for the tenth edition of The Cytokine Handbook are complete.

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2 Cytokine genetics – Polymorphisms, functional variations and disease associations Grant Gallagher 1, Joyce Eskdale 1 and Jeff L. Bidwell 2 1

University of Medicine and Dentistry of New Jersey, Newark, NJ, USA 2 University of Bristol, Bristol, UK

There was once an old sailor my grandfather knew, who had so many things that he wanted to do, that whenever he thought it was time to begin, he couldn’t because of the state he was in. A.A. Milne The degree to which antigens induce an immune response varies markedly between individuals. Much of this variation is determined by the combination of antigen presenting molecules, both class-I and classII MHC, that the individual is expressing and the range of T cell receptor structures that have accumulated. Together, these determine whether and how well that individual’s immune system will see any given antigen and it will be apparent that the random nature of T cell and B cell receptor construction means that even monozygotic twins will show variation in this respect, or at least have this potential. These aspects of the immune system have formed the backbone of the science of immunogenetics and continue to do so. Immunology, and particularly immunogenetics, continues to be an expanding field. First, as sequence differences are discovered in promoters and coding regions of immunologically relevant genes – such as cytokines and their receptors – the breadth of immune variation between individuals has become apparent. Second, as genome scans of

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

autoimmune, infectious and malignant diseases define loci of pathogenic importance, specific genomic regions automatically become of interest in immunogenetics, even though they may not contain any ‘obviously’ immunological candidate genes. Third, although normal variation in the immune system can be important in disease development in the presence of contributing factors, certain rare mutations can themselves be fundamental in causing disease. Such variations may be associated with susceptibility to certain diseases or contribute to severity and progression. In addition, the same disease may have different contributing actions in one ethnic group compared with another and of course, certain polymorphisms may exist only in one ethnic group (or be absent from that group). Over the last ten years then, it has become obvious that aspects of the immune system other than the MHC also show genetic variation. These variations have been noted in the whole range of immunologically important molecules, from adhesion molecules

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and other cell-surface structures to cytokines and other soluble signals. Thus, the science of immunogenetics has moved into a new ‘post-HLA’ phase and it is clear that the vast variation of the immune system must impact on all theatres of immunological activity, from the daily response to minor environmental antigens, through infectious and autoimmune diseases to malignancy, and man-made immunological insults, such as vaccination and transplantation. The importance of cytokines to the immune system cannot be understated: they are soluble immunomodulatory proteins which are active on a vast array of target cells, generally but not wholly within the immune and haematopoietic systems. Their actions are mediated through highly specific cell-surface receptors and usually result in gene activation with subsequent mitotic, activation, suppression or differentiation effects. Often they act in concert or in complex interactive loops which can be positive or negative and furthermore, these effects may differ with the target cell. It is small wonder then, that cytokines and particularly inflammatory cytokines, have received the lion’s share of attention and this chapter will focus on these molecules. Cytokines are produced by a wide range of cell types and have often been broadly classified as ‘monokines’ (produced by cells of the monocyte lineage) or ‘lymphokines’ (produced by cells of the lymphocyte lineages). This simplistic classification has been variously replaced (e.g. TH1 and TH2 cytokines), but any classification belies the highly complex network in which cytokines always seem to work. For example, they may induce or repress their own expression, as well as that of other cytokines. In addition many are pleiotropic, affecting several cell-types and there is a poorly understood functional redundancy between individual cytokines which nonetheless can have individual functions. These properties of cytokines continue to complicate efforts to analyse both the function of individual cytokines and the influence of cytokine gene polymorphisms on gene expression and disease. As will be seen by the tables presented at the end of this chapter (Bidwell et al., 1999, 2001; Haukim et al., 2002), a significant amount of work has been conducted in the field of cytokine genetics. As in any field, these are not all the greatest of studies. However, we leave it to you, the reader, to decide for yourself which

represent work of most importance to your particular area. We have illustrated this chapter with examples of cytokine genetic studies in human disease. Broadly speaking however, the rationale for studying cytokine gene polymorphisms is as follows:

• to improve our understanding of the origin and mechanism of human disease

• to identify novel diagnostic markers of susceptibility, severity and outcome

• to identify novel therapeutic targets and suitable patients for immunomodulatory treatments

• to identify novel intervention strategies (enhanced vaccination, for example). Such examples are predicated upon the assumption that cytokine levels do vary between individuals, so it is worthwhile to ask if this is the case and if, being so, diseases are affected. The question of whether a person’s genetic make-up or living environment contributes more to their risk of disease has been an important one for some time. In 1988 Sorensen and colleagues (Sorensen et al., 1988) demonstrated clearly that adopted individuals carried a risk of death from infectious causes that was equivalent to that of their natural, rather than adoptive, parents. In so doing they established unequivocally that premature death in adults from infectious causes had a strong genetic background. This is seen nowadays in the context of the wide genetic variation known to exist in the immune system. Often, manifestations of this are routinely accepted. For example, everyone knows people who shake off colds and flu easily and others who seem to have them all winter. TNF and latterly IL-10, have received particular attention in infectious disease. A range of experiments in murine models, together with studies on human material in vitro, demonstrate that variations in IL-10 production can profoundly alter response to, and outcome from, infectious disease. In the mouse, experimental diminution or removal of IL-10 has been achieved by the administration of monoclonal antibodies which neutralise IL-10, or by genetically knocking out the IL-10 gene. The complimentary augmentation of IL-10 levels has been accomplished by injecting recombinant IL-10 or by using IL-10 transgenic mice. Such experiments are designed to explore the extremes of IL-10 levels (i.e. none, or receptorsaturating) and are therefore models for trends in

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immune responses that might be expected to result from natural, genetically defined, differences in cytokine levels. In these contexts, a range of bacterial and other infections have been studied. Typical results include studies on Lysteria monocytogenes which demonstrate that withdrawing IL-10 by antibody neutralization (Wagner et al., 1994) or by genetic knockout (Dai et al., 1997) led to marked increase in disease resistance and improved outcome while increasing the amount of IL-10 present during an infection, either by rIL-10 administration (Kelly and Bancroft, 1996) or in IL-10 transgenic animals (Groux et al., 1999), led to worse disease and poorer outcome. Similar results exist for experimental infection with Streptococcus pneumoniae with high levels of IL-10 supporting increased disease development in mice and IL-10 absence allowing the mice to resist infection (van der Poll et al., 1996). IL-10 also affects responses to mycobacterial infections (Murray et al., 1997). Although responses to viruses have been less frequently studied, Vaccinia virus replication was dramatically impaired in IL-10 knock-out animals (van den Broek et al., 2000) and Herpes simplex-associated skin pathology is ameliorated by application of IL-10 topically (Tumpey et al., 1994). A more complete summary of these experiments is described in the review of Moore et al., (2001), but in general, these experiments suggest that bacterial infections are worse in the context of high IL-10 levels and less severe in a relative absence of IL-10, while the opposite may be true of viral infections. Similar work has been done for tumour necrosis factor (TNF). TNF is considered to be an important mediator of protection from parasitic, bacterial and viral infection (Vassalli, 1992). Once again using Lysteria monocytogenes as a model organism, it has been shown that withdrawing TNF with neutralizing monoclonal antibodies is extremely detrimental (Havell, 1989). However, the effects of high or low TNF are not clear cut and appear to vary with the infection – for example, withdrawing TNF protects from endotoxaemic death (Buetler et al., 1985) while elevated TNF levels are associated with a much poorer outcome in human malaria (Kwiatowski et al., 1990). Conversely, TNF knock-out mice are completely susceptible to mycobacterial infections, developing uncontrolled, fatal infections (Jacobs et al., 2000). These experimental studies come together to show clearly that levels of

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both IL-10 and TNF are important in determining the course and outcome of infectious disease. Even as recently as 1995, genetic studies had demonstrated that markers existed which could be related to differential TNF levels (e.g. Pociot et al., 1992), and many studies had also looked closely at IL1; these have been extensively reviewed elsewhere and will not be discussed in detail here. No such markers existed for IL-10 and indeed it was not widely accepted that cytokines in general would display genetically defined intra-individual differences. Clearly however, a wealth of experimental evidence supported the concept that differences in susceptibility to and severity of diseases might well have their roots in genetically defined differences in the ability to produce important cytokines. Parallel studies in human infection supported this concept. These are perhaps best demonstrated by the elegant studies of Westendorp et al., (1997a, 1997b; van Dissel et al., 1998). They showed that low TNF production was associated with a ten-fold risk of fatal outcome from meninococcal meningitis, while high IL-10 levels were associated with a twenty-fold risk of fatality. Despite the recognized ability of TNF to induce IL-10 levels from monocytes and IL-10 to down-regulate TNF, these two factors were independent, that is the levels of one cytokine were not dependent upon those of the other. These studies complemented earlier reports of high IL-10 levels being associated with fatal bacterial infections in man. Recently, it has been shown to affect macrophage responses during mycobacterial infections (Murray et al., 1997). Furthermore, the severity to which meningitis progresses is associated with serum IL-10 levels, such that high serum IL-10 was observed in patients with a poor or fatal outcome, while patients who had mild disease and a good prognosis had lower serum IL-10 levels (e.g. Lehmann et al., 1995). Recent studies have demonstrated clearly that levels of TNF and IL-10 vary between individuals in this way, and that highly informative genetic markers exist with which to examine the heritable basis of this. In the TNF locus, a complex pattern of microsatellite alleles and SNP alleles form a system which demonstrates that three families of haplotypes exist, associated with TNF secretion (Weissensteiner and Lanchbury, 1997). Notwithstanding the relationship between the TNF locus and the class-I and class-II MHC alleles

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(Gallagher et al., 1997), TNF genetics are not merely a subset of the greater MHC. TNF secretion following LPS stimulation has been shown clearly to vary independently of the MHC (Pociot et al., 1992), and it can be demonstrated that disease associations which involve the TNF locus are independent of (although complementary to) the MHC (Rood et al., 2000; Caballero et al., 2000). While many studies examine the SNP alleles (Wilson et al., 1997; Majetschak et al., 1999; Negoro et al., 1999), in fact, microsatellite alleles or combinations thereof, may provide more accurate information. Thus, the TNFd4 microsatellite allele (Weissensteiner and Lanchbury, 1997) and the TNFa2 microsatellite allele (Pociot et al., 1992) offer excellent resolution of the bewildering array of SNP allelic possibilities. Indeed, the TNFa2d4 haplotype has been associated with a range of chronic inflammatory disorders against disparate MHC backgrounds (Plevy et al., 1996; Mattey et al., 1999). While much work has concentrated on TNF and IL-10, other studies have demonstrated functional genetic variation in other cytokines. The range of genetic markers in human cytokine genes is very large and these have been used to study functional or disease associations, but only in single populations (Bidwell et al., 1999, 2001; Mullighan et al., 1999). Some of the better studies are on genes with relevance to infection. For example, a promoter polymorphism in the IL-6 gene defines high or low producers in both resting and stimulated conditions (Fishman et al., 1998). In addition, chemokines and their receptors (Gonzalez et al., 2001; Paxton et al., 2001) have also been studied recently and proven to be both polymorphic and informative. These studies have been accompanied by a very small number of reports which specifically address the question of ethnic variation within the distribution of polymorphic elements in cytokine (and related) genes. These studies have shown that the geographic/ethnic distribution of alleles in a number of genes does in fact vary (Cox et al., 2001; Gonzalez et al., 2001; Padyukov et al., 2000). Thus, early studies which concentrated on demonstrating individual differences in cytokine production, or the association of higher (or lower) cytokine production with disease have in more recent years developed to confirm these differences and indicate their genetic origins. So, what is the evidence that cytokine polymor-

phisms can help in the understanding of human disease? The influence of cytokine gene polymorphisms on gene expressions and disease has largely been addressed as two separate subjects and only a few studies have integrated those. Two excellent examples come in atopy/asthma. A number of SNPs exist in the IL-13 promoter (Pantelidis et al., 2000), at least one of whch (C-T at 1055; van der Pouw Kraan et al., 1999) affects the binding of a specific transcription factor to the IL-13 promoter, altering its function such that the TT genotype is associated with increased levels of IL-13 production in allergic asthma patients (Ahmed et al., 2000; Liu et al., 2000; Howard et al., 2001). Asthma provides an addition a example of where functional cytokine genetics studies have been fruitful. In the promoter of RANTES, lies a G-A SNP at position 403. The 403.A allele is associated with increased susceptibility to both atopy and asthma, with homozygosity for 403.A being associated with a 6.5-fold increase in prevalence of responsive airway obstruction (Fryer et al., 2000). In a parallel study, it was shown to produce/destroy a GATA site and in the presence of the GATA site not only was RANTES promoter activity increased in transfection studies, but the extra GATA site was strongly associated with atopy in general, supporting the previous asthma study (Nickel et al., 2000). The RANTES gene locus is also associated with rates of HIV progression (Paxton et al., 2001). One of the most interesting series of studies in human cytokine genetics has involved the autoimmune disease systemic lupus erythematosus (SLE) and the cytokine IL-10. One of the earliest observations following the discovery of IL-10 was that it appeared to be over-produced in SLE. While many symptoms can come together to provide a diagnosis of systemic lupus erythematosus (SLE), it is usually held to be a disease characterized by autoantibody production. IL-10 is recognized to stimulate the proliferation of human B cells and their secretion of all classes of immunoglobulin (Rousset et al., 1992). IL-10 was originally defined as a cytokine able to alter the balance of murine TH1/TH2 activity, in favour of the TH2–type response (Fiorentino et al., 1991) and it has several properties which would appear to encourage lupus autoimmunity. One critical aspect of this is its ability to diminish macrophage activation and antigen presentation, thereby directly and indirectly

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inhibiting T-cell function (Enk et al., 1993; de Waal Malefyt et al., 1993). In particular, IL-10 can function as a negative autocrine regulator of TNF production (Wanidworanun and Strober, 1993). IL-10 may also promote inflammatory responses through its potent stimulation of B cell proliferation and differentiation (Rousset et al., 1992). Raised IL-10 levels have been reported in several autoimmune states (Llorente et al., 1994), originating from both B cells and monocytes. Also, in vitro studies have shown that hypergammaglobinaemia in SLE is IL-10 dependent (Llorente et al., 1995) and it has been reported to protect B cells from apoptosis (Levy and Brouet, 1994). In addition, the ability of IL-10 to induce anergy in T cells and (Taga et al., 1993) may relieve some elements of the normal control over B cell function, if suppression is reduced (it may be of interest that conversely, IL-10 can function as a growth factor for gamma/delta-positive human T cells; Pawelec et al., 1995). Increased IL-10 production may therefore contribute to SLE by direct effects on B cell autoantibody production and survival and it is of interest that a recent study has suggested that the region of IL-10:IFN-c secreting cells in SLE is an indicator of disease severity (Hagiwara et al., 1996). It has also been shown that removing IL-10 with antiIL-10 antibodies slows the development of murine autoimmunity (Ishida et al., 1994). This may well be through a mechanism which prevents the activation to autoreactivity of Ly-1 (ie, CD5) positive B cells (Ishida et al., 1992), themselves thought to be a major source of B cell-derived IL-10 (O’Garra et al., 1992). In addition, the lingering question of whether lupus is a TNF-deficiency disorder is supported by anecdotal reports that rheumatoid arthritis patients receiving anti-TNF therapy may, in exceptional circumstances, develop autoantibody specificities characteristic of lupus. There have also been reports that experimental lupus can be treated in some respects by the administration of TNF. Thus, the well-characterized ability of IL-10 to directly down-regulate TNF production (Wanidworanun and Strober 1993) is also implicated as a mechanism by which high levels of IL-10 could be permissive for lupus development. IL-10 secretion levels do vary between indivduals and this can be associated with disease outcome. Most dramatically, this has been shown in studies on meningitis patients (Westendorp et al., 1997a), where individuals with genetically high IL-10 secretion fared very

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poorly in comparison to those patients with low IL-10 secretion. Increased levels of IL-10 in mononuclear cells from lupus patients has been demonstrated in numerous studies (Llorente et al., 1993, 1994; Hagiwara et al., 1996; Houssiau et al., 1995; Al-Janadi et al., 1996). Indeed, unaffected primary and secondary family members of lupus patients produce over five times as much IL-10 than do control subjects (Llorente et al., 1997). Taken together, these findings are consistent with high production of IL-10 functioning as one of a limited number of primary genetic defects which are permissive for lupus, but it also clearly demonstrates that high IL-10 secretion is not in itself causative of this condition. Certainly, all of the molecular abnormalities that have been described in lupus patients could not be directly the consequence of alleles in the gene of the gene product being assayed. The most parsimonious model is that most of the cytokine abnormalities are secondary, meaning that the observed abnormality is the result of something else that is causing them. A few, however, represent intrinsic abnormalities that predispose to lupus and ‘cause’ the others. Expression levels of IL-10 are good candidates for such intrinsic abnormalities. Having been found in family members suggests that it would also have been found in the lupus patients before disease onset and part of the milieu which governs the immune response to the putative ‘lupus-causing’ environmental factor toward lupus autoimmunity. The region immediately upstream of the human IL10 gene is highly polymorphic, with two dinucleotide repeats and many single base substitutions. The interest in this gene is such that several groups of workers have investigated these polymorphic elements independently. The two microsatellites were described first in the literature by Eskdale and co-workers (Eskdale and Gallagher, 1995; Eskdale et al., 1996), while Fabio Cominelli’s group first described the 1082 A/G substitution marker (Tountas and Cominelli, 1996). The two additional single base substitutions were first described by Turner and colleagues (1997) and independently characterized by Eskdale et al. (1997a); Turner et al. also independently identified the Cominelli A/G SNP, now often referred to as ‘IL101082’. One of these microsatellite loci (IL10.G) has shown an altered distribution of allele frequencies in lupus patients in comparison to controls (Eskdale et al., 1997b), with evidence that associations between

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microsatellite alleles and the class of autoantibody present also exist. While this latter observation remains to be verified, several groups have followed and confirmed the basic association between the IL10.G microsatellite and the presence of SLE (Mehrian et al., 1998; D’Alfonso et al., 2000). A followup study in SLE families, which would have the potential to reveal linkage, is still awaited, but it is of interest that one recent study also finds association between this microsatellite and Sjogren’s syndome, wich shares many lupus-like features with SLE (Hulkkonen et al., 2001). SNPs studied in IL-10 have also proved informative in lupus (Lazarus et al., 1997; Mok et al., 1998; Gibson et al., 2001) and further more distal markers continue to be discovered (Kube et al., 2001). Thus, there is ample evidence that an enormous genetic variation occurs in human cytokine genes, but only a small proportion of the studies carried out have demonstrated a functional role for these markers. A similar small proportion of the disease association studies carried out have been confirmed in independent, replicate populations and in only a few cases have disease-associated SNPs also been shown to exert a functional effect – so far! This should encourage us, rather than discourage us, to continue as a research community to use and investigate these markers and to consider cytokine genes as highly informative reservoirs of genetic variation in the immune system. However, these exciting developments in the understanding of cytokine genetics should be interepreted with a note of caution, to prevent us from being seduced into thinking that only markers within individual cytokine genes can control differences in inter-individual cytokine secretion. This is not so. Other elements which can influence the expression of cytokine (and other?) genes should not be forgotten. For example, the rigour with which Fishman et al. (1998) approached the measurement of IL-6 production in their control subjects, demonstrates the importance of the natural metabolic variation which occurs daily. This supported earlier studies, for example that of Petrovsky and Harrison (1997a), who showed that the LPS induction of IL-10 and IFN-gamma varied throughout the day, observing that the IFN-gamma/IL-10 ratio peaked early in the morning and concluding that both cortisol and melatonin could regulate diurnal immune variation. Although much has been made of the requirement for

caution when interpreting genetic data from the TNF cluster without due consideration of the MHC and linkage disequilibrium, MHC effects on cytokines off chromosome 6 have not been as well documented. The evidence has begun to emerge, however. A study in 1997 (Petrovsky and Harrison, 1997b) demonstrated that secreted levels of IFN-gamma varied markedly with class-II alleles, in an MLR. DR1, DR2 and DR6 were associated with high IFN-gamma secretion while DR3, DR4 and DR7 were associated with lower IFN-gamma production. Similar conclusions were drawn for those DQ alleles in linkage disequilibrium with the DR alleles noted above. This pattern was reversed for TNF secretion (i.e. DR3 was high TNF and so on), mirroring earlier work by Pociot et al. (1992) who demonstrated a DR-based hierarchy of TNF secretion which was of greater magnitude than the TNF-allele results for which they are more usually remembered. Similar data are available for other aspects of the immune system, for example antibody production (Mineta et al., 1996). In this regard, DR3 has received the greatest attention. T cell activation varies in DR3-positive individuals, perhaps because of diminished CD69 expression (Candore et al., 1995), as do cytokines themselves (Caruso et al., 1996) particularly in regard to autoimmune DR3 positive subjects (Lio et al., 1997). Apoptosis may differ because these individuals have diminished expression of CD95/FAS (Stasi et al., 1997) and indeed lower total lymphocyte counts have been described in association with B8-DR3 (Caruso et al., 1997). Little insight to the mechanism of these various effects by the class-II on immune function was available until recently, when it was demonstrated that different class-II molecules varied in the efficiency with which they transduce signals from CD4 across the cell membrane, and that this variation is carried with the intracellular portion of the class-II molecule (Fleury et al., 1996). As if this were not confusing enough, the age of the donors themselves has been shown to affect T cell activation (Lio et al., 1996) through various mechanisms. In conclusion, the genetic effect seen to be acting on cytokine production, and implicating them as diseaseassociated loci in their own right, are complicated by the MHC and age. How well we as a research community deal with these complications will determine how efficiently the influence of cytokine immunogenetics on disease is elucidated.

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TABLE 2.1 List of human cytokine gene polymorphisms Gene

Polymorphism

First author, year

GM-CSF GM-CSF-Ra GM-CSF-Ra GM-CSF-Ra GM-CSF-Ra GM-CSF-Ra GM-CSF-Rb

codon 117 C → T nt1148 G →A nt199 C → G nt428 A → G nt640 A → G nt824 C → T nt1306 C → T (Ser426)

GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb GM-CSF-Rb IFNc IFNc IFNc IFNc IFNc IFNc IFNaR IFNaR IFNa IFNa (IFNA10) IFNa (IFNA10)

nt1835 C → A nt1968 G → T nt1972 G → A nt1982 G → A nt2427 G → A nt301 C → T (Cys91) nt773 G → C (Glu249Glu) nt962 G → A (Asp312Asn) Exon 4, 5199 A → T Exon 4, 5272 A → G Intron 3, 2459 A → G Intron 3, 2671 T → C Intron 3, 3177 T → C Intron 3, 3273 G → A 18G → A 408C → T Dinucleotide repeat nt1265 A → C nt991 (60*) T → A Cys20Stop (Sau3A I)

IFNa (IFNA17)

nt1101 (170*) A → C

IFNa (IFNA17) IFNa (IFNA17)

nt1453 C → T nt1482 (551*) T → G Ile184Arg (Ssp I RFLP)

IFNa (IFNA17) IFNa (IFNA2)

nt171insA (Nla III RFLP) nt1068 G → A

IFNa (IFNA2)

nt1101 G → A

IFNaR IFNb IFNb IFNc IFNc

HindIII RFLP 3’ MspI RFLP nt153 C → T 333 C → T Intron 1, (CA) repeat

IFNcRI IL-10

TaqI RFLP 1082 G → A

IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10

1255 C → T 1349 A → G 2013 A → G 2050 G → A 2100 C → A 2739 A → G 2763 C → A 2769 A → G 2776 A → G

Tagiev, 1995 Wagner, 1994 Wagner, 1994 Wagner, 1994 Wagner, 1994 Wagner, 1994 Freeburn, 1996 Freeburn, 1998 Freeburn, 1996 Freeburn, 1996 Freeburn, 1996 Freeburn, 1996 Freeburn, 1996 Freeburn, 1998 Freeburn, 1998 Freeburn, 1998 Iwasaki, 2001 Iwasaki, 2001 Iwasaki, 2001 Iwasaki, 2001 Iwasaki, 2001 Iwasaki, 2001 Muldoon, 2001 Muldoon, 2001 Kwiatkowski, 1992 Golovleva, 1996 Golovleva, 1996 Miterski, 1999 Golovleva, 1996 unconfirmed Miterski, 1999 Golovleva, 1996 Golovleva, 1996 Miterski, 1999 Miterski, 1999 Golovleva, 1996 unconfirmed Miterski, 1999 Golovleva, 1996 unconfirmed Miterski, 1999 Vielh, 1990 Riggin, 1982 Miterski, 1999 Giedraitis, 1999 Gray, 1983 Ruiz-Linares, 1993 Hauptschein, 1992 Tounas, 1996 Turner, 1997 D’Alfonso, 2000 D’Alfonso, 2000 D’Alfonso, 2000 Gibson, 2001 Gibson, 2001 D’Alfonso, 2000 Gibson, 2001 D’Alfonso, 2000 Gibson, 2001

BASIC CYTOKINE BIOLOGY

26

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.1 (continued ) Gene

Polymorphism

First author, year

IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-10

2849 G → A 3’ flanking region, 117 after stop codon C → T 3533 A → T 3575 T → A 3715 A → T 5’ distal (CA) repeat (IL10.R) 5’ proximal (CA) repeat (IL10.G) 5402 C → G 592 C → A

IL-10 IL-10 IL-10 IL-10 IL-10

6208 G → C 657 A → G 6752 A → T 7400 (3bp deletion) 819 C → T

IL-10 IL-10 IL-10 IL-10 IL-10 IL-10 IL-11 IL-12 (p35) IL-12 (p35) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-12 (p40) IL-13

851 A → G 8531 G → A 8571 C → T Exon 1, 78 G → A (Gly15 → Arg) Intron 3, 19 C → T Intron 3, 953 T → C 5’ dinucleotide repeat 916 C → T 916 C → T 1287 G → T 1287 G → T Exon 7, 16117 G → C Exon 8, 16974 A → C Intron 1, 3696 A → G Intron 1, 3757 T → C Intron 1, 4572, TG insertion Intron 1, 4793 C → G Intron 2, 8798 TAA repeat Intron 2, 8930 A → G Intron 2, 8944 A → G Intron 3, 9910 G → A Intron 4, 11244 A → T Intron 4, 11563 AT repeat Intron 7, 16521 A → C nt1188 (5’UTR) A → C 1055 C → T

IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13 IL-13Ra

3’ UTR, 2525 G A 3’ UTR, 2749 3’ UTR, 2580 C A 3’-UTR, nt2043 G → A 3’-UTR, nt2579 C → A 5’ promoter, 1512 A C additional Gln residue, position 98 Exon 4, 2044 G A Intron 1, nt543 G → C Intron 3, 1923 C T Intron 3, nt1922 C → T nt 4257, G → A nt1050 C → T

Gibson, 2001 Donger, 2001 D’Alfonso, 2000 Gibson, 2001 Gibson, 2001 Eskdale, 1996 Eskdale, 1995 Kube, 2001 Eskdale, 1997 Turner, 1997 Kube, 2001 D’Alfonso, 2000 Kube, 2001 Kube, 2001 Eskdale, 1997 Turner, 1997 D’Alfonso, 2000 Kube, 2001 Kube, 2001 Donger, 2001 Donger, 2001 Donger, 2001 Bellingham, 1998 Pravica, 2000 Pravica, 2000 Pravica, 2000 Pravica, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Huang, 2000 Hall, 2000 van der Pouw Kraan, 1999 Laundy, 2000 Graves, 2000 Graves, 2000 Graves, 2000 Pantelidis, 2000 Pantelidis, 2000 Graves, 2000 McKenzie, 1993 Graves, 2000 Pantelidis, 2000 Graves, 2000 Pantelidis, 2000 Liu, 2000 Ahmed, 2000

BASIC CYTOKINE BIOLOGY

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.1 (continued ) Gene

Polymorphism

First author, year

IL-13Ra IL-16 IL-18 IL-18 IL-18 IL-18 IL-18 IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1Ra IL-1RI IL-1RI IL-1RI IL-1RI IL-1RI IL-1RI IL-1RI IL-1RI IL-1RI IL-1a IL-1a IL-1a IL-1a

1050 C → T 295 T → C 113 T → G 127 C → T 137 G → C 607 C → A 656 G → T 2016 T → C Intron 2 86bp VNTR nt11100 T → C (MspA1I) nt1731 G → A nt1821 G → A nt1868 A → G nt1887 G → C nt1934 T → C nt8006 T → C (MspI) nt8061 C → T (MwoI) nt9589 A → T (SspI) 2 PstI RFLPs Exon 1C, 140 A → T Exon 1C, 52 C → A (BsrB I) Exon 1C, 97 G → A (Sty I) Intron 1A, 1622 (of AF302042) G → A (Hinf I) Intron 1A, 701 (of AF302042) G → A (Pst I) Intron 1B, 52 (of AF146426) A → G (Msp I) Intron 1C,  1498 (of AF302043) T → C (Alu I) Intron 1C, 976 (of AF302043) T → C (BstF5 I) (TTA) repeat 4345 T → G 889 Dinucleotide repeat

IL-1a IL-1b IL-1b IL-1b IL-1b IL-1b IL-2 IL-2 IL-2 IL-2 IL-2 IL-2R IL-2R IL-2R IL-2Ra IL-2Rb IL-3 IL-3 IL-3 IL-3 IL-3 IL-3 IL-3 IL-3

Intron 6, 46bp VNTR 3953 (nt5887) C → T (TaqI) 35 T → C (AluI) 511 G → A (AvaI) nt3263 C → T nt5810 A → T (BsoFI) 166 330 Allele A: 122bp dinucleotide repeat Dinucleotide repeat exon 1 nt742 T → G Allele 0: 165 bp dinucleotide repeat Allele 2: 169 bp dinucleotide repeat Allele 9: 147 bp dinucleotide repeat TaqI RFLP Dinucleotide repeat 131 C → T 5 C → T 16 C → T 211 C → A BglII RFLP Enhancer nt232 Enhancer nt236 Enhancer nt283

Ahmed, 2000 Nakayama, 2000 Giedraitis, 2001 Giedraitis, 2001 Giedraitis, 2001 Giedraitis, 2001 Giedraitis, 2001 Kornman, 1998 Tarlow, 1993 Guasch, 1996 Langdahl, 2000 Langdahl, 2000 Langdahl, 2000 Langdahl, 2000 Langdahl, 2000 Guasch, 1996 Guasch, 1996 Guasch, 1996 Bergholdt, 1995 Sitara, 1999 Sitara, 2000 Sitara, 2000 Bergholdt, 2000 Bergholdt, 2000 Sitara, 1999 Bergholdt, 2000 Bergholdt, 2000 Zuliani, 1990 Kornman, 1998 Kornman, 1998 Todd, 1991 Epplen, 1994 Bailly, 1993 Pociot, 1992 Guasch, 1996 di Giovine, 1992 Langdahl, 2000 Guasch, 1996 John, 1998 John, 1998 Khani-Hanjani, 2001 Epplen, 1994 Denny, 1997 Khani-Hanjani, 2001 Khani-Hanjani, 2001 Khani-Hanjani, 2001 Cottrell, 1994 Brewster, 1991 Jeong, 1998 Jeong, 1998 Jeong, 1998 Jeong, 1998 Jaquet, 1989 Jeong, 1998 Jeong, 1998 Jeong, 1998

BASIC CYTOKINE BIOLOGY

27

28

CYTOKINE GENETICS

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POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.1 (continued ) Gene

Polymorphism

First author, year

IL-4 IL-4 IL-4 IL-4 IL-4 IL-4 IL-4 IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4R IL-4Ra IL-4Ra IL-4Ra IL-4Ra IL-4Ra IL-4Ra

33 C → T 34 C → T 524 C → T 590 C → T (BsmFI) Intron 2 dinucleotide repeat Intron 2, 70bp VNTR Intron 3, (GT) repeat nt1124 A → C nt1167 G → T nt1216 T → C nt1218 C → T nt1224 T → C nt1232 C → T nt148 A → G nt1902 G → A (R576Q) nt2281 T → C nt426 C → T nt747 C → G nt864 T → C 1914 T → C 3223 C → T 890 T → C CAAAA repeat (5–7) Exon 11, 1654 G → A (V554 → I) nt1124 A → C (E375A)

IL-4Ra

nt1216 T → C (C406R)

IL-4Ra

nt148 A → G (I50V)

IL-4Ra

nt1682 T → C (S478P) (previously S503P)

IL-4Ra

nt1902 G → A (R551Q) (previously R576Q)

IL-4Ra

nt2281 T → C (S761P)

IL-5Ra

80 G → A (MaeIII)

IL-5Ra IL-6 IL-6

Dinucleotide repeat (CA)n repeat 174 G → C (NlaIII)

IL-6

3’ (AT)-rich minisatellite

IL-6 IL-6 IL-6 IL-6

5’ (AT)-tract (5 alleles) 572 G → C 597 G → A BglII RFLP

IL-6 IL-6 IL-6 IL-6R IL-8

MspI RFLP nt565 G → A (FokI) XbaI RFLP (CA)n repeat HindIII RFLP

Suzuki, 1999 Takabayashi, 1999 Borish, 1994 Walley, 1996 Marsh, 1994 Mout, 1991 Mout, 1991 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Hershey, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Deichmann, 1997 Hackstein, 2001 Hackstein, 2001 Hackstein, 2001 Hackstein, 2001 Lozano, 2001 Deichmann, 1997 Deichmann, 1999 Deichmann, 1997 Deichmann, 1999 Deichmann, 1997 Deichmann, 1999 Deichmann, 1997 Deichmann, 1999 Kruse, 1999 Hershey, 1997 Deichmann, 1999 Deichmann, 1997 Deichmann, 1999 Kollintza, 1998 Kollintza, 1998 Epplen, 1994 Tsukamoto, 1998 Olomolaiye, 1997 Olomolaiye, 1998 Fishman, 1998 Bowcock, 1989 Murray, 1997 Fishman, 1998 Terry, 2000 Terry, 2000 Fugger, 1989 Blankenstein, 1989 Fugger, 1989 Fishman, 1998 Linker-Israeli, 1996 Tsukamoto, 1998 Fey, 1993

BASIC CYTOKINE BIOLOGY

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.1 (continued ) Gene

Polymorphism

First author, year

IL-9 LTa (TNFb) LTa (TNFb) RANTES RANTES RANTES TGFa TGFb1

Dinucleotide repeat AspHI RFLP Intron 1, NcoI RFLP (Thr26Asn) (TNFB*1Asn26; TNFB*2Thr26) 109 T → C 28 C → G 403 A → G TaqI RFLP 509

TGFb1

800

TGFb1 TGFb1 TGFb1 TGFb1 TGFb1

988 nt713–8delC nt72 unspecified nt788 C → T nt869 (Leu10Pro)

TGFb1

nt915 (Arg25Pro)

TGFb1 TGFb2 TGFb2 TGFb2 TNFRSF1A (p55) TNFRSF1A (p55) TNFRSF1A (p55) TNFRSF1B (p75) TNFRSF1B (p75) TNFRSF1B (p75) TNFRSF1B (p75) TNFRSF1B (p75) TNFRSF1B (p75) TNFRSF1B (p75) TNFa TNFa TNFa TNFa TNFa TNFa TNFa TNFa

R124S 4 RFLPs, SSCPs 5’UTR (4bp insertion – ACCA) Exon 1 (SNP G → A) 383 A → C (BglII) nt36 A → G (MspA1 I) Intron 1, 11-allele polymorphic microsatellite marker 3’-UTR SSCP ‘5/6’ 3’-UTR SSCP ‘7/8’ exon 10, nt1663 A → G exon 10, nt1668 T → G exon 10, nt1690 C → T exon 6, M196R exon 6, nt676 C → T 70 G → A TNFa 163 G → A 238 G → A 308 G → A (TNF1G; TNF2A) 376 G → A 574 856 (*-857)

TNFa

862 (*-863)

TNF

TNFa, b, c, d, e microsatellites

Polymeropoulos, 1991 Ferencik, 1992 Messer, 1991 Rink, 1996 Azzawi, 2001 al Sharif, 1999 Hajeer, 1999 Hayward, 1987 Cambien, 1996 Awad, 1998 Cambien, 1996 Awad, 1998 Cambien, 1996 Langdahl, 1997 Awad, 1998 Langdahl, 1997 Cambien, 1996 Awad, 1998 Cambien, 1996 Awad, 1998 Stewart, 1999 Nishimura, 1993 Alansari, 2001 Alansari, 2001 Pitts, 1998 Pitts, 1998 Eskdale, 2000 Kaufman, 1994 Kaufman, 1994 Pantelidis, 1999 Pantelidis, 1999 Pantelidis, 1999 Komata, 1999 Pantelidis, 1999 Brinkman, 1997 TNFa Brinkman, 1997 D’Alfonso, 1994 Wilson, 1993 Wilson, 1997 Brinkman, 1997 Uglialoro, 1998 Uglialoro, 1998 *Higuchi, 1998 Uglialoro, 1998 *Higuchi, 1998 Nedospasov, 1991 Udalova, 1993

Full references are provided in the appropriate Genes and Immunity reviews and supplements (Bidwell et al., 1999, 2001; Haukim et al., 2002).

BASIC CYTOKINE BIOLOGY

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30

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POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.2 In vitro expression studies Gene

Polymorphism and allele (or haplotype) Expression

First author, year (note 1)

IFNc IFNc IL-10 IL-10 IL-10 IL-10 IL-10

(CA)n intron 1 (all alleles) (CA)n intron 1 (allele 2) 1082 A, 819 C, 592 C 1082 A, 819 T, 592 A 1082 G, 819 C, 592 C 1082A, 819T, 592A 1082G, 819C, 592C

No effect Increased Decreased Decreased Increased Decreased Increased

IL-10 IL-10 IL-10 IL-13 IL-18 IL-1Ra IL-1Ra

R 2, G 14 R3 R 3, G 7 1055 T 607 homozygous for C, 137 homozygous for G intron 2 86bp VNTR (allele 2) Intron 2 86bp VNTR, (allele 2)

Increased Decreased Decreased Increased Increased (not statistically significant) Decreased Increased

Cartwright, 1999 Pravica, 1999 Turner, 1997 Turner, 1997 Turner, 1997 Crawley, 1999 Crawley, 1999 Maurer, 2000 Eskdale, 1998 Eskdale, 1998 Eskdale, 1998 van der Pouw Kraan, 1999 Giedraitis, 2001

IL-1a

Intron 6, 46bp VNTR

Related To VNTR Allele

IL-1b IL-4Ra IL-6 IL-6

Increased Reduced sIL-4R Increased Increased Decreased

Terry, 2000

Increased

Terry, 2000

IL-6

3953 (nt5887) T 3223 C → T 174 G 3’ (AT)-rich minisatellite (790, 792, 808 and 820bp alleles) 597A, 572G, 373A8/T12, 174G haplotype 597G, 572G, 373A9/T11, 174G haplotype IL-6 –174 G ( C (NlaIII)

Tountas, 1999 Tarlow, 1993 Danis, 1995 Hurme, 1998 Bailly, 1993 Bailly, 1993 Pociot, 1992 Hackstein, 2001 Fishman, 1998 Linker-Israeli, 1999

Kilpinen, 2001

LT

Intron 1, NcoI RFLP

LT LT LT  TNFa

TGFb1

Intron 1, NcoI RFLP: TNFB*1 (Asn26) Intron 1, NcoI RFLP: TNFB*2 (Thr26) Intron 1, NcoI RFLP: TNFB*1 (Asn26), TNFa6 Intron 1, NcoI RFLP: TNFB*2 (Thr26), TNFa2 nt915 (Arg25)

Increased in neonates, not in adults No effect on LT secretion Increased Decreased Decreased

TNFa TNFa TNFa TNFa TNFa

489, 308, 1031, 863, 857 70 G → A 1031 238 A 238 G → A

No difference No effect Increased Increased No effect

TNFa TNFa

238, allele 2 244, 238

Decreased Increase (in certain cell lines)

IL-6 IL-6

LT  TNFa

Pociot, 1993 Messer, 1991 Messer, 1991 Pociot, 1993

Increased

Pociot, 1993

Increased

Awad, 1998 Awad, 1998 Kaijzel, 2001 Uglialoro, 1998 Higuchi, 1998 Grove, 1997 Pociot, 1995 Huizinga, 1997 Kaijzel, 1998 Uglialoro, 1998 Kaluza, 2000 Bayley, 2001

BASIC CYTOKINE BIOLOGY

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

31

TABLE 2.2 (continued ) Gene

Polymorphism and allele (or haplotype) Expression

First author, year (note 1)

TNFa

308

No effect

TNFa

308 (TNF2)

Increased

TNFa

308 (TNF2)

Increased

TNFa TNFa TNFa

308 (TNF2) 308, 376 376 G → A

No effect No effect No effect

TNFa TNFa TNFa TNFa TNFa TNFa TNFa TNFa TNFa TNF TNF TNF TNF TNF TNF

509 T 574 856 (*857) 856 (*857) 857T 862 (*863) 862 (*863) 863 A 863 A a a2 c d3 a13 a2 and a9

Increased No effect Increased No effect Increased Increased No effect Decreased (31%) Increased No effect on LT secretion Decreased No effect on LT secretion Increased Decreased Increased

Pociot, 1993 Turner, 1995 Huizinga, 1997 Uglialoro, 1998 Huang, 1999 Kroeger, 2000 Maurer, 1999 Wilson, 1997 Galbraith, 1998 Sotgiu, 1999 Bayley, 2001 Huizinga, 1997 Kaijzel, 1998 Luedecking, 2000 Uglialoro, 1998 Higuchi, 1998 Uglialoro, 1998 Hohjoh, 2001 Higuchi, 1998 Uglialoro, 1998 Skoog, 1999 Hohjoh, 2001 Pociot, 1993 Derkx, 1995 Pociot, 1993 Turner, 1995 Obayashi, 1999 Obayashi, 1999

Full references are provided in the appropriate Genes and Immunity reviews and supplements (Bidwell et al., 1999, 2001; Haukim et al., 2002).

BASIC CYTOKINE BIOLOGY

32

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POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 In vivo disease association studies Cytokine and polymorphism

Disease

Association

First author, year (note 1)

EPO-R nt5964 C → G

Primary familial and congenital polycythaemia Early-onset pauciarticular juvenile chronic arthritis Multiple sclerosis Acute myeloid leukaemia

Yes

Kralovics, 1998

No

Epplen, 1995

No No

Epplen, 1997 Freeburn, 1998

Acute myeloid leukaemia

No

Freeburn, 1998

Acute myeloid leukaemia

No

Freeburn, 1998

Acute myeloid leukaemia

No

Freeburn, 1998

Early-onset pauciarticular juvenile chronic arthritis Multiple sclerosis Multiple sclerosis Multiple sclerosis

No

Epplen, 1995

Yes (protection) Yes (susceptibility) No

Miterski, 1999 Miterski, 1999 Miterski, 1999

Multiple sclerosis

No

Miterski, 1999

Multiple sclerosis

Yes (susceptibility)

Miterski, 1999

Multiple sclerosis Rheumatoid arthritis

No No

Miterski, 1999 Pokorny, 2001

Atopic asthma

Yes (Japanese children) Yes (increased severity) Yes (increased frequency of allele 5; decreased frequency of allele 2) Yes (in conjunction with TNF widtype) No

Nakao, 2001

FGF1–a (GT)n 5’-UTR FGF1–a (GT)n 5’-UTR GM-CSF-Rb nt1306 C → T (Ser426) GM-CSF-Rb nt301 C → T (Cys91) GM-CSF-Rb nt773 G → C (Glu249Glu) GM-CSF-Rb nt962 G → A (Asp312Asn) IFNa (CA)n intron 1 IFNa (GT)n allele 02 IFNa (GT)n allele 07 IFNa (IFNA10) nt991 (60*) T → A Cys20Stop (Sau3A I) IFNa (IFNA17) nt1482 (551*) T → G Ile184Arg (Ssp I RFLP) IFNa (IFNA17) nt171insA (Nla III RFLP) IFNb nt153 C → T IFNc CA(13) intron A microsatellite IFNc (CA)n intron 1 IFNc (CA)n intron 1

Graft-versus-host-disease

IFNc (CA)n intron 1

Grave’s disease

IFNc (CA)n intron 1

Hay fever

IFNc (CA)n intron 1

Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Lung allograft fibrosis Multiple sclerosis

IFNc (CA)n intron 1 IFNc (CA)n Intron 1 IFNc (CA)n intron 1 IFNc (CA)n intron 1 IFNc (CA)n intron 1 IFNc (CA)n Intron 1 IFNc (CA)n Intron 1 IFNc (CA)n intron 1 IFNc (CA)n Intron 1 (126 bp repeat) IFNc (CA)n Intron 1 and IL-10 1082 G → A IFNc 333 C → T IFNc-R1 (Val14Met) IFNc-R1 (Val14Met), IFNc-R2 Gln64/Arg64

Multiple sclerosis Multiple sclerosis Rejection of renal transplant Systemic lupus erythematosus Rheumatoid arthritis Renal transplant rejection Multiple sclerosis SLE Atopic asthma

Cavet, 2001 Siegmund, 1998

Nieters, 2001 Pociot, 1997

Yes

Awata, 1994

Yes

Jahromi, 2000

Yes No No No (Europeans) No

Awad, 1998 Epplen, 1997 Wansen, 1997 He, 1998 Dai, 2001 Goris, 1999 Pelletier, 2000

No

Lee, 2001

Yes (susceptibility and severity) Yes

Khani-Hanjani, 2000

No Yes No (Japanese children)

BASIC CYTOKINE BIOLOGY

Asderakis, 1998 Giedraitis, 1999 Tanaka, 1999 Nakao, 2001

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IFNc-R1 Met14/Val14 genotype and IFNc-R2 Gln64/Gln64 genotype IFNc-R Val14Met

Systemic lupus erythematosus

Yes (development)

First author, year (note 1) Nakashima, 1999

Systemic lupus erythematosus Chronic Graft-versus-host disease Early-onset Periodontal disease Inflammatory bowel disease and ulcerative colitis Multiple sclerosis Psoriasis Systemic lupus erythematosus UVB-induced immunosuppression Reactive arthritis Graft-versus-host disease in allogeneic bone marrow transplantation HLA-identical bone marrow transplantation Rheumatoid arthritis Systemic lupus erythematosus Asthma (elevated IgE) Acute rejection in orthotopic liver transplantation Asthma severity End-stage liver disease Multiple sclerosis Psoriasis Rheumatoid arthritis Rheumatoid arthritis Type I autoimmune hepatitis Epstein-Barr virus infection Joint destruction Recurrence of hepatitis C in liver transplant recipients Recurrent spontaneous abortions Cutaneous malignant melanoma Renal transplant rejection Inflammatory bowel disease Rejection of renal transplant Ulcerative colitis

Yes

Tanaka, 1999

Yes

Takahashi, 2000

No

Hennig, 2000

No

Parkes, 1998

No Yes Yes No

He, 1998 Asadullah, 2001 Eskdale, 1997 Mehrian, 1998 Allen, 1998

Yes (protective) Yes

Kaluza, 2001 Middleton, 1998

Yes (increased Graftversus-Host disease) Yes No

Cavet, 1999

Yes No

Hobbs, 1998 Bathgate, 2000

Yes No No No No No No Yes (susceptibility)

Lim, 1998 Bathgate, 2000 Maurer, 2000 Reich, 1999 Hajeer, 1998 Cantagrel, 1999 Cookson, 1999 Czaja, 1999 Helminen, 1999

Yes Yes

Huizinga, 2000 Tambur, 2001

No

Karhukorpi, 2001

Yes

Howell, 2001

No Yes (decreased frequency) Yes

Marshall, 2000 Tagore, 1999

IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G) IL-10 (IL10.G10 and G12) IL-10 (IL10.G12–G15)

IL-10 (IL10.G12–G15) IL-10 (IL10.R) IL-10 (IL10.R) IL-10 571 C → A IL-10 -1082 IL-10 1082 IL-10 1082 IL-10 1082 IL-10 1082 IL-10 1082 IL-10 1082 IL-10 1082 IL-10 1082 A IL-10 1082 A IL-10 1082 A IL-10 1082 A IL-10 –1082 A IL-10 –1082 A IL-10 1082 G IL-10 1082 G IL-10 1082 G IL-10 1082 G/A

Renal transplantation outcome

Yes (decreased frequency) Yes

BASIC CYTOKINE BIOLOGY

Eskdale, 1998 Eskdale, 1997

Pelletier, 2000 Tagore, 1999 Poole, 2001

33

34

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POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

IL-10 1082, 819, 592 haplotype IL-10 1082, 819, 592 haplotype IL-10 1082, 819, 592 haplotype

Chronic cutaneous lupus erythematosus Multiple sclerosis

No

van der Linden, 2000

No

Rejection of Paediatric Heart Transplant

IL-10 1082, 819, 592 haplotype IL-10 1082A, 592A

Rheumatoid arthritis and Felty’s syndrome Inflammatory bowel disease Primary sclerosing cholangitis Rheumatoid arthritis (IgA RFve,IgG RFve) Coronary artery disease and Myocardial Infarction EBV infection

Yes (high IL-10 production is protective) No

Pickard, 1999 Maurer, 2000 Awad, 2001

IL-10 1082A, 819C, 592C haplotype IL-10 1082A, 819C, 592C haplotype IL-10 1082A, 819T, 592A haplotype IL-10 1082A, 819T, 592A haplotype IL-10 1082A, 819T, 592A haplotype IL-10 1082A, 819T, 592A haplotype IL-10 1082A, 819T, 592A haplotype IL-10 1082G, 819C, 592C haplotype IL-10 1082G, 819C, 592C haplotype, also occurs in combination with IL-10 1082A, 819T, 592A haplotype IL-10 592 IL-10 592 IL-10 –592 C → A IL-10 –627 C → A IL-10 –627 C → A IL-10 –627, 1117 IL-10 819 IL-10 Exon 1, 78 G → A (Gly15 → Arg) IL-10 Intron 3, 19 C → T IL-10 Intron 3, 953 T → C IL-10 3’ flanking region, 117 after stop codon C → T Il-12 (p40) 3’UTR nt1188 A → C

Coakley, 1998

No

Klein, 2000

No

Donaldson, 2000

Yes

Hajeer, 1998

No

Koch, 2001

Yes (protective)

Helminen, 2001

Juvenile rheumatoid arthritis Response of chronic hepatitis C to IFNa therapy Systemic lupus erythematosus nephritis (Chinese) Systemic lupus erythematosus Primary Sjogren’s syndrome

Yes (involvement of 4 joints) Yes (improved response)

Crawley, 1999

Yes

Mok, 1998

Yes (Ro)

Lazarus, 1997

Yes (susceptibility)

Hulkkonen, 2001

Primary biliary cirrhosis Type I autoimmune hepatitis Sudden infant death syndrome Advanced liver disease Primary sclerosing cholangitis Inflammatory bowel diseases Type I autoimmune hepatitis Myocardial infarction

No No Yes

Zappala, 1998 Cookson, 1999 Czaja, 1999 Summers, 2000

Yes Yes

Grove, 2000 Mitchell, 2001

No

Aithal, 2001

No No

Cookson, 1999 Czaja, 1999 Donger, 2001

No

Hall, 2000

Yes

van der Pouw Kraan, 1999 Liu, 2000 Ahmed, 2000

IL-13 1055 C → T

Rheumatoid arthritis, Multiple sclerosis Asthma

IL-13, nt 4257 G → A IL-13Ra 1050 C → T

Atopic dermatitis Atopic asthma

IL-13R130Q IL-18 607, 137

Asthma Multiple sclerosis

Yes No (Japanese population) No No

BASIC CYTOKINE BIOLOGY

Edwards-Smith, 1999

Leung, 2001 Giedraitis, 2001

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-1R1 Pst I-A

Insulin-dependent diabetes mellitus Bone loss in inflammatory bowel disease Acute myeloid leukaemia (secondary) Acute Myocardial infarction Alcoholic hepatic fibrosis (Japanese) Alopecia areata

No

First author, year (note 1) Kristiansen, 2000

Yes (protective)

Schulte, 2000

No

Langabeer, 1998

No

Iacoviello, 2000

Yes

Takamatsu, 1998

Yes (severity) Yes

Tarlow, 1994 Cork, 1995 Keen, 1998

No

McKibbin, 2000

weak Yes (protective) Yes No

Hurme, 1998 Cullup, 2001 Blakemore, 1995 Mühlberg, 1998 Cuddihy, 1996 Hamajima, 2001 Liu, 1997

IL-1Ra (240 bp allele) IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR

Bone loss (early postmenopausal) Corneal melting in systemic vasculitis EBV seronegativity Graft-versus-host disease Grave’s disease Grave’s disease and Grave’s ophthalmopathy H.pylori Henoch-Schonlein nephritis Inflammatory bowel disease Inflammatory bowel disease

No (susceptibility) Yes No

No

Hacker, 1997 Hacker, 1998 Mansfield, 1994 Bioque, 1996 Louis, 1996 Kristiansen, 2000

Yes

Pociot, 1994

Yes

Blakemore, 1996

Yes No

Clay, 1994 Bellamy, 1998

No

Yes

IL-1Ra intron 2 86bp VNTR

Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus, Non-insulindependent diabetes mellitus nephropathy Lichen sclerosis Malaria (P. falciparum): severity Multiple sclerosis

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR

Multiple sclerosis Multiple sclerosis

No Yes

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR (A1/A2 genotype) IL-1Ra intron 2 86bp VNTR

Myasthenia gravis Osteoporosis Perinuclear ANCA ulcerative colitis Polymyositis and Dermatomyositis Rheumatoid arthritis Rheumatoid arthritis Single vessel coronary disease Sjögren’s syndrome Spondylarthropathies

No No Yes

Huang, 1996 Epplen, 1997 Semana, 1997 Wansen, 1997 Feakes, 2000 Crusius, 1995 de la Concha, 1997 Huang, 1996 Bajnok, 2000 Papo, 1999

No

Son, 2000

No No Yes

Cantagrel, 1999 Perrier, 1998 Francis, 1999

Yes No

Perrier, 1998 Djouadi, 2001

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR

BASIC CYTOKINE BIOLOGY

35

36

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-1Ra intron 2 86bp VNTR

Systemic lupus erythematosus Systemic lupus erythematosus Type I autoimmune hepatitis Ulcerative colitis Myasthenia gravis

No

No Yes

Blakemore, 1994 Suzuki, 1997 Cookson, 1999 Czaja, 1999 Bouma, 1999 Huang, 1998

Alcoholism

Yes (Spanish)

Pastor, 2000

Juvenile idiopathic inflammatory myopathies Multiple sclerosis

Yes (Caucasians)

Rider, 2000

Yes

Sciacca, 1999

Primary biliary cirrhosis

Yes (homozygotes)

Arkwright, 2001

Corneal melting in vasculiti Multiple myeloma

No

McKibbin, 2000

No

Zheng, 2000

Osteoporosis

Yes

Langdahl, 2000

Osteoporotic fractures

Yes

Langdahl, 2000

CD4 count in HIV

No

Witkin, 2001

Chronic obstructive pulmonary disease Early onset sporadic Alzheimer’s disease Gastric cancer

No

Ishii, 2000

Yes

Rebeck, 2000

Yes

Machado, 2001

Yes

El-Omar, 2001

Yes

IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR IL-1Ra intron 2 86bp VNTR & IL-1b 3953 exon 5 IL-1Ra intron 2 86bp VNTR (A1 allele) IL-1Ra intron 2 86bp VNTR (A1 allele) IL-1Ra intron 2 86bp VNTR (A1 allele) IL-1Ra intron 2 86bp VNTR (A1 allele) IL-1Ra intron 2 86bp VNTR (A1/A2/A3 alleles) IL-1Ra intron 2 86bp VNTR (A1A1/A3 alleles) IL-1Ra intron 2 86bp VNTR (A1A1/A3 alleles) IL-1Ra intron 2 86bp VNTR (A1A1/A3 alleles) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele)

Yes No

First author, year (note 1) Danis, 1995

Gastric cancer from H.pylori Idiopathic recurrent miscarriage Iga nephropathy

Yes

Tempfer, 2001 Unfried, 2001 Shu, 2000

Ischaemic heart disease

No

Manzoli, 1999

Juvenile idiopathic arthritis Multivessel coronary disease Nephropathia epidemica

Yes

Vencovsky, 2001

No

Francis, 1999

Yes (protective)

Makela, 2001

Yes (with susceptibility, Not severity) No

Boiardi, 2000 Donaldson, 2000

Yes

Fang, 1999

Single-vessel coronary disease Stenosis after angioplasty

Yes

Francis, 1999

Yes (protective)

Francis, 2001

Systemic lupus erythematosus

Yes (in LD with HLA DR17, DQ2)

Tjernstrom, 1999

Polymyalgia rheumatica Primary sclerosing cholangitis Severe sepsis

BASIC CYTOKINE BIOLOGY

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

37

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) IL-1Ra intron 2 86bp VNTR (A2 allele) and IL-1b C511T haplotype IL-1Ra intron 2 86bp VNTR (A2 allele) and IL-1b 3953 (allele 2) IL-1Ra intron 2 86bp VNTR (A2 allele) and IL-1b 3953 (allele 2) IL-1Ra intron 2 86bp VNTR (A3 allele) IL-1Ra intron 2 86bp VNTR A2()/IL-1b 3953 A1() IL-1Ra nt8061 C → T (MwoI) IL-1Ra, 2018 (allele 2) IL-1Ra, 2018 (allele 2)

Tuberculin (Mantoux) reactivity Ulcerative colitis

Yes (reduced)

Ulcerative colitis

Yes

Ureaplasma urealyticum vaginal colonization Vulvar vestibulitis

Yes (negative association) Yes

Jeremias, 2000

Vulvar vestibulitis

Yes

Jeremias, 2000

Decline in lung function

Yes

Joos, 2001

Early-onset periodontitis

Parkhill, 2000

Multiple sclerosis

Yes (combined genotypes increase risk for smokers) Yes (progression)

Juvenile idiopathic inflammatory myopathies Tuberculous pleurisy

Yes (AfricanAmericans) Yes

Wilkinson, 1999

Ulcerative colitis Fibrosing alveolotis Pouchitis following ileal pouch-anal anastomosis Restenosis after coronary stenting Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Early-onset pauciarticular juvenile chronic arthritis Juvenile chronic arthritis Multiple sclerosis Rheumatoid arthritis Alzheimer’s disease Early-onset Alzheimer’s disease Juvenile chronic arthritis Juvenile rheumatoid arthritis Multiple sclerosis Periodontal disease Single and multivessel coronary disease Alzheimers H. pylori infection Rheumatoid arthritis Periodontitis Severity of periodontitis and antibody response to microbiota Periodontitis

Yes Yes Yes

Stokkers, 1998 Whyte, 2000 Carter, 2001

Yes (protective)

Kastrati, 2000

Yes Yes

Pociot, 1994 Metcalfe, 1996 Bergholdt, 1995

No

Epplen, 1995

No No No Yes Yes

Donn, 1999 Epplen, 1997 Gomolka, 1995 Du, 2000 Grimaldi, 2000

No Yes

Donn, 1999 McDowell, 1995

No Yes

Ferri, 2000 Shirodaria, 2000

No Yes No No Yes Yes

Francis, 1999 Nicoll, 2000 Hamajima, 2001 Bailly, 1995 McDevitt, 2000 Papapanou, 2001

Yes

Kornman, 1997 Kornman, 1998

IL-1Ra, 2018 (allele 2) IL-1RI IL-1RI RFLP-A IL-1a (CA)n intron 5 IL-1a (CA)n intron 5 IL-1a (CA)n intron 5 IL-1a (CA)n intron 5 IL-1a 889 IL-1a 889 IL-1a 889 IL-1a 889 IL-1a 889 IL-1a 889 IL-1a 889 IL-1a 889 (T allele) IL-1a 889 T → C IL-1a intron 6 IL-1a 4845 IL-1b 3953 IL-1a 4845 IL-1b 3953 IL-1b

No (Spaniards)

BASIC CYTOKINE BIOLOGY

First author, year (note 1) Wilkinson, 1999 Gonzalez Sarmiento, 1999 Tountas, 1999 Jeremias, 1999

Schrijver, 1999 Rider, 2000

38

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

IL-1b  IL-1Ra

Inflammatory bowel disease Multiple sclerosis Adult periodontitis

Yes

Bioque, 1995 Heresbach, 1997 Kantarci, 2000 Galbraith, 1999

Alopecia areata

Polymyalgia rheumatica

Yes (in combination Galbraith, 1999 with KM loci) Yes (in conjunction with a Licastro, 2000 T,T genotype in ACT gene) Yes (white South Mwantembe, 2001 Africans) Yes (shortened Barber, 2000 survival) No Boiardi, 2000

Severe sepsis

No

Fang, 1999

Silicosis

No

Yucesoy, 2001

Single and multivessel coronary disease Spondylarthropathies

No

Francis, 1999

No

Djouadi, 2001

Type I autoimmune hepatitis Wegener’s granulomatosis

No No

Cookson, 1999 Czaja, 1999 Huang, 2000

Primary biliary cirrhosis

Yes

Arkwright, 2001

Idiopathic recurrent miscarriage Inflammatory bowel disease Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus (DR3–/DR4–) Insulin-dependent diabetes mellitus (with nephropathy) Joint destruction in rheumatoid arthritis Localised juvenile periodontitis Low-grade squamous intraepithelial lesions Multiple myeloma

No

Hefler, 2001

No

Hacker, 1998

No

Lanng, 1993

No

Kristiansen, 2000

Yes

Pociot, 1992

Yes

Loughrey, 1998

Yes (increased)

Buchs, 2001

No (AfricanAmericans) Yes

Walker, 2000

No

Zheng, 2000

Multiple sclerosis

No

Wansen, 1997

Myasthenia gravis

Yes

Huang, 1998

Peptic ulcer disease

Yes (severity)

Periodontitis

No (in population of Chinese heritage)

Garcia-Gonzalez, 2001 Armitage, 2000

IL-1b 3953 (nt5887) C → T IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI) IL-1b 3953 (nt5887) C → T (TaqI): Allele 1,1 (homozygous) IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele, IL-1RN VNTR IL-1b 3953 (nt5887) C → T (TaqI): T allele

Alzheimer’s disease Inflammatory bowel disease Pancreatic cancer

No Yes

BASIC CYTOKINE BIOLOGY

Majeed, 1999

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

IL-1b 3953 (nt5887) C → T Periodontitis (TaqI): T allele IL-1b 3953 (nt5887) C → T (TaqI): T allele and IL-1a 4845 Periodontitis IL-1b 3953 (nt5887) C → T Primary sclerosing (TaqI): T allele cholangitis IL-1a 3953 (nt5887) C → T Rheumatoid arthritis (TaqI): T allele IL-1b 3953 (nt5887) C → T Ulcerative colitis (TaqI): T allele IL-1b 3953 (nt5887) C → T Ulcerative colitis (TaqI): T allele IL-1b 3953 (nt5887) C → T Wegener’s granulomatosis (TaqI): T allele IL-1b 511 C → T Gastric cancer IL-1b 511 C → T Gastric cancer IL-1b 511 C → T Multiple sclerosis IL-1b 511 C → T Temporal lobe epilepsy IL-1b 511 G → A (AvaI) and Alcoholic liver disease 3953 (nt5887) C → T (TaqI) IL-1b 511 G → A (AvaI) and Inflammatory bowel 3953 (nt5887) C → T (TaqI) disease IL-1b 511 G → A (AvaI), 3953 Osteoporotic fractures (nt5887) C → T (TaqI), 3877 G → A IL-1b 511 G → A Brain alterations in schizophrenics IL-1b 511 G → A Early onset of Parkinson’s disease IL-1b 511 G → A Localization related epilepsy IL-1b 511 G → A Low bone mass in inflammatory bowel disease IL-1b 511 G → A (AvaI) Chronic obstructive pulmonary disease IL-1b 511 G → A (AvaI) EBV seronegativity IL-1b 511 G → A (AvaI) Hippocampal sclerosis IL-1b 511 G → A (AvaI) IL-1b 511 G → A (AvaI)

Insulin-dependent diabetes mellitus Meningococcal disease

IL-1b 511 G → A (AvaI) IL-1b 511 G → A (AvaI)

Nephropathia epidemica Parkinson’s disease

IL-1b 511 G → A (AvaI) IL-1b 511 G → A (AvaI) IL-1b 511 G → A (AvaI)

Polymyalgia rheumatica Rheumatoid arthritis Single and multivessel coronary disease Temporal lobe epilepsy with hippocampal Multiple sclerosis

IL-1b 511 G → A (AvaI) IL-1b 511 G → A (AvaI), IL-1a –889, 3953 C → T, IL-1Ra VNTR IL-1b 511 G → A (AvaI), IL-1a –889, IL-1Ra VNTR IL-1b 31 C → T

Association

First author, year (note 1)

Yes

Gore, 1998

Yes No

Socransky, 2000 Donaldson, 2000

Yes (predictive of erosive disease) No

Cantagrel, 1999 Bouma, 1999

Yes

Stokkers, 1998

No

Huang, 2000

No Yes No No Yes (in Japanese alcoholics) Yes

Kato, 2001 Machado, 2001 Ferri, 2000 Buono, 2001 Takamatsu, 2000

No

Langdahl, 2000

Yes

Meisenzahl, 2001

Increased severity for homozygotes of allele 1 Yes

Nishimura, 2000 Peltola, 2001

Yes

Muldoon, 2001

No

Ishii, 2000

Yes Possible association in Japanese study. No

Hurme, 1998 Kanemoto, 2000

Increased severity for homozygotes with either allele Yes (protective) No (although age of onset in homozygotes was lower) No No No

Read, 2000

Nemetz, 1999

Kristiansen, 2000

Makela, 2001 Nishimura, 2001 Boiardi, 2000 Cantagrel, 1999 Francis, 1999

Possible association for A homozygotes No

Luomala, 2001

Schizophrenia

Yes

Katila, 1999

Gastric cancer from H. pylori

Yes

El-Omar, 2001

BASIC CYTOKINE BIOLOGY

Kanemoto, 2000

39

40

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-1b 31 C → T IL-2 (CA)n 3’-flanking region

Yes (susceptibility) No No

Parkes, 1998

IL-2 (CA)n 3’-flanking region

H. pylori infection Early-onset pauciarticular juvenile chronic arthritis Inflammatory bowel disease Multiple sclerosis

First author, year (note 1) Hamajima, 2001 Epplen, 1995

No

IL-2 (CA)n 3’-flanking region IL-2 (CA)n 3’-flanking region IL-2Rb (GT)n 5’-UTR IL-2Rb dinucleotide repeat

Rheumatoid arthritis Ulcerative colitis Multiple sclerosis Schizophrenia

No weak No No

IL-2Rc

Severe combined immunodeficiency disease*

Yes

IL-3 16 C → T IL-4 –34 C → T IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI)

Yes No weak Yes

IL-4 590 C → T (BsmFI)

Rheumatoid arthritis Atopic eczema Asthma and atopy Asthma and atopy (Japanese) Autoimmune thyroid disease Renal allograft rejection Acquisition of syncytiuminducing variants of HIV-1 in patients Asthma

Epplen, 1997 He, 1998 Gomolka, 1995 Parkes, 1998 Epplen, 1997 Nimgaonkar, 1995 Tatsumi, 1997 Clark, 1995 Pepper, 1995 Puck, 1995 O’Marcaigh, 1997 Puck, 1997 Puck, 1997 Wengler, 1998 Fugmann, 1998 Yamada, 2001 Elliott, 2001 Walley, 1996 Noguchi, 1998 Kawashima, 1998 Hunt, 2000

IL-4 590 C → T (BsmFI)

Asthma

IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI)

Atopic eczema Atopy, asthma, rhinitis

IL-4 590 C → T (BsmFI)

Childhood atopic asthma

IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI)

Crohn’s disease IgE levels Insulin-dependent diabetes mellitus Renal transplantation outcome Rheumatoid arthritis IgE levels

IL-2 (CA)n 3’-flanking region

IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI)

IL-4 590 C → T (BsmFI) IL-4 590 C → T (BsmFI) IL-4 intron 2 dinucleotide repeat IL-4 intron 2 dinucleotide repeat IL-4 Intron 2, 70bp VNTR IL-4 Intron 2, 70bp VNTR IL-4 Intron 3, (GT) repeat IL-4 Intron 3, (GT) repeat IL-4 Intron 3, (GT) repeat IL-4 Intron 3, 70bp VNTR (RP1 allele)

Minimal change nephropathy Multiple sclerosis Myasthenia gravis Chronic polyarthritis Multiple sclerosis Myasthenia gravis Rheumatoid arthritis

Yes No Increased (In Japanese HIV-1 patients) Increased in fatal/ near fatal asthma No (Kuwaiti Arabs) Yes (in US and Japanese) No Yes (Japanese infant population) No (Japanese population) Yes No No

Klein, 2001 Dizier, 1999 Jahromi, 2000

Yes

Poole, 2001

No No

Cantagrel, 1999 Dizier, 1999

No

Parry, 1999

Yes No Yes (protective) No No Yes

Vandenbroeck, 1997 Huang, 1998 Buchs, 2000 He, 1998 Huang, 1998 Cantagrel, 1999

BASIC CYTOKINE BIOLOGY

Cartwright, 2001 Nakayama, 2000 Sandford, 2000 Hijazi, 2000 Elliott, 2001 Zhu, 2000 Takabayashi, 2000

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-4Ra nt148 A → G (I50V) IL-4Ra nt148 A → G (I50V) IL-4Ra nt148 A → G (I50V)

Atopy/asthma Childhood atopic asthma Atopic disease

No Yes Yes

IL-4Ra nt1902 G → A (R576Q) Asthma IL-4Ra nt1902 G → A (R576Q) Atopic disease IL-4Ra nt1902 G → A (R576Q) Chronic polyarthritis IL-4Ra nt1902 G → A (R551Q, Atopic disease/asthma previously R576Q) IL-4Ra nt1902 G → A (R551Q, Childhood atopic asthma previously R576Q) IL-4Ra val50ile, gln576arg, Inflammatory bowel A3044G, G3289A disease IL-4Ra nt1682 T → C (S478P) Atopy/asthma (previously S503P) IL-4Ra nt1902 G → A (R551Q) Atopy/asthma (previously R576Q) IL-4Ra nt1902 G → A (R551Q) Crohn’s disease (previously R576Q), IL-4 –34 C → T IL-4Ra nt1902 G → A (R551Q, Adult atopic dermatitis previously R576Q) IL-4Ra nt1902 G → A (R551Q, Minimal change previously R576Q) nephropathy IL-4Ra, Q576R Mastocytosis IL-4Ra, S786P Asthma IL-5Ra (GA)n 3’-UTR Early-onset pauciarticular juvenile chronic arthritis IL-5Ra (GA)n 3’-UTR Multiple sclerosis IL-5Ra (GA)n 3’-UTR Rheumatoid arthritis IL-6 (130 bp allele) Bone loss in inflammatory bowel disease IL-6 (CA)n repeat (allele 1) Female menopause IL-6 (intron 4G) and TNF-R2 1690 C allele IL-6 –174 , 597 IL-6 –174 C → G IL-6 –174 C → G IL-6 –174 C → G IL-6 –174 C → G IL-6 –174 C → G (NlaIII) IL-6 –174 C → G (NlaIII)

Idiopathic pulmonary fibrosis Multiple sclerosis Insulin dependent diabetes mellitus Lipid abnormalities Rejection of renal transplant Asymptomatic carotid artery atherosclerosis Kaposi sarcoma

IL-6 –174 G → C (NlaIII)

Systemic-onset juvunile chronic arthritis Abdominal aortic aneurysms

IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII)

Alzheimer’s disease Alzheimer’s disease Ankylosing spondylitis

IL-6 –174 G → C (NlaIII)

Graft-versus-host disease

No effect on severity Yes No No (Japanese)

First author, year (note 1) Noguchi, 1999 Takabayashi, 2000 Mitsuyasu, 1998 Izuhara, 1999 Sandford, 2000 Hershey, 1997 Buchs, 2000 Noguchi, 1999

No

Takabayashi, 2000

No

Olavesen, 2000

Yes

Kruse, 1999

Yes Yes

Kruse, 1999 Rosa-Rosa, 1999 Aithal, 2001

Yes

Oiso, 2000

No

Parry, 1999

Yes (protective) No No

Daley, 2001 Andrews, 2001 Epplen, 1995

No No Yes

Epplen, 1997 Gomolka, 1995 Schulte, 2000

Yes (bone mineral density) Yes

Tsukamoto, 1999 Pantelidis, 2001

No Yes

Fedetz, 2001 Jahromi, 2000

Yes Yes

Fernandez-Real, 2000 Reviron, 2001

Yes

Rauramaa, 2000

Yes (increased susceptibility in HIV-infected men) Yes

Foster, 2000

Predictor of future Cardiovascular mortality No No No Yes (increased severity)

BASIC CYTOKINE BIOLOGY

Fishman, 1998 Jones, 2001 Bagli, 2000 Bagli, 2000 Collado-Escobar, 2000 Cavet, 2001

41

42

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

IL-6 –174 G → C (NlaIII)

Inflammatory bowel disease Multiple myeloma Myasthenia gravis Osteoporosis Primary Sjogren’s syndrome Renal allograft rejection Renal allograft rejection Systemic lupus erythematosus Systemic lupus erythematosus Lipid abnormalities

No

First author, year (note 1) Klein, 2001

No No Yes (susceptibility) No

Zheng, 2000 Huang, 1999 Ferrari, 2001 Hulkkonen, 2001

No Yes No

Cartwright, 2001 Marshall, 2001 Linker-Israeli, 1999

Yes (clinical features)

Schotte, 2001

Yes Yes

Fernandez-Real, 2000 Murray, 1997

No Yes

Huang, 1999 Linker-Israeli, 1996

Yes (susceptibility: Caucasians and African-Americans) Yes (protection: Caucasians) Yes (susceptibility: Caucasians) Yes (protection: African-Americans) Yes

Linker-Israeli, 1999

IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) IL-6 –174 G → C (NlaIII) G allele IL-6 3’ (AT)-rich minisatellite IL-6 3’ (AT)-rich minisatellite IL-6 3’ (AT)-rich minisatellite IL-6 3’ (AT)-rich minisatellite (792bp allele)

Bone loss (bone mineral density) Myasthenia gravis Systemic lupus erythematosus Systemic lupus erythematosus

IL-6 3’ (AT)-rich minisatellite (796bp and 828bp alleles) IL-6 3’ (AT)-rich minisatellite (808bp and 820bp alleles) IL-6 3’ (AT)-rich minisatellite (828bp allele) IL-6 3’ (AT)-rich minisatellite and IL-6 – 174 G → C (NlaIII) haplotype IL-6 3’ flanking region (9 alleles)

Systemic lupus erythematosus Systemic lupus erythematosus Systemic lupus erythematosus Alzheimer’s disease

IL-6 3’ flanking region (C allele) IL-6 3’ UTR IL-6 BglII IL-6 MspI & BglII

Multiple sclerosis Low bone mineral density Rheumatoid arthritis Rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, systemic lupus erythematosus Mean age of onset of rheumatoid arthritis Multiple sclerosis Cardiac transplant rejection Ankylosing spondylitis Ankylosing spondylitis Atopic asthma

IL-6: 174, 622 IRF-1 (GT)n intron 7 LTa (TNFb) Asp HI LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP (2/2 genotype) LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP

Multiple sclerosis

Atopy Autoimmune thyroiditis Bronchial hyperreactivity in asthma Cardiac transplant rejection Chronic lymphocytic leukaemia Disease progression in sepsis (neonates)

Linker-Israeli, 1999 Linker-Israeli, 1999 Linker-Israeli, 1999 Bagli, 2000

Yes (allele determines course and onset) No No No No

Schmidt, 2000 Takacs, 2000 Blankenstein, 1989 Fugger, 1989

Yes

Pascual, 200

No No No Yes Yes (females)

Epplen, 1997 Abdallah, 1999 Verjans, 1991 Fraile, 1998 Trabetti, 1999

No No No

Castro, 2000 Chung, 1994 Li Kam Wa, 1999

No Yes (advanced stage)

Abdallah, 1999 Demeter, 1997

No

Weitkamp, 2000

BASIC CYTOKINE BIOLOGY

Vandenbroeck, 2000

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP

Gastric cancer Grave’s disease Hashimoto’s disease Hyperinsulinaemia in coronary artery disease Idiopathic membranous nephropathy Inflammatory bowel disease Insulin resistance Insulin-dependent diabetes mellitus

Yes (survival) via LD with HLA? No Yes

First author, year (note 1) Shimura, 1995 Badenhoop, 1992 Badenhoop, 1990 Braun, 1998

via LD with HLA?

Medcraft, 1993

No

Xia, 1995

Yes (decreases) via LD with HLA?

Yes Yes

Hayakawa, 2000 Badenhoop, 1989 Badenhoop, 1989 Badenhoop, 1990 Jenkins, 1991 Pociot, 1991 Yamagata, 1991 Ilonen, 1992 Feugeas, 1993 Pociot, 1993 Vendrell, 1994 Whichelow, 1996 Shimura, 1994 Hagihara, 1995 Fugger, 1990 He, 1995 Zelano, 1998 Vendrell, 1995

No No via LD with HLA? No

Barber, 1999 Messer, 1991 Fugger, 1989 Bernal, 1999

Yes

Kankova, 2001

No via LD with HLA?

LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP

LTa (TNFb) intron 1 NcoI RFLP

Lung cancer

Yes (survival)

LTa (TNFb) intron 1 NcoI RFLP

Multiple sclerosis and optic neuritis Myasthenia gravis Non-insulin-dependent diabetes mellitus (hypertriglyceridaemia) Pancreatic cancer Primary biliary cirrhosis Primary biliary cirrhosis Primary sclerosing cholangitis Proliferative diabetic retinopathy Rheumatoid arthritis Rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, systemic lupus erythematosus, Sjogren’s Sarcoidosis Severe posttraumatic sepsis Severe sepsis Severe sepsis Spontaneous abortion Systemic scleroderma Type 1 respiratory failure Wegener’s granulomatosis Behcet’s disease

No

No Yes Yes Yes (Non-survival) No Yes Yes (in homozygotes) No Yes (NcoI)

Vinasco, 1997 Fugger, 1989 Atsumi, 1992 Bettinotti, 1993 Campbell, 1994 Vandevyver, 1994 Somoskovi, 1999 Majetschak, 1999 Stuber, 1995 Fang, 1999 Laitinen, 1992 Pandey, 1999 Waterer, 2001 Huang, 2000 Mizuki, 1992

Alcoholic brain atrophy

Yes

Yamauchi, 2001

Myasthenia gravis (early onset) Prolonged clinical course of sarcoidosis Childhood immune thrombocytopenia

Yes

Skeie, 1999

Yes

Yamaguchi, 2001

Yes (as part of haplotype with FCGR3B)

Foster, 2001

LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP

LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP LTa (TNFb) intron 1 NcoI RFLP & EcoRI LTa (TNFb) intron 1 NcoI RFLP (Allele 1) LTa (TNFb) intron 1 NcoI RFLP (Allele 1) LTa (TNFb) intron 1 NcoI RFLP (Allele 1) LTa (TNFb) intron 1 NcoI RFLP (Allele 2)

BASIC CYTOKINE BIOLOGY

43

44

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

LTa (TNFb) intron 1 NcoI RFLP (rare B1 allele) LTa (TNFb) intron 1 NcoI RFLP, TNFa, b, c NFa11

Plaque psoriasis

Yes

Multiple sclerosis

No

Proliferative diabetic retinopathy Atopy and Asthma HIV and asthma HIV transmission Cleft lip and cleft palate Cleft lip Multiple sclerosis Systemic sclerosis Alzheimer’s disease

Yes (high risk allele)

RANTES 403 G → A RANTES 403 G → A RANTES 403 G → A TGFa Allele 4, TGFb Allele 2 TGFa TaqI RFLP TGFb1 509 C → T TGFb1 509 C → T, 800 G → A TGFb1 –800, 509, nt788 C → T (T263I) TGFb1 nt509 C → T TGFb1 nt509 C → T TGFb1 nt509 C → T TGFb1 nt509 C → T and/or TGFb1 nt 869 T → C TGFb1 nt713–8delC TGFb1 nt713–8delC TGFb1 nt713–8delC TGFb1 nt713–8delC TGFb1 nt713–8delC TGFb1 nt788 C → T (T263I) TGFb1 nt788 C → T (T263I) TGFb1 nt788 C → T (T263I) TGFb1 nt800 G → A TGFb1 nt800 G → A TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro) TGFb1 nt869 (Leu10Pro), TGFb1 nt915 (Arg25Pro) TGFb1 nt869 (Leu10Pro), TGFb1 nt915 (Arg25Pro) TGFb1 nt915 (Arg25Pro)

Asthma (elevated IgE) Coronary artery disease and hypertension Plasma levels of TGFb1 Susceptibility to osteoporosis Diabetic nephropathy Insulin-dependent diabetes mellitus Osteoporosis Osteoporosis Osteoporosis in betathalasemia patients Coronary artery disease and hypertension Diabetic nephropathy Insulin-dependent diabetes mellitus Coronary artery disease and hypertension Plasma levels of TGFb1 Bone mineral density Coronary artery disease and hypertension End-stage heart failure due to cardiomyopathy Graft vascular disease (in recipient) Multiple sclerosis Ossification of the posterior longitudinal ligament Osteoporosis and spinal osteoarthritis Postmenopausal osteoporosis (Japanese) Recurrence of Hepatitis C in liver transplant recipients Acute rejection in orthotopic liver transplantation End-stage liver disease Atopic dermatitis

First author, year (note 1) Vasku, 2000

Yes Yes Yes Yes No Yes (with HLA-DR2) No No

Roth, 1994 Vandevyver, 1994 Kumaramanickavel, 2001 Fryer, 2000 Marshall, 2001 McDermott, 2000 Tanabe, 2000 Scapoli, 1998 Green, 2001 Zhou, 2000 Luedecking, 2000

Yes No

Hobbs, 1998 Syrris, 1998

Yes Yes (Japanese)

Grainger, 1999 Yamada, 2001

No No

Pociot, 1998 Pociot, 1998

Yes Yes No

Langdahl, 1997 Bertoldo, 2000 Perrotta, 2000

No

Syrris, 1998

Yes No

Pociot, 1998 Pociot, 1998

No

Syrris, 1998

Yes Yes No

Grainger, 1999 Yamada, 2001 Syrris, 1998

Yes

Holgate, 2001

Yes

Holweg, 2001

Yes (in association with HLA-DR2) Yes (Japanese patients)

Kamiya, 2001

Yes

Yamada, 2000

Yes

Yamada, 1998

Yes

Tambur, 2001

No

Bathgate, 2000

No

Bathgate, 2000

Yes

Arkwright, 2001

BASIC CYTOKINE BIOLOGY

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

45

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

TGFb1 nt915 (Arg25Pro)

Coronary artery disease and hypertension Fibrotic lung disease and lung allograft fibrosis Hypertension Idiopathic dilated cardiomyopathy Renal failure after clinical heart transplantation Cardiac transplant vasculopathy Severity of diabetic nephropathy Spinal osteophytosis Ovarian carcinogenesis

No

TGFb1 nt915 (Arg25Pro) TGFb1 nt915 (Arg25Pro) TGFb1 nt915 (Arg25Pro) TGFb1 nt915 (Arg25Pro) TGFb1 nt915 (Arg25Pro) (homozygous for G) TGFb1 T29C (Leu10Pro) TGFb1 T29C (Leu10Pro) TGFb–RI missense mutations in exons 2,3,4 and 6 TGFb–RI, exon 5 (frameshift mutation at codons 276–277) TGFb–RII codon 389 C(T TGFb1 T29C (Leu10Pro) TGFb1 T29C (Leu10Pro) TNFa 1031, 863 and TNFRSF1A 383 TNFa TNFa TNFa, TNFb TNFa, TNFb, TNFc, TNFd TNFa/b TNFa1 and a7 TNFa10 TNFa10 TNFa10b4 TNFa11 TNFa11 TNFa11 TNFa11, b4 TNFa12 TNFa13 TNFa1b5 TNFa1b5, a2b1, a2b3, a7b4, a6b5 TNFa2 TNFa2 TNFa2 TNFa2 TNFa2 TNFa2 TNFa2 TNFa2 and TNF –238 (G allele)

Yes No

Awad, 1998 Awad, 1998 Li, 1999 Tiret, 2000

Yes

Baan, 2000

Yes

Densem, 2000

No

Akai, 2001

Yes Yes

Yamada, 2000 Chen, 2001

Ovarian carcinogenesis

Yes

Wang, 2000

Early onset colorectal cancer Breast cancer Myocardial infarction Human T-cell lymphotropic virus-1 associated myelopathy Cardiac transplant rejection Multiple sclerosis Multiple sclerosis

Yes

Shin, 2000

Yes (elderly white) Yes (males) No (Japanese patients)

Ziv, 2001 Yokota, 2000 Nishimura, 2000

No Yes (118bp allele) Yes, via LD with HLA?

Abdallah, 1999 McDonnell, 1999 Sandberg-Wollheim, 1995 Matthias, 1998 Martinez, 2000 Hajeer, 2000 Kunstmann, 1999

Pharyngeal cancer Rheumatoid arthritis Basal cell carcinoma Helicobacter pyloriassociated duodenal ulcers IgA deficiency Multiple sclerosis Cervical cancer Multiple sclerosis Rheumatoid arthritis (severity) Multiple sclerosis Progession to type-1 diabetes form adultonset diabetes Systemic sclerosis Multiple sclerosis Insulin-dependent diabetes mellitus Campylobacter jejunirelated Guillain-Barré syndrome Celiac disease Colorectal cancer IgA deficiency Multiple sclerosis Myasthenia gravis Rheumatoid arthritis Alzheimer disease

Yes

First author, year (note 1) Syrris, 1998

No Yes Yes Yes (males: negative association) Yes (protective) Yes Yes (In association with HLA) Yes Yes (in LD with HLA-DRB1) Yes Yes Yes (Japan) Yes via LD with HLA?

De la Concha, 2000 Allcock, 1999 Ghaderi, 2001 Ghaderi, 2000 Lucotte, 2000 Mu, 1999 Allcock, 1999 Obayashi, 2000

Yes

Takeuchi, 2000 Allcock, 1999 Monos, 1995 Hajeer, 1996 Ma, 1998

Yes Yes No Yes, via LD with HLA? Yes Yes Yes (later onset)

Metcalfe, 1996 Gallagher, 1998 De la Concha, 2000 Epplen, 1997 Hjelmstrom, 1998 Gomolka, 1995 Perry, 2001

BASIC CYTOKINE BIOLOGY

46

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

TNFa2, a6

Insulin-dependent diabetes mellitus Celiac disease

Yes

TNFa2, b3 TNFa2, b3 TNFa2, b4, d5 TNFa2, b4, d5 TNFa3 TNFa4 TNFa6 TNFa6 TNFa6 TNFa6 TNFa6, b5, c1, d3, e3 TNFa7, a11 TNFa9 TNFa9 TNFb2 TNFb3 TNFb3, d4, d5 TNFc TNFc TNFc1 TNFc2 TNFd TNFd3 TNFd3 TNFd3d3 TNFd4 TNFd4 and d6 TNFd4 and d6 TNFd7 TNFRSF1A (p55) C52F TNFRSF1B TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R TNFRSF1B (p75) exon 6, M196R

Giant cell arteritis and polymyalgia rheumatica Basal cell carcinoma Basal cell carcinoma Gastric cancer Proliferative diabetic retinopathy Early-onset pauciarticular juvenile chronic arthritis Helicobacter pyloriassociated gastric ulcers Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Early onset of multiple sclerosis Insulin-dependent diabetes mellitus (early onset) Renal transplant rejection Parkinson’s disease Laryngeal cancer Clozapine-induced agranulocytosis Rheumatoid arthritis Ulcerative colitis (progression) Rheumatoid arthritis HIV disease progression Multiple sclerosis Cardiac transplant rejection Graft-versus-host disease in allogeneic bone marrow transplantation HLA-identical bone marrow transplantation Renal transplant rejection Basal cell carcinoma Basal cell carcinomas Colorectal cancer TNF receptor-associated periodic syndromes Schizophrenia Crohn’s disease Narcolepsy Rheumatoid arthritis Rheumatoid arthritis SLE (Japanese) Systemic lupus erythematosus Systemic lupus erythematosus Thai adult malaria sensitivity

First author, year (note 1) Pociot, 1993

via LD with HLA-DQ2 haplotypes Yes

Polvi, 1998

Yes Yes Yes Yes (low risk allele)

Hajeer, 2000 Hajeer, 2000 Saito, 2001 Kumaramanickavel, 2001 Epplen, 1995

Yes

Mattey, 2000

Yes (females: negative association) Yes (Peru) Yes (with HLA-DRB1 shared epitope) Yes Yes

Mulcahy, 1996 Boiko, 2000

Yes

Obayashi, 1999

Yes Yes (reduced risk of disease) Yes Yes

Asano, 1997 Kruger, 2000 Matthias, 1998 Turbay, 1997

Yes Yes

Bali, 1999 Bouma, 1999

Yes Yes No Yes Yes

Mulcahy, 1996 Khoo, 1997 McDonnell, 1999 Turner, 1995 Middleton, 1998

Yes (early mortality)

Cavet, 1999

Yes Yes Yes Yes Yes

Asano, 1997 Hajeer, 2000 Hajeer, 2000 Saito, 2001 McDermott, 1999

No No No No Yes Yes No No

Wassink, 2000 Kawasaki, 2000 Hohjoh, 2000 Shibue, 2000 Barton, 2001 Komata, 1999 Al-Ansari, 2000 Sullivan, 2000 Lee, 2001

No

Hananantachai, 2001

BASIC CYTOKINE BIOLOGY

Kunstmann, 1999 Castro, 2001 Mattey, 1999

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

TNFRSF1B 3’-UTR SSCP ‘7/8’ TNFRSF1B 3’-UTR SSCP ‘7/8’

Grave’s disease Insulin-dependent diabetes mellitus Systemic lupus erythematosus Familial combined hyperlipidemia Autoimmune diseases accompanied by vasculitis Coronary artery disease

No No

First author, year (note 1) Rau, 1997 Rau, 1997

No

Sullivan, 2000

Yes

Geurts, 2000

No

Takahashi, 2001

Yes

Benjafield, 2001

Systemic lupus erythematosus TNFa -863C/A and -308G/A Coronary artery disease and myocardial infarction TNFa 488A Common variable immunodeficiency TNFa -1031 HTLV-1 uveitis TNFa -1031 Kawasaki disease TNFa -1031 (C allele) Crohn’s disease TNFa -1031 (C allele) Opthalmopathy in Grave’s disease TNFa -1031 (C allele) Parkinson’s disease TNFa -1031 (C allele) Systemic juvenile chronic arthritis TNFa -1031, -863, -857, -308, -238 Psoriatic arthritis TNFa -1031C, -863A haplotype Insulin-dependent diabetes mellitus TNFa -1031C, -863A, -857C Crohn’s disease TNFa -163 Non-insulin-dependent diabetes mellitus TNFa -238 Alcoholic steatohepatitis TNFa -238 Ankylosing spondylitis

No

Tsuchiya, 2000

No

Koch, 2001

Yes

Mullighan, 1997

Yes No Yes Yes

Seki, 1999 Kamizono, 1999 Negoro, 1999 Kamizono, 2000

Yes (early onset) Yes

Nishimura, 2001 Date, 1999

No No

Hamamoto, 2000 Hamaguchi, 2000

Yes No

Kawasaki, 2000 Hamann, 1995

Yes No (German populations) Yes ( in HLA-B27 negative patients) No No No Yes (protective)

Grove, 1997 Milicic, 2000

No Yes Yes No

Abdallah, 1999 Höhler, 1998 Höhler, 1998 Epplen, 1995

No Yes No No Yes No

Jacob, 1999 Day, 1998 Kamizono, 1999 Epplen, 1997 Huizinga, 1997 Hamann, 1995

No No

Galbraith, 1998 Bernal, 1999

Yes (males) No Yes (erosion) Yes (joint destruction)

Reich, 1999 Vinasco, 1997 Brinkman, 1997 Kaijzel, 1998

TNFRSF1B 3’-UTR SSCP ‘7/8’ TNFRSF1B Intron 4 (CA repeat) TNFRSF1B(p75) exon 6, M196R TNFRSF1B, Intron 4 (CA16 allele in microsatellite) TNFRSF1B, nt168 (K56K)

TNFa -238

Ankylosis spondylitis

TNFa -238 TNFa -238 TNFa -238 TNFa -238

Antiphospholipid syndrome Antiphospholipid syndrome Brucellosis Cancers (gastric, uterine, renal and cervical) Cardiac transplant rejection Chronic active hepatitis C Chronic hepatitis B Early-onset pauciarticular juvenile chronic arthritis Early-onset psoriasis Insulin resistance (decreased) Kawasaki disease Multiple sclerosis Multiple sclerosis Non-insulin-dependent diabetes mellitus Periodontitis (adult) Primary sclerosing cholangitis Psoriasis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis

TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238 TNFa -238

BASIC CYTOKINE BIOLOGY

Gonzalez, 2001 Bertolaccini, 2001 Bertolaccini, 2001 Caballero, 2000 Jang, 2001

47

48

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

TNFa -238

Scarring trachoma (Chlamydial) Silicosis

No

Conway, 1997

Higher for severe form, lower for moderate form No

Yucesoy, 2001

TNFa -238 TNFa -238 TNFa -238 (A allele) TNFa -238 (G/A ), -308 (G/A) TNFa -238, -244, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, -308 TNFa -238, TNFa TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308

Type I autoimmune hepatitis Ankylosing spondylitis Insulin resistance syndrome Chaga’s disease Ankylosing spondylitis Carbamazepine hypersensitivity reactions Hepatitis C-induced cirrhosis Meningococcal disease Multiple sclerosis Pneumoconiosis Systemic lupus erythematosus (Whites and Black S. African) Systemic lupus erythematosus (Italians) Actinic prurigo Acute rejection in orthotopic liver transplantation Adult asthma Adult asthma Alcoholic steatohepatitis Ankylosing spondylitis Atherosclerosis Atherosclerosis Atopic asthma Atopy, asthma, rhinitis Bipolar affective puerperal psychosis Body fat content Bronchial hyperreactivity in asthma Brucellosis Cardiac transplant rejection Cardiac transplant rejection Celiac disease Celiac disease Cerebral malaria Chronic active hepatitis C Chronic hepatitis B Chronic lymphocytic leukaemia Chronic lymphocytic leukaemia Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease Corneal melting in systemic vasculitis

Yes (via LD with HLA-B27) No (Danish populations) No No Yes, via LD with HLA Yes (histological severity) No No

Cookson, 1999 Czaja, 1999 Kaijzel, 1999 Rasmussen, 2000 Beraun, 1998 Fraile, 1998 Pirmohamed, 2001

Yes (TNFa -308) No, via LD with HLA?

Yee, 2000 Westendorp, 1997 Lucotte, 2000 Anlar, 2001 Zhai, 1998 Rudwaleit, 1996

No

D’Alfonso, 1996

No Yes

Carey, 1998 Bathgate, 2000

No Yes (but No phenotypic difference) No No No No No No No

Louis, 2000 Thomas, 2001 Grove, 1997 Verjans, 1994 Keso, 2001 Wang, 2000 Trabetti, 1999 Zhu, 2000 Middle, 2000

Yes (AA genotype) Yes, via LD with HLA?

Hoffstedt, 2000 Li Kam Wa, 1999

Yes (1/2 genotype) No No via LD with HLA? Yes Yes No No No

Caballero, 2000 Turner, 1995 Abdallah, 1999 Manus, 1996 de la Concha, 2000 McGuire, 1994 Höhler, 1998 Höhler, 1998 Wihlborg, 1999

Yes

Demeter, 1997

No

Higham, 2000

No

Higham, 2000 Ishii, 2000 Keatings, 2000

Yes (Homozygous for A allele) No

BASIC CYTOKINE BIOLOGY

McKibbin, 2000

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

49

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

TNFa -308 TNFa -308 TNFa -308

Coronary heart disease Dermatitis herpetiformis Early-onset pauciarticular juvenile chronic arthritis Graft-versus-host disease in allogeneic bone marrow transplantation Hepatitis C-related liver failure HIV-encephalitis

No via LD with HLA? No

Herrmann, 1998 Wilson, 1995 Epplen, 1995

No

Mayer, 1996 Middleton, 1998

Yes (TNF2) No No No (TNF2)

Rosen, 1999 Sato-Matsumura, 1998 Wihlborg, 1999 Tiret, 2000

No

Stirnadel, 1999

trend No No No No, via LD with HLA?

Louis, 1996 Day, 1998 Lee, 2000 da Sliva, 2000 Pociot, 1993 Wilson, 1993 Deng, 1996 Allen, 2000 Kamizono, 1999 Roy, 1997 Roy, 1997 Clay, 1996 Fargion, 2001

TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308

TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308

Hodgkin’s disease Idiopathic dilated cardiomyopathy Infant malarial infection and morbidity Inflammatory bowel disease Insulin resistance Insulin resistance and obesity Insulin resistance syndrome Insulin-dependent diabetes mellitus Irritant contact dermatitis Kawasaki disease Leprosy Leprosy Lichen sclerosus Liver damage in hereditary haemochromatosis Metabolic syndrome Multiple myeloma Multiple sclerosis

Yes No No (tuberculoid) Yes (lepromatous) No Yes (protective)

Nephropathia epidemica Non-insulin-dependent diabetes mellitus Obesity Pancreatic cancer Periodontitis (adult) Polycystic ovaries Primary sclerosing cholangitis Rejection of paediatric heart transplant Renal allograft rejection Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis, Systemic lupus erythematosus Sarcoidosis Scarring trachoma Schizophrenia Sclerosing cholangitis Septic shock Severe malarial and other infections Severe sepsis Silicosis

Yes No

No No No

Yes No No No Yes Yes (low TNF is protective) No No Yes (Nodular disease) Yes, via LD with HLA?

Lee, 2000 Zheng, 2000 He, 1995 Epplen, 1997 Wingerchuck, 1997 Huizinga, 1997 Kanerva, 1998 Hamann, 1995 Herrmann, 1998 Barber, 1999 Galbraith, 1998 Milner, 1999 Bernal, 1999 Awad, 2001

No Yes Yes Yes Yes Yes

Cartwright, 2001 Lacki, 2000 Vinasco, 1997 Wilson, 1994 Danis, 1995 Somoskovi, 1999 Conway, 1997 Boin, 2001 Bathgate, 2000 Mira, 1999 Wattavidanage, 1999

No Yes

Stuber, 1995 Yucesoy, 2001

BASIC CYTOKINE BIOLOGY

50

CYTOKINE GENETICS

–

POLYMORPHISMS , FUNCTIONAL VARIATIONS AND DISEASE ASSOCIATIONS

TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

TNFa -308

Subacute systemic lupus erythematosus System lupus erythematosus

Yes

Werth, 2000

Yes (TNF2 genotypes independent of DR3) Yes, via LD with HLA?

Rood, 2000

TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 TNFa -308 (G → A) and LTa (TNFb) NcoI (A → G) TNFa -308 (G/A or A/A) TNFa -308 (TNF1) TNFa -308 (TNF1) TNFa -308 (TNF1) TNFa -308 (TNF1) TNFa -308 (TNF1) TNFa -308 (TNF1) and LTa (allele 2) haplotype TNFa -308 (TNF1/1) TNFa -308 (TNF1/2) TNFa -308 (TNF1/2) TNFa -308 (TNF1/2) TNFa -308 (TNF1/2) TNFa -308 (TNF1/2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2)

TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2)

Systemic lupus erythematosus and nephritis (Koreans) Systemic lupus erythematosus (African-Americans) Systemic lupus erythematosus (Chinese) Type 1 autoimmune hepatitis UVB-induced immunosuppression Venous thromboembolism Wegener’s granulomatosis Coronary atherothrombotic disease Rejection in renal transplants Adult periodontitis Ankylosing spondylitis Childhood immune thrombocytopenia Helicobacter pylori-associated duodenal ulcers Idiopathic dilated cardiomyopathy Leprosy Primary biliary cirrhosis Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease Corneal melting in vasculitis Parkinson’s disease Thai adult malaria severity Acute graft-versus-host disease Alveolitis in farmers’ lung Ankylosing spondylitis Ankylosing spondylitis Asthma Atopy Bone cancer Breast carcinoma Cardiac sarcoidosis Childhood asthma Chronic beryllium disease Crohn’s disease (steroid-dependent) Delayed-type hypersensitivity reaction in the skin of borderline tuberculoid leprosy patients Erythema nodosum Excessive fat accumulation (females) Fibrosing alveolitis

Yes

Kim, 1995 Kim, 1996 Sullivan, 1997

No, via LD with HLA?

Fong, 1996

Yes, via LD with HLA? No

Cookson, 1999 Czaja, 1999 Allen, 1998

No No Possible

Brown, 1998 Huang, 2000 Padovani, 2000

Yes Yes (advanced disease) Yes Yes (as part of haplotype with FCGR3A) Yes (females) (increased risk) No

Reviron, 2001 Galbraith, 1999 McGarry, 1999 Foster, 2001 Kunstmann, 1999 Tiret, 2000

Yes (susceptibility)

Shaw, 2001

Yes (late stage disease) No

Jones, 1999 Higham, 2000 Teramoto, 2001 Sakao, 2001

Yes No Yes No Yes Yes No (protective haplotype in HLA-B27 positive s) Yes (German populations) Yes Yes No (in children) Yes Yes Yes (UK/Irish populations) Yes Yes

McKibbin, 2000 Kruger, 2000 Hananantachai, 2001 Takahashi, 2000 Schaaf, 2001 Rudwaleit, 2001

Yes

Moraes, 2001

Yes Yes (TNF2 homozygotes)

Labunski, 2001 Hoffstedt, 2000

Yes

Whyte, 2000

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Milicic, 2000 Chagani, 1999 Castro, 2000 Patio-Garcia, 2000 Mestiri, 2001 Takashige, 1999 Winchester, 2000 Maier, 2001 Louis, 2000

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TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

Association

First author, year (note 1)

TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2)

Infection with H. pylori caga Leprosy Malaria morbidity and childhood morbidity Meliodosis Mortality from septic shock Multiple sclerosis Myasthenia gravis Myasthenia gravis (early onset) Neuritis in leprosy Obesity

Yes Yes (protective) Yes

Yea, 2001 Santos, 2000 Aidoo, 2001

TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) TNFa -308 (TNF2) -1031, -863, -857 and -237 TNFa -308 (TNF2) and LTa (allele2) haplotype TNFa -308 (TNF2) and LTa (TNFb) NcoI TNFa -308 (TNF2) and LTa G allele TNFa -308 (TNF2), TNF –238 TNFa -308 and IL-10 1082 G → A TNFa -308 and IL-10 1082 G → A TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 and LTa (TNFb) NcoI TNFa -308 G/A TNFa -308 G/A TNFa -308, -238, TNFa2 (2–1–2 haplotype) TNFa -308, TNF B TNFa -376 G ( A TNFa -376 G ( A TNFa -376 G ( A TNFa -376 G ( A TNFa -376 G ( A TNFa -850

Yes Yes No Yes (in LD with HLA?) Yes Yes (heterozygotes) Yes (Caucasian populations) Primary biliary cirrhosis Yes Primary biliary cirrhosis Yes (negative association) Primary sclerosing cholangitis Yes Pulmonary sarcoidosis (Lofgren)Yes (in LD with HLA?) Recurrent pregnancy loss Yes Rejection of renal transplant Yes Spontaneous preterm birth Yes Subacute cutaneous lupus Yes erythamatosus Systemic lupus erythematosus Yes Ulcerative colitis Yes Palmoplantar pustulosis No

Nuntayanuwat, 1999 Tang, 2000 Maurer, 1999 Huang, 1999 Skeie, 1999 Sarno, 2000 Brand, 2001

Rood, 2000 Hirv, 1999 Niizeki, 2000

Leprosy

Yes (protective)

Shaw, 2001

Chronic obstructive pulmonary disease Septic shock

No (in Caucasoid individuals) Yes

Patuzzo, 2000

Ankylosing spondylitis Cardiac transplant rejection Renal transplant rejection

No (English populations) Yes Yes, TNF( -308 alone

Acute pancreatitis Ankylosing spondylitis Cardiac transplant rejection Multiple sclerosis Multiple sclerosis

No No No No Yes (HLA-independent)

Non-insulin-dependent diabetes mellitus Narcolepsy

No

Powell, 2001 Kaijzel, 1999 Abdallah, 1999 Huizinga, 1997 Fernandez-Arquero, 1999 Hamann, 1995

No

Kato, 1999

Tanaka, 1999 Gordon, 1999 Mitchell, 2001 Swider, 1999 Reid, 2001 Pelletier, 2000 Roberts, 1999 Millard, 2001

Waterer, 2001

Milicic, 2000 Turner, 1997 Sankaran, 1998 Sankaran, 1998 Asthma Yes Moffatt, 1997 Asthma (childhood) Yes Albuquerque, 1998 Asthma and atopy (Italians) Yes (LTa (TNFb) NcoI only) Trabetti, 1999 Colorectal cancer Yes (b NcoI only) Park, 1998 Congestive heart failure No Kubota, 1998 Dermatitis herpetiformis Yes Messer, 1994 Hairy cell leukaemia No Demeter, 1997 Mucocutaneous leishmaniaisis Yes Cabrera, 1995 Multiple sclerosis Yes (development) Mycko, 1998 Non-Hodgkin’s lymphoma Yes Warzocha, 1998 (outcome) Early onset periodontitis No Shapira, 2001 Obesity and insulin resistance No Romeo, 2001 Alzheimer’s disease Yes Collins, 2000

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TABLE 2.3 (continued ) Cytokine and polymorphism

Disease

TNFa -850 C( T TNFa -850 C( T TNFa -857 TNFa -857 (T allele) TNFa -857 (T allele)

Alzheimer’s disease Vascular dementia Kawasaki disease Crohn’s disease Insulin-dependent diabetes mellitus TNFa -857 (T allele) Insulin-dependent diabetes mellitus TNFa -857 (T allele) Narcolepsy TNFa -857 (T allele) Non-insulin-dependent diabetes mellitus TNFa -857 (T allele) Progression to adult T-cell leukaemia from human T-cell lymphotropic virus-1 TNFa -857 (T allele) Rheumatoid arthritis TNFa -857 (T allele) Systemic juvenile chronic arthritis TNFa -857 (T allele), TNFRSF1B Human T-cell lymphotropic (exon 6, T → G), LTa (TNFb) NcoI virus-1 associated myelopathy TNFa -863 HTLV-1 uveitis TNFa -863 Kawasaki disease TNFa -863 (A allele) Crohn’s disease TNFa -863 (A allele) Ophthalmopathy in Grave’s disease TNFa -863 (A allele) Systemic juvenile chronic arthritis TNFa -863 (A allele), Diabetes TNFa -1031 (C allele)

Association

First author, year (note 1)

Yes (with apolipoprotein E) Yes No Yes No

McCusker, 2001 McCusker, 2001 Kamizono, 1999 Negoro, 1999 Hamaguchi, 2000

No

Hamaguchi, 2000

Yes Hohjoh, 1999 Possible association for Kamizono, 2000 obese –857 T homozygotes Yes Tsukasaki, 2001 Yes Yes

Seki, 1999 Date, 1999

Yes (Japanese patients)

Nishimura, 2000

Yes No Yes Yes

Seki, 1999 Kamizono, 1999 Negoro, 1999 Kamizono, 2000

Yes

Date, 1999

No

Hamaguchi, 2000 Kamizono, 2000

Full references are provided in the appropriate Genes and Immunity reviews and supplements (Bidwell et al., 1999, 2001; Haukim et al., 2002.

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3 The phylogeny of cytokines Chris J. Secombes1 and Pete Kaiser 2 1

University of Aberdeen, Aberdeen, UK

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Institute for Animal Health, Compton, UK

Truth is ever to be found in the simplicity, and not in the multiplicity and confusion of things. Sir Isaac Newton (1642–1727)

INTRODUCTION The largest dichotomy in animal defences is seen in early vertebrates (Secombes and Pilström, 2000). Only jawed vertebrates possess anticipatory immune responses characterized by the presence of lymphocytes with antigen specific receptors (Ig and TCR), that can undergo clonal proliferation in response to specific antigens. In addition, all jawed vertebrates possess both class I and class II MHC genes (Stet et al., 1998; Bartl, 1998), necessary for antigen presentation to T cells. Lymphocyte heterogeneity appears to exist throughout the jawed vertebrates, with conclusively demonstrated T and B cell subpopulations being present in amphibians and higher vertebrates (Horton and Ratcliffe, 1998). In fish it has been more difficult to confirm the thymic-dependence of T cells, although cells functionally equivalent to T and B cells are clearly present. Vertebrates also possess a variety of nonspecific cellular and humoral defences based

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

upon phagocytes, natural killer cells and a large array of soluble molecules (Yano, 1996; Secombes, 1996). Invertebrates similarly possess nonspecific cellular and soluble defences, many of which are the forerunners to those seen in vertebrates (Söderhall et al., 1996). That cytokines are required to initiate and regulate immune responses is well established in mammals. Intuitively, this seems a likely scenario in all animals with complex cellular defences, whether or not such defences are anticipatory in nature. This does not imply that all mammalian cytokines will be present in all animals. Indeed, those released from T cells that act primarily on lymphocytes may prove to be unique to the jawed vertebrates. On the other hand, cytokines released from cells of the nonspecific defences or that are important in their regulation may well be universal, or have a functionally equivalent analogue in ‘lower’ animals. In this chapter, what is known about non-mammalian cytokines is reviewed.

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INTERFERONS Interferons (IFNs) are cytokines able to inhibit virus replication and in mammals they are subdivided into type I and type II IFN, with the former consisting of IFN-a (leukocyte), IFN-b (fibroblast), IFN-x and IFN-s (trophoblast). With the exception of IFN-s, type I IFN are induced by cells infected with virus and probably any cell type can produce them. In contrast, type II IFN is induced following antigen or mitogen (ConA, PHA) stimulation of T cells and has a wide spectrum of biological activities, including macrophage activation and up-regulation of class II MHC antigens, hence the alternative term immune IFN. Although some studies report antiviral activity in invertebrates (Washburn et al., 1996; Beckage, 1996) and even the presence of functional interferon consensus response elements (ISRE) (Georgel et al., 1995), to date IFN activity appears to be unique to vertebrates. The possible divergence of IFN-a from IFN-b at the level of the reptiles has also been indicated. However, to date only in birds has IFN been cloned and sequenced outwith mammals.

Type I interferon Fish It is well established that fish fibroblast or epithelial cell lines (Gravel and Marlsberger, 1965; DeSena and Rio, 1975) and isolated leukocytes (Snegaroff, 1993; Rogel-Gaillard et al., 1993; Congleton and Sun, 1996) can secrete IFN in response to virus infection and poly I:C. This IFN activity is acid (pH 2) stable, relatively temperature resistant but destroyed by trypsin treatment, all properties typical of a type I IFN. In addition, this activity is species-specific with respect to the cells being protected but non-specific with regard to the challenge virus, i.e. it can provide protection against a virus unrelated to that which induced it. Similarly, in vivo studies have shown that IFN activities with biological properties identical to those described above can be detected in the serum of fish following natural or experimental viral infection, injection with poly I:C or DNA vaccination with viral G proteins (Dorson et al., 1975; Kim et al., 2000). IFN activity is detectable within 1 day post-infection,

begins to decrease by 4 days and is undetectable by day 14. The IFN is protective in vivo, as demonstrated by an increased survival of rainbow trout (Oncorhynchus mykiss) alevins bath-challenged with viral haemorrhagic septicaemia (VHS) virus following an injection of IFN-containing serum, compared with fish injected with control serum (DeKinkelin et al., 1982). The IFN activity is short-lived following passive transfer, being undetectable 6–8 h post-injection. Physicochemical analysis of these in vitro- and in vivo-induced IFNs has shown large differences. Serum IFN has a molecular weight of 26 kDa (Dorson et al., 1975), whereas fibroblast IFN has a molecular weight of 18 kDa and 94–100 kDa (DeSena and Rio, 1975; Liang et al., 2001). Cross-hybridization studies with a human IFN-b cDNA probe suggest that IFN-b genes are present in bony fish (Wilson et al., 1983; Tengelsen et al., 1991) but studies with a human IFN-a cDNA probe have failed to reveal hybridization (Wilson et al., 1983). Although IFN genes have not been sequenced in fish, a number of IFN-induced genes have been cloned. These include Mx (Leong et al., 1998), IRFs (Yabu et al., 1998), Vig-1 (Boudinot et al., 1999) and Vig-2 (Boudinot et al., 2001) genes. The promoter of the rainbow trout Mx gene has been sequenced and shown to contain an ISRE together with GAAA and TATA boxes (Collet and Secombes, 2001). A promoter-LUX reporter construct containing 594 bp of the 5 flanking region of the Mx gene was shown to induce strong luciferase expression when transfected cells were stimulated with poly I:C (Collet and Secombes, 2001).

Amphibia Cross-hybridization studies with a human IFN-b cDNA probe suggest that IFN-b sequences exist in frog (Xenopus laevis) DNA (Wilson et al., 1983). No hybridization occurs with a human IFN-a cDNA probe, as in bony fish.

Reptiles Tortoise (Testudo graeca) kidney and peritoneal cell cultures and turtle (Terrapene carolina) heart cell cultures secrete factors with IFN activity in response to virus infection or poly I:C (Galabov, 1981; Mathews and Vorndam, 1982). The tortoise IFN is acid stable (pH 2), relatively heat resistant, but sensitive to

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trypsin treatment. As in fish, protection of cell lines against viral CPE by reptilian IFN is species-specific, but nonspecific with regard to the challenge virus. Cross-hybridization of a human IFN-a cDNA probe with lizard (Lacerta viridis) DNA reveals faint but consistently labelled DNA fragments (Wilson et al., 1983). Thus, different classes of type I IFN are possibly present in reptiles.

Birds Interferon was first described in the chicken as an antiviral activity in conditioned media (CM) from chorioallantoic membranes which had been exposed to inactivated influenza virus (Isaacs and Lindenmann, 1957), and subsequently purified by Lampson et al. (1963) as a 20–34 kDa protein. Despite this early work, it was to be a further three decades before the first chicken IFN gene was cloned. Chickens have at least 10 type I IFN genes present in their genome (Sick et al., 1996). Of these, three intronless genes have been sequenced and are predicted to encode proteins of 193 amino acids (aa) in length (with 31 aa signal sequences), with four potential Nglycosylation sites and an estimated MW of 19 kDa (Sekellick et al., 1994; Sick et al., 1996). These genes have 24% aa identity with mammalian IFN-a, 20% with mammalian IFN-b and only 3% with mammalian IFN-c, and have been termed IFN1. An additional intronless single copy gene has also been sequenced (Sick et al., 1996). Its predicted gene product has 57% aa identity with chicken IFN1, and has been termed IFN2. The mature protein is 176 aa in length (signal peptide of 27 aa) with an estimated MW of 20 kDa. A turkey type I IFN gene has been cloned which encodes a predicted signal peptide and mature protein of 30 and 162 aa, respectively (Suresh et al., 1995), with an estimated MW of 18.85 kDa. This gene would appear to be the turkey homologue of IFN1, with 91% and 82% identity at the nucleotide (nt) and aa levels, respectively. Similarly, in ducks an intronless type I IFN gene has been sequenced which encodes a predicted 30 aa signal peptide and a 161 aa mature protein (Schultz et al., 1995). It has 50% identity to IFN1 and 61% identity to IFN2. The type I IFN genes in mammals are on autosomes. In both the duck and the chicken, the type I genes described above are on the Z (sex) chromosome

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(Nanda et al., 1998). Opinion is still divided as to whether IFN1 and IFN2 truly represent the chicken orthologues of mammalian IFN-a and IFN-b. IFN1 represents a multigene family, whereas IFN2 is a single copy gene (compare IFN-a and IFN-b in mammals). Recent phylogenetic analyses suggest that the presumed gene duplication giving rise to IFN1 and IFN2 in the chicken occurred after the divergence of birds and mammals, and that the duplication giving rise to mammalian IFN-a and IFN-b was an independent evolutionary event (Roberts et al., 1998; Hughes and Roberts, 2000). However, earlier phylogenetic analyses of mammalian type I IFN genes suggested that the gene duplication event occurred before the divergence of mammals and birds (De Mayer and De Mayer-Guinard, 1988). Further, the structures of the promoters of IFN1 and IFN2, and their differential responses to inducers of type I IFN (Sick et al., 1998) also suggest they are the chicken homologues of mammalian IFN-a and IFN-b. IFN1 (but not IFN2) mRNA expression in vivo is upregulated by oral administration of the imidazoquinoline S-28463, which selectively induces mammalian IFN-a. The IFN2 promoter has both IRF-1 and NF-jB binding sites, as does the promoter of mammalian IFN-b, whereas the IFN1 promoter has IRF-1 sites, but no NF-jB site (compare mammalian IFN-a). This has led Lowenthal et al. (2001) to propose a nomenclature for the avian IFN proteins, with IFN1 and IFN2 to be known as IFN-a and IFN-b respectively. This nomenclature will be used henceforth in this chapter. The potential of type I avian IFN to act as an immunomodulator and a vaccine adjuvant is now being assessed. Recombinant IFN-a, administered in drinking water, can ameliorate the effects of challenge with Newcastle disease virus (NDV) (Marcus et al., 1999). The ability of chicken IFN-a to act as a vaccine adjuvant when co-delivered as DNA with NDV genes in fowlpox (FPV) vectors, in both chickens (Karaca et al., 1998) and turkeys (Rautenschlein et al., 2000), is unclear. Although FPV-NDV-IFN-a vaccinated chickens did not suffer from the weight loss seen in FPV or FPV-NDV-vaccinated chickens, they produced lower NDV-specific antibody titres than FPV-NDVvaccinated chickens (Karaca et al., 1998). When similar vaccine trials were conducted in turkeys, the presence of IFN-a elicited earlier production of anti-NDV antibodies than FPV-NDV vaccines alone,

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but otherwise had no obvious adjuvant effect (Rautenschlein et al., 2000). Both recombinant and plasmid-encoded IFN-a induced improved antibody responses to tetanus toxoid (TT) (as a bacterial model antigen) in adult chickens, but not in day-old birds (Schijns et al., 2000). Antibody responses were not augmented, however, when IFN-a was co-injected with inactivated Infectious Bursal Disease virus antigen into either adult or day-old chickens (Schijns et al., 2000). Recombinant IFN-a has also been shown to have a protective effect on the induction of Rous sarcoma virus (RSV)-induced tumours in chickens (Plachy et al., 1999).

Type II interferon Fish In rainbow trout IFN activity is present in 48 h CM from ConA-pulsed head kidney leukocytes (Graham and Secombes, 1990). IFN activity in these crude preparations is significantly decreased following exposure to acid conditions (pH 2) or relatively high temperatures (60°C), suggesting it is a type II IFN. Fractionation of the supernatants reveals IFN activity in two protein peaks with molecular weights of 19 and 32 kDa, with most activity being in the former peak. Exposure of the 19 kDa peak to acid conditions (pH 2), 60°C for 1 h and trypsin completely removes the antiviral activity. The IFN activity co-elutes with MAF activity in this species, which is also acid- and temperature-sensitive. Whether these two activities (MAF and IFN) are really due to the same molecular species awaits further study.

Birds The cDNA for chicken IFN-c, cloned by Digby and Lowenthal (1995), encodes a predicted protein of 164 aa (a signal peptide of 19 aa and a mature protein of 145 aa), with two potential N-glycosylation sites and an estimated MW for the mature protein of 16.8 kDa. It shares 35% and 32% identity at the aa level with equine and human IFN-c respectively, but only 15% with chicken type I IFN. Chicken IFN-c is a single copy gene composed of four exons with remarkable structural similarity to its mammalian homologues (Kaiser et al., 1998b). A number of potential regulatory

signals similar to those found in mammals have been identified in the promoter, in each intron and in the 3 UTR (Kaiser et al., 1998b). The gene maps to chicken chromosome 1, with synteny with human chromosome 12 (Guttenbach et al., 2000). The coding sequences and partial intron sequences of IFN-c from four other galliforms (guinea fowl, ringnecked pheasant, Japanese quail and turkey) were recently determined (Kaiser et al., 1998a). The coding regions of IFN-c are highly conserved amongst the galliforms (93.5–96.7% and 87.8–97.6% at the nt and aa levels, respectively). This high degree of overall identity at the predicted primary aa sequence level of the protein, including the deduced IFN-c receptorbinding motifs, suggested that IFN-c may be crossreactive among these species. This has since been shown to be the case for turkey and chicken IFN-c (Lawson et al., 2001). The cDNA for duck IFN-c has 80% nt identity and 67% predicted aa identity with chicken IFN-c (Huang et al., 2001). Comparative protein modelling suggested that the predicted three-dimensional structures of chicken and duck IFN-c were similar, and subsequent experiments with recombinant proteins showed that the two proteins were functionally crossreactive (Huang et al., 2001). As with mammalian IFN-c, native chicken IFN-c has potent macrophage activating factor activity that is heat- and pH-labile (Lowenthal et al., 1995). Recombinant chicken IFN-c expressed from E. coli or COS cells were poor antiviral agents but strongly stimulated NO secretion and expression of MHC class II in macrophages (Weining et al., 1996). However, baculovirus-derived recombinant chicken IFN-c, as well as stimulating macrophages, also had antiviral activity (Lambrecht et al., 1999), and thus is probably a more suitable recombinant for studies into the function of avian IFN-c. Again similarly to mammals, chicken type I and type II IFN act synergistically (Sekellick et al., 1998), both in terms of antiviral activity and in their ability to activate macrophages. Anti-chicken IFN-c mAb have been produced following gene-gun immunization of mice, and used to develop a quantitative capture ELISA specific for chicken IFN-c (Lambrecht et al., 2000). One of these mAb, 1E12, neutralizes the biological effects of both chicken (Lambrecht et al., 2000) and turkey IFN-c (Lawson et al., 2001).

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Initial experiments have shown that IFN-c has potential as a vaccine adjuvant, in both chickens and turkeys. Turkeys were vaccinated in ovo with recombinant FPV (rFPV) expressing the fusion and hemagglutinin-neuraminidase glycoproteins of NDV, as well as type I or type II IFN (Rautenschlein et al., 1999). The rFPV-NDV-IFN-c construct induced the onset of anti-NDV antibody production 1 week earlier than other vaccine constructs (1 week post hatch). The rFPV-NDV-IFN-c construct was also the most protective vaccine against NDV challenge. Lowenthal et al. (1998) developed a model system to measure the adjuvant potential of chicken IFN-c, using sheep red blood cells (SRBC) as a model antigen. Priming with IFN-c and SRBC gave enhanced primary and secondary IgG antibody responses compared with those in birds that received SRBC alone. When IFN-c was coadministered, a 10-fold lower level of antigen was sufficient to give similar antibody responses to birds receiving SRBC alone. Treatment with IFN-c also resulted in an increase in the proportion of birds that responded to antigen challenge. When TT was used as a model antigen (Schijns et al., 2000), IFN-c had similar effects to those described above for IFN-a. Native chicken IFN, shown to be a mixture of both type I and type II IFN (Heller et al., 1997), inhibits Marek’s disease virus (MDV) replication, as measured by plaque assays, and suppression of virus-specific proteins in MDV-infected chicken cells. The interferon/interleukin-10 receptor gene cluster has recently been cloned in the chicken (Reboul et al., 1999). In mammals, the cluster includes both chains of the type I IFN receptor (IFNAR1 and IFNAR2), and one of the chains of both the IFN-c-receptor (IFNGR2) and the IL-10 receptor (IL10R2). To date in the chicken IFNAR1, IFNAR2 and IL10R2 have been identified, in the same order and transcriptional orientation as in mammals, with 36%, 28% and 42% predicted aa identities with their human homologues, respectively. The region of the chicken genome where IFNGR2 is predicted to lie has yet to be sequenced.

INTERLEUKINS The term interleukin was introduced to describe cytokines able to act on leukocytes in a specific manner. The number of interleukins discovered has

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been increasing steadily, with possibly more receiving interleukin status in the near future. Outside of mammals the majority of cloned cytokines with interleukin activity have been isolated from birds, although IL-1 genes have now been isolated in most vertebrate groups. The biological activity and sources of these molecules are now considered in more detail.

Interleukin-1 Fish An IL-1b gene has been cloned in many species of bony fish (Zou et al., 1999a; Bird et al., 1999a; Fujiki et al., 2000), with two IL-1b genes being present in some species (Pleguezuelos et al., 2000; Engelsma et al., 2000). IL-1b has also been cloned and sequenced in a single cartilaginous fish species, the lesser spotted dogfish (Bird et al., 1999b). In rainbow trout, the full length cDNA is 1325 nt and translates into a 260 aa precursor, with three potential glycosylation sites at positions 6, 12 and 142. Unlike the situation with mammalian IL-1bs but in common with all nonmammalian IL-1bs sequenced to date, there is no clear IL-1b converting enzyme (ICE or caspase 1) cut site (immediately after a conserved aspartic acid) for processing of the precursor molecule. Nevertheless, alignment with known genes reveals that a double alanine (at positions 95/96) aligns with the first two residues of the mammalian mature peptide, and is a fairly typical start to the mature peptide N-terminus. In addition, immediately upstream are two basic amino acids (Arg93 and Arg94) which present a potential cleavage site for several known enzyme convertases. This predicts that the trout mature peptide contains 166 aa, with an estimated size of 20 kDa. Indeed, production of this predicted mature peptide as a recombinant molecule in E. coli has confirmed it is bioactive, and capable of increasing phagocytosis and IL-1b, COX2 and MHC class II expression in trout phagocytes (Hong et al., 2001). Similarly, a predicted carp IL-1b mature peptide has been produced as a recombinant molecule and shown to have adjuvant activities, with respect to antibody production, in a bacterial vaccine (Yin and Kwang, 2000a). IL-1b expression in fish leukocytes has been shown to be inducible by stimulation with LPS in vitro, and by LPS and infection with Gram-negative bacteria in

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vivo (Zou et al., 1999b, 2000a). Expression is clearly detectable in the spleen, kidney, gills and blood, with lower levels present in the liver. Typically expression at these sites is correlated with expression of incompletely spliced transcripts, containing 1–2 introns (Zou et al., 1999b). Expression is influenced by temperature, with low culture temperatures (4°C) inhibiting expression (Zou et al., 2000a). Expression is also inhibited by preincubation with cortisol before LPS stimulation. The IL-1b gene can have a different organization in different fish species, with most of the variation occurring at the 5 (precursor) end of the gene. In carp, as with mammals, there is a 7 exon/6 intron arrangement, with the first exon being untranslated (Engelsma et al., 2001). However, in rainbow trout there is a 6 exon/5 intron organization, with a coding exon missing equivalent to exon 2 or 3 of mammals (Zou et al., 1999b). The dogfish IL-1b gene also has a 6 exon/5 intron organization, but here it is the 5 untranslated exon that is missing (Wang et al., 2000). Some interesting difference are also apparent when comparing the genomic sequence of species with two IL-1b genes. For example, in rainbow trout the IL-1b2 cDNA has two 9 nt deletions relative to the IL-1b1 cDNA (Pleguezuelos et al., 2000). Whilst one of these is indeed missing from the genomic sequence, the second is due to an altered splicing of the 3 end of intron 3, which deletes 9 nt from the cDNA but keeps the remaining molecule in frame. The IL-1RI and IL-1RII have also been cloned and sequenced in fish, in Atlantic salmon (Subramaniam et al., 2002) and rainbow trout (Sangrador-Vegas et al., 2000), respectively. The type I receptor cDNA contains a 1716 nt open reading frame (ORF), and has two variant 5 UTRs. There is a 14 nt common proximal 5 UTR immediately upstream of the ORF, with either a 78 nt or a 122 nt distal UTR. This suggests that as in mammals (Sims et al., 1995), the fish IL-1RI may be under the control of multiple promoters. The translated protein is predicted to contain a signal peptide, three extracellular Ig domains, a short transmembrane domain and an intracellular region with characteristic Toll/IL-1R (TIR) signalling domains. The receptor had a wide tissue distribution, and was up-regulated by treatment with LPS in the kidney, spleen, liver and gills. The type II IL-1R contains a 1323 nt ORF, with relatively short 5 (75 nt) and 3 (151 nt) UTRs. The

predicted protein also contains a signal peptide, three Ig domains and a transmembrane region, but the intracellular domain is short and consists of only 24 aa. This suggests that as in mammals the type II IL-1R may not signal and may act as a decoy receptor. Expression studies showed that the IL-1RII was also widely distributed, being most abundant in the kidney, blood and spleen. The IL-1RII could also be up-regulated, in kidney leukocytes, after stimulation in culture with LPS/TNFa.

Amphibia Within amphibia, the IL-1b gene has been cloned in only X. laevis to date (Zou et al., 2000b). Here the cDNA consists of 1462 nt, that translate in a single reading frame to give a predicted 238 aa IL-1b molecule. The molecule shows 47–49% nt identity with mammalian and chicken IL-1bs, with lower identity to fish sequences (40–42%). Like other non-mammalian IL-1b genes, there is no clear ICE cut site, despite the presence of caspase-1 in this species (Nakajima et al., 2000), although many features of the molecule suggest it is produced as a precursor. Expression studies have shown that the IL-1b transcript is detectable in the liver, spleen, kidney and brain of control Xenopus, and that expression can be up-regulated by injection of LPS or stimulation of splenocytes in vitro with LPS.

Birds A cDNA encoding the chicken homologue of mammalian IL-1b was recently cloned by expression screening (Weining et al., 1998). In mammalian cells, CXC chemokines are strongly induced by proinflammatory cytokines, such as IL-1. Weining et al. (1998) showed that LPS-stimulated HD11 cells (Beug et al., 1979), a chicken macrophage cell line, secreted a bioactivity that stimulated the synthesis of a chicken CXC chemokine, K60 (Sick et al., 2000). Upon screening an activated HD11 cDNA library using this assay, they identified a cDNA clone of 1107 nt followed by a poly(A) tail. The ORF encoded a predicted polypeptide of 267 aa which had 25% and 30% similarity to human IL-1b and IL-1 receptor antagonist (IL-1ra), respectively, but only 13% similarity to human IL-1a. Further study of the predicted polypeptide suggested that this was in fact the chicken homologue of mam-

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malian IL-1b. The predicted protein lacked a typical signal peptide, but contained an N-terminal region resembling that of the mammalian IL-1b propeptide, with stretches of basic aa and negatively charged residues that are found at corresponding positions in the human IL-1b propeptide. The predicted protein was also similar in length to the human IL-1b precursor, some 100 aa longer than the uncleaved form of human IL-1ra. In mammals, the IL-1b propeptide is cleaved by ICE after a conserved aspartic acid residue. This aspartic acid residue is lacking in the predicted chicken IL-1b aa sequence, making it difficult to predict the N-terminus of mature chicken IL-1b. Based on comparisons of IL-1b protein sequences, Weining et al. (1998) expressedahistidine-taggedfragmentofchickenIL-1b, containing all aa residues downstream of Ala106, in E.coli.This recombinant protein was shown to be active both in vitro, using the K60 bioassay described above, and in vivo, via its ability to induce serum corticosterone post-intravenous injection of adult chickens. Chicken IL-1b has a similar gene structure (five exons and four introns in the coding region of the gene) to its mammalian and fish homologues, with the exception that there is only one apparent exon encoding the 5 UTR, as opposed to two exons in mammals and bony fish (Kaiser et al., 2001). Overall, the gene is approximately one quarter the size of mammalian IL-1b genes. Chicken IL-1b maps to one end of chromosome 2, linked to the microsatellite marker LEI0031 (Kaiser et al., 2001). However, there is no obvious synteny with the chromosomal location of either human or mouse IL-1b (which both map to the centre of chromosome 2 in their respective species), possibly due to the low number of defined markers in this region of the chicken genomic map. This is the first example of a chicken cytokine gene being nonsyntenic with its mammalian orthologues (see elsewhere in this chapter). The recent isolation of several BACs (P. Kaiser, unpublished results) from the Wageningen BAC library (Crooijmans et al., 2000) should allow the promoter of IL-1b, and also the extent of the IL-1 family in the chicken, to be determined by direct sequencing. The role of IL-1b in the chicken’s immune response to disease is now being determined. From mammalian models, it is to be expected that many avian infections will result in a pro-inflammatory response

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mediated, in part, by IL-1b. Expression of IL-1b mRNA in the gut has been shown to increase 80-fold 7 days after Eimeria tenella infection, using a quantitative RT-PCR technique (Laurent et al., 2001). IL-1b expression was also increased, but to a lesser degree, following E. maxima infection. In a viral infection, IL-1 activity was increased in macrophage supernatants from birds suffering from poult enteritis and mortality syndrome (PEMS) (Heggen et al., 2000). Conversely, following Salmonella invasion in an in vitro cell culture system, IL-1b mRNA expression was generally decreased (Kaiser et al., 2000). IL-1b mRNA levels, of course, do not necessarily reflect release of biologically active protein. In terms of adjuvant activity, recombinant IL-1b had a minimal effect when delivered with TT as a model antigen to either day-old or 3-week-old chickens (Schijns et al., 2000). The chicken type-I IL-1 receptor (IL-1RI) (Guida et al., 1992) has 60% aa identity with human and mouse IL-1RI. Its intracellular domain is most conserved, with 76% and 79% aa identity with mouse and human IL-1RI respectively, suggesting conservation of signalling functions. The extracellular binding region of the receptor contains three immunoglobulin domains, as in mammalian IL-1RI, but the sequence identity is lower than in the cytoplasmic domain. Klasing and Peng (1987) expressed the ligand-binding domain of the chicken IL-1RI (soluble (s) IL-1RI) in yeast, and then raised polyclonal antisera to the recombinant in rabbits. The recombinant sIL-1RI partially neutralized (by up to 77%) the thymocyte costimulation activity of CM from LPS-stimulated HD11 cells. Antisera against the sIL-1RI also neutralized the IL-1 bioactivity in CM, presumably by binding to the extracellular domain of the membrane-bound IL-1RI on thymocytes. This also demonstrated that up to 30% of the thymocyte costimulation activity in the CM was due to another cytokine. Lawson et al. (2000) demonstrated that, for example, recombinant chicken and turkey IL-2 have thymocyte costimulation activity.

Interleukin-2 Fish Whilst CM from fish leukocyte cultures stimulated by mitogen (PHA) or alloantigen (MLR) appear to have

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IL-2-like activity, in that they can induce the proliferation of purified PHA-activated lymphoblasts (T-cell blasts) (Caspi and Avtalion, 1984; Grondel and Harmsen, 1984), to date IL-2 has not been cloned in fish. Nevertheless, one component of the trimeric IL-2R has been cloned in rainbow trout, the gamma chain (cC) (Wang and Secombes, 2001), which is a common subunit of many IL receptors (e.g. IL-4, -7, -9, -15 and -21). This cDNA contains 2291 nt, coding for an ORF of 1029 nt, with a 72 nt 5 UTR and a 1190 nt 3 UTR. The predicted translation has 28–30% aa identity to mammalian cC sequences, and contains an identifiable signal peptide and transmembrane domain. Within the extracellular domain are six conserved cysteine residues and the W-S-X-W-S motif typical of haemopoietin receptors. The largest difference to the mammalian molecules is a lack of conserved tyrosine residues in the trout intracellular domain.

Amphibia As in fish, CM from amphibian leukocytes stimulated with mitogens (PHA, ConA) have IL-2-like activity in that they are costimulatory in a thymocyte proliferation assay, induce the proliferation of PHA-induced lymphoblasts and support the growth of alloreactive T cell lines (Watkins and Cohen, 1987; Haynes et al., 1992; Koniski and Cohen, 1994). Attempts to purify this growth factor in Xenopus have shown it has a molecular weight of 16 kDa, and lectin affinity chromatography indicates it has a 3-D configuration of carbohydrates similar to human IL-2 (Watkins and Cohen, 1985; Haynes and Cohen, 1993a). However, to date no sequence information exists.

Reptiles In the snake Spalerosophis diadema, CM from ConAstimulated splenocytes enhance the ConA-induced proliferation of splenic lymphoblasts (El Ridi et al., 1986). Fractionation of these CM reveals activity in two peaks, with molecular weights of 15 kDa and 39–42 kDa. Only the low-molecular-weight protein is detectable by SDS-PAGE, which suggests that the high-molecular-weight molecule is possibly a polymer of the former. The pI values of these molecules lie in the ranges 5.5–5.8 and 6.4–6.6.

Birds The chicken IL-2 cDNA was cloned via expression screening for T cell proliferative activity. It encodes a predicted protein of 143 aa, with a signal sequence of 22 aa and a mature protein of 121 aa (Sundick and Gill-Dixon, 1997). The predicted protein has almost equal identity with mammalian IL-2 and mammalian IL-15 (e.g. 24.5% and 23.8% identity with bovine IL-2 and IL-15, respectively). However, unlike mammalian IL-2, but like mammalian IL-15, the predicted chicken protein has four conserved cysteines that form two intrachain disulphide bonds (Rothwell et al., 2001a). The cDNA was only definitively shown to encode chicken IL-2 when the gene structure, promoter structure and genetic location were determined (Kaiser and Mariani, 1999). The exon:intron structure of chicken IL-2 corresponds almost exactly to those of mammalian IL-2s. The cDNA contains five repeats of the ‘instability’ motif ATTTA in the 3 UTR in exon 4. Chicken IL-2 is a single copy gene, with neither structural nor promoter polymorphisms identified. The promoter contains a number of predicted regulatory sequences similar to those found in mammals, but lacks the mammalian NF-jB and octamer binding sites. The gene maps to chromosome 4, linked to the gene for annexin V, with synteny with mouse chromosome 3 and human chromosome 4. Glycosylation is not required for the bioactivity of recombinant chicken IL-2 (Stepaniak et al., 1999). Endogenous IL-2 occurs in vitro as a monomer of 14.2 kDa and is secreted by splenocytes within 4 h of ConA stimulation (Stepaniak et al., 1999). Mutational analysis of chicken IL-2 showed that Asp17 is a critical N terminal contact site for binding to the putative chicken IL-2R, and that removal of C terminal aa gave proteins with decreased bioactivity as a function of the kind and number of aa removed (Kolodsick et al., 2001). Unlike the turkey IFNs, turkey IL-2 has low identity with its chicken homologue (69.93% at the aa level) (Lawson et al., 2000). In contrast, the promoters of the two avian IL-2 genes share a high degree of identity (95.7% over 380 nt). Phylogenetic analysis shows that the avian IL-2s have diverged to a greater extent than IL-2s from closely related mammalian species. Considering the low level of aa identity, including aa residues known to be important in binding IL-2 in mammals, it is perhaps surprising that both turkey

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and chicken IL-2 cross-react in functional bioassays. Despite this cross-reactivity, two mAbs raised against chicken IL-2 specifically recognize and neutralize chicken IL-2, but not turkey IL-2 (Rothwell et al., 2001b). Another panel of mAbs can neutralize chicken IL-2 and one of these, with a rabbit polyclonal antisera against chicken IL-2, has been used to develop a capture ELISA (Miyamoto et al., 2001). The growth of cd T cells was preferentially promoted when ConA-activated chicken spleen cells were cultured in the presence of recombinant chicken IL-2 (Choi and Lillehoj, 2000). The cd T cells displayed a high level of spontaneous cytotoxicity against LSCCRP9 tumour cells, an avian NK cell target. To investigate the role of cd T cells and IL-2 in coccidiosis, Choi and Lillehoj (2000) orally infected chickens with E. acervulina and thereafter monitored IL-2 mRNA expression in the spleen and duodenum, and duodenal cd T cell levels. Following both primary and secondary infections, there was a significant enhancement of both cd T cells in the gut and IL-2 mRNA transcripts in the gut and spleen. There have been a number of chicken EST libraries established recently (Abdrakhmanov et al., 2000; Tirunagaru et al., 2000). These libraries contain ESTs (e.g. Acc. Nos BG713313 and BG713418) similar to the mammalian IL-2R c chain.

Interleukin-3 and colony-stimulating factors (CSF) IL-3 has yet to be cloned outside of the mammals. Nevertheless, biological cross-reactivity of mammalian IL-3 has been noted in some species and factors with CSF activity have been described and a number of CSF-R cloned.

Fish Anti-IL-3 sera have been used to detect antigenically cross-reactivemoleculesintheseraofvirus-challenged trout by ELISA (Ahne, 1993). Serum CSF activity is present in rainbow trout following injection with LPS (Kodama et al., 1994), and in carp following injection with Freund’s complete adjuvant (Moritomo et al., 1994). In addition, carp macrophage CM have CSF activity (Yoshikawa et al., 1994) and a recently developed goldfish cell line requires autologous condi-

65

tioned medium for optimal proliferation (Wang et al., 1995). Although no CSF sequences are yet known in fish, the M-CSF receptor (CSF1R) has been cloned in the puffer fish (Fugu rubripes) (How et al., 1996), zebra fish (Danio rerio) (Parichy et al., 2000) and rainbow trout (Wang et al., 2001). In puffer fish, the gene consists of 21 exons and has 39% amino acid homology with human CSF1R, with the kinase domain having much higher homology (63%). The gene is linked tandemly in a head-to-tail array with the PDGFRb gene (see Transforming growth factors).

Birds Chicken IL-3 has yet to be cloned, and few reports have described IL-3-like activity in the chicken. Purified human IL-3 stimulated anion secretion in chicken intestine (Chang et al., 1990), but the mechanism of action underlying this response remains unknown. Polyclonal antisera against human IL-3 seemed to recognize some components of solubilized basal lamina of chicken ovarian follicles in Western blot (Asem et al., 2000). However, as the authors acknowledge, a positive reaction obtained with an antibody in Western blot does not prove that a particular protein exists in the chicken ovarian basal lamina, particularly bearing in mind the generally low level of identity between chicken and mammalian cytokines, and the lack of cross-reactivity of other anti-human cytokine antibodies with their chicken homologues. Chicken myelomonocytic growth factor (MGF), necessary for the survival and growth of normal and transformed avian myeloid precursor cells (Leutz et al., 1984; Metz et al., 1991), has significant aa identity with both mammalian IL-6 (40%) and G-CSF (52–56%) (Leutz et al., 1989; Sterneck et al., 1992). The predicted protein consists of 201 aa, with a 23 aa signal peptide. Expression of constructs containing the MGF promoter is restricted to myelomonocytic cells, and is activated by kinases (Sterneck et al., 1992). The MGF gene has five exons and four introns (Sterneck et al., 1992), as do mammalian IL-6 and G-CSF. However, since the recent cloning of chicken IL-6 (Schneider et al., 2001) (see below), it is obvious that MGF is not the chicken homologue of IL-6, as has been claimed in some reports. Stem cell factor (SCF) has been cloned in both the

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THE PHYLOGENY OF CYTOKINES

chicken and Japanese quail (Zhou et al., 1993; Petitte and Kulik, 1996). They share 98% aa identity with each other, but only 53% identity with mammalian SCF. Other factors with CSF activity have been observed, but their specific identity remains unknown. For example, serum CSF activity is detectable during and immediately after coccidial infection in chickens (Byrnes et al., 1993b), and stromal cell lines secrete factors that induce the proliferation and differentiation of precursor cells in embryonic and haematopoietic tissues (Obranovich and Boyd, 1996; Siatskas et al., 1996). Kogut et al. (1997) demonstrated the presence of a G-CSF-like factor in lymphokines (ILK) from T cells of birds immunized against Salmonella enteritidis by Western blot using a goat anti-human G-CSF polyclonal antisera. Pretreatment of the ILK with the antisera totally abolished its G-CSF-like activity.

Interleukin 6 Invertebrates Concentrated coelomic fluids from the starfish A. forbesi have been found to contain a factor that supports the growth of a murine IL-6 dependent B cell hybridoma (B9 cells), and that is recognized in Western blots with a polyclonal anti-human IL-6 serum (Beck and Habicht, 1996). This factor has a molecular weight of 30 kDa and a pI of 5.5. Isolated coelomocytes can release this factor after stimulation with LPS, with a peak release after 12 h. Furthermore, IL-6R MoAb have been shown to bind to axial organ cells in the sea star A. rubens, by FACS analysis (Legac et al., 1996).

Fish Anti-IL-6 sera have been used to detect antigenically cross-reactive molecules in the sera of viruschallenged carp and trout (Ahne, 1993) and in supernatants from ConA stimulated trout blood leukocytes (Ahne, 1994), by ELISA.

Birds Following the identification of a partial chicken IL-6 cDNA in one of the EST libraries, the full-length cDNA was cloned using suppression subtractive hybridiza-

tion (SSH) technology (Schneider et al., 2001). Chickens were orally treated with the synthetic immune modifier S-28463, which in mammals strongly induces expression of IFN-a, tumor necrosis factor (TNF), IL-1, IL-6 and IL-8 (Tomai et al., 1995), and IFN-a in the spleens of chickens (Karaca et al., 1998; Sick et al., 1998). SSH permits the identification of differentially expressed genes, and was used to identify mRNAs induced in the spleens of chickens treated with S-28463. One of the resulting cDNAs encoded a predicted protein with 35% aa identity with human IL-6 (Schneider et al., 2001). The mature protein is 194 aa in length, with a putative signal peptide of 47 aa. Recombinant chicken IL-6, produced in E. coli, monkey COS-7 cells or chicken LMH cells (a chicken hepatoma cell line), induced proliferation of the IL-6-dependent murine hybridoma cell line 7TD1. When injected into chickens, it induced an increase in serum corticosterone levels. The majority of the chicken IL-6 gene structure has been determined (Kaiser et al., 2001) and is very similar in structure to mammalian IL-6 genes, with similar numbers of aa encoded by each exon. IL-6 may have an important role in determining the course of Salmonella infections in the chicken (Kaiser et al., 2000). A chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host-specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. Several cytokines, including IL-6, were measured by quantitative RT-PCR and bioassay. Invasion of S. typhimurium and S. enteritidis caused an eight- to ten-fold increase in production of IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produce a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid. IL-6 is also produced during both murine and chicken Eimeria infections (Lynagh et al., 2000). IL-6 activity, similarly to IL-1, was increased in macrophage supernatants from birds suffering from PEMS (Heggen et al., 2000). A further member of the IL-6 family of cytokines

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INTERLEUKINS

has been described in birds – growth promoting activity (GPA), which is thought to be equivalent to ciliary neurotrophic factor (CNTF) (Koshlukova et al., 1996). Cultured chick ciliary ganglion neurons possess receptors capable of binding both GPA and human CNTF, but GPA is up to five times more potent than human CNTF in promoting chick neuronal survival (Koshlukova et al., 1996). In mammals, the receptors for the IL-6 family of cytokines (IL-6, CNTF, IL-11, leukaemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and oncostatin M (OSM)) all share a common receptor component, gp130. The cDNA encoding the chicken homologue of gp130 was recently cloned (Geissen et al., 1998). It encodes a predicted protein of 918 aa, with a 23 aa signal sequence, and has 61% aa identity with human gp130. It contains a characteristic cytokine receptor domain, including the WSXWS motif (Bazan, 1990). The 15 cysteines conserved in mammalian gp130 are also conserved in chicken gp130, as are seven of eleven potential N-glycosylation sites. In the intracellular region, various signalling motifs are also conserved. Monoclonal antibodies have now been raised against chicken gp130 (Horiuchi et al., 2001a).

Interleukin 8 and chemokines Interleukin 8 belongs to the family of low molecular weight cytokines that are potent chemoattractants for leukocytes, termed chemokines. They are subdivided into subgroups based upon the number of cysteine residues at the N-terminus, and whether the cysteines are separated by amino acids, e.g. the C, CC, CXC and CX3C subgroups. Outwith mammals, the biological activity of chemokines has been recognized for many years, but only recently have the genes begun to be cloned.

Fish IL-8, a CXC chemokine, has been cloned in lamprey (Lampetra fluviatilis), rainbow trout and Japanese flounder, suggesting it is present in all vertebrates. In lamprey, the cDNA encodes for a 101 aa protein, with 40% aa identity to chicken IL-8 and 32–33% to mammalian IL-8 (Najakshin et al., 1999). In the Japanese flounder, the IL-8 cDNA consists of 884 nt, with a 330 nt ORF encoding a 109 aa protein, the largest seen to

67

date for an IL-8 protein (Lee et al., 2001). The predicted protein showed 35–36% aa identity with mammals, but only 25% identity with the lamprey sequence. IL-8 expression was detectable in the spleen and kidney of LPS-stimulated fish, and after LPS stimulation of leukocytes in culture, but no constitutive expression was observed. In rainbow trout, the IL-8 cDNA is 918 nt with a 294 nt ORF encoding 97 aa (Laing et al., 2002). The trout molecule has 56% aa identity and 66% nt identity to the flounder sequence, with similar homologies to mammalian and lamprey genes. The trout IL-8 transcript shows low constitutive expression in many tissues, and both LPS and poly I:C are able to up-regulate its expression in a trout macrophage cell line. The trout IL-8 gene is 1824 nt, and contains three introns, as in birds and mammals. In all the fish IL-8-like sequences, there is no ELR motif immediately upstream of the CXC domain. This motif is associated with the ability to attract and activate neutrophils. In flounder this triplet is SLH, in trout DLR, and in lamprey GGR. It remains to be determined whether these conservative substitutions are also crucial for neutrophil activation in these species. Another CXC chemokine cloned recently in fish is a cIP-like chemokine (Laing et al., 2001a). The rainbow trout ORF encodes a 100 aa protein, within a gene that contains three introns. This protein has approximately 30% aa identity to a range of CXC chemokines. However, exon 2, coding for much of the functional domain, has 37% aa identity with CXCL10 (cIP-10). A CXC chemokine that resembles SCYB14/BRAK (52–54% aa identity to known mammalian genes) has also been sequenced in fish (Long et al., 2000), and is again a 100 aa protein. In zebra fish this gene is expressed in the nervous system during development, suggesting it may have roles beyond traditional leukocyte attraction. Several CC chemokines have also been cloned in fish. For example CK1 is a CC chemokine from rainbow trout, that consists of a 824 nt transcript encoding a 100 aa protein with a 27 aa signal peptide (Dixon et al., 1998). The trout CK1 has six Cys residues that suggest it is a member of the C6-b chemokines, with which it has highest homology. The gene, however, is composed of four exons which is not typical of mammalian CC chemokines. Another CC chemokine, from

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THE PHYLOGENY OF CYTOKINES

carp, is more similar to monocyte chemotactic proteins (Fujiki et al., 1999). This molecule consists of a 101 amino acid protein with a 26 aa signal peptide, and has 27% aa similarity with human MCP1, MCP2 and MCP3. A third CC chemokine has been discovered in the Japanese flounder, that is short relative to the other two molecules (only 90 aa) and has highest homology to eotaxin (37% aa identity) (Kono and Sakai, 2001). Chemokine receptors have also been cloned in fish, in most cases from EST-type studies. CXCR with homology to both CXCR1 and CXCR2 have been cloned in rainbow trout (Zhang et al., in press) and carp (Fujiki et al., 1999). Whilst in carp two different receptors exist, with 33% aa identity, whether they correspond exactly to CXCR1 and CXCR2 is not clear. In trout the single CXCR1/2 gene sequenced to date does not have equal aa identity to both carp genes (53% versus 36%), suggesting the carp molecules probably arose from an ancient gene duplication that preceded the separation of these groups of fish. The CXCR3 (Laing et al., 2001b) and CXCR4 (Daniels et al., 1999; Fujiki et al., 1999; Alabyev et al., 2000) genes have also been cloned in bony fish, with the CXCR4 sequences having 59–67% aa identity with the equivalent mammalian genes. In sturgeon CXCR4 expression is high in the spleen, detectable in the liver but absent from the ovary. A CCR has also been cloned in rainbow trout, with highest homology to CCR7 (Daniels et al., 1999). The transcript is 2287 nt, encoding an ORF of 368 aa, and there is constitutive expression of this molecule in a wide variety of tissues.

Amphibia Whilst no chemokine genes have been discovered in amphibia to date, a CXCR4 gene has been sequenced (Moepps et al., 2000). The encoded receptor was expressed in insect cells, and shown to be responsive to SDF-1a and SDF-1b. During development of the Xenopus embryo, CXCR4 gene expression could be detected in the nervous system and in the dorsal lateral plate (the first site of hematopoiesis).

Birds The number of chicken chemokines and chemokine receptors now cloned has increased rapidly in the last

few years (for gene and predicted protein details see Table 3.1). There are other potential chicken chemokines and chemokine receptors in the EST libraries that will not be discussed here. Rather than cover all of the chemokines, this section will concentrate on the chicken CXC chemokine IL-8/CAF, and recent results describing the mapping of chicken chemokine genes. The chicken CXC chemokine 9E3/CEF4 was the first non-mammalian cytokine cDNA to be cloned (Bedard et al., 1987; Sugano et al., 1987). The protein encoded by this cDNA has 51% aa identity with human IL-8 (Barker et al., 1993) and 45% identity with human GRO-a (Stoeckle and Barker, 1990). All three cytokines are members of the ELR CXC chemokine subfamily, and as such could be expected to be involved in angiogenesis. Consistent with this, 9E3/CEF4 has been shown to play a role in wound healing (MartinsGreen et al., 1991), and can initiate the woundhealing cascade in vivo (Martins-Green and Feugate, 1998). It is also chemotactic for chicken peripheral blood mononuclear cells and mitogenic for fibroblasts (Barker et al., 1993). Based on these biological activities, 9E3/CEF4 was variously described as the chicken homologue of IL-8 (Barker et al., 1993) or GRO-a (Martins-Green et al., 1991). However, more recently it was proposed that this chemokine be called the chicken chemotactic and angiogenic factor (CAF) (Martins-Green and Feugate, 1998). At the gene level, 9E3/CEF4 is very similar to human IL-8 (Kaiser et al., 1999). The exon:intron structure of 9E3/CEF4 corresponds almost exactly to that of human IL-8 and differs from those of other known mammalian CXC chemokine genes, including GRO-a. Genetic distance analysis also suggests that this gene encodes chicken IL-8. A number of potential regulatory sequences similar to those found in human IL-8, but not in human GRO-a, have also been identified in the 9E3/CEF4 promoter. This evidence suggested that 9E3/CEF4 is the avian orthologue of IL-8 (Kaiser et al., 1999). On balance, in terms of its biological activity 9E3/CEF4 is best described as CAF, although it may still represent the chicken equivalent of mammalian IL-8. Recent mapping experiments have suggested that the chicken may have a reduced chemokine repertoire compared with mammals. Hughes et al. (2001) recently identified and mapped three novel chicken

BASIC CYTOKINE BIOLOGY

TABLE 3.1 Chicken chemokines and chemokine receptors Predicted protein Chemokine/ chemokine receptora

Signal peptide (aa)

Mature peptide (aa)

Identity (aa)

Lymphotactin (C)

42/25b

72

27.8% with human 33% with murine

MIP-1b (CC)

21

61

57% with human MIP-1b 52% with human MIP-1a

Exons

References

1

Rossi et al., 1999; Hughes and Bumstead, 2000a

19

Petrenko et al., 1995; Hughes and Bumstead, 1999; Hughes et al., 2001

9

Hughes et al., 2001

19

Sick et al., 2000; Hughes et al., 2001

3

19

Hughes et al., 2001

50% with mammalian MIP family

3

19

Hughes et al., 2001

87/88d

51% with human IL-8 45% with human GRO-a

4

4

Bedard et al., 1987; Sugano et al., 1987; Barker et al., 1993; Kaiser et al., 1999

20

84

67% with chicken IL-8/CAF 50% with mammalian CXC

4

Sick et al., 2000; Hughes and Bumstead, 2000b

CXCR1

?e

?e

67% with human CXCR1 65% with human CXCR2

Li et al., 2000

CXCR4

?f

?f

82% with human CXCR4

Liang et al., 2001

LARC (ah189) (CC)

23

77

58% with human LARC

K203 (CC)

21

68

43% with chicken MIP-1b 50% with human MIP-1b

RANTES (ah294) (CC)

23

68

64% with human RANTES

ah221

23

68

IL-8/CAFc (CXC)

15/16d

K60 (CXC)

a b c d e f

3

Chromosome

Nomenclature used reflects that most common in the literature. The cDNA has two potential translation initiation sites – one giving a signal sequence of 42 aa, the other one of 25 aa (Rossi et al., 1999). The true identity of this chicken chemokine is unclear, see text. The true start of the mature protein is unclear. The cDNA has two potential translation initiation sites, one yielding a mature protein of 425 aa, the other one of 380 aa. The cDNA encodes a protein of 359 aa.

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CC chemokines (ah189, ah221 and ah294 – see Table 3.1). Genetic distance analysis of the known human, mouse and chicken CC chemokines showed parallels between phylogenetic grouping and chromosomal positions for the human and chicken CC chemokines (Hughes et al., 2001). Three chicken CC chemokines, ah294, MIP-1b and K203 (see Table 3.1) closely group with the mammalian MIP-like cluster on human chromosome 17. ah221 (see Table 3.1) lies in a cluster with the human MCP chemokines. Other chicken chemokines form distinct branches with their mammalian counterparts. These are reflected in their chromosomal positions. The chicken CXC chemokines, located on chicken chromosome 4 (Kaiser et al., 1999; Hughes and Bumstead, 2000b), form a well-defined cluster with the CXC chemokines on human chromosome 4. ah189 (chicken chromosome 9, Hughes et al., 2001) and human LARC (chromosome 2) are also syntenic, as are human (chromosome 1) and chicken (chromosome 1, Hughes and Bumstead, 2000a) lymphotactin. It therefore appears that the major branching of the chemokine family occurred before the divergence of birds and mammals. The chicken genome seems to contain representatives of, but not as many members as, most of the mammalian CC chemokine clusters. In humans and mice the CXC chemokine cluster, and the CC chemokine MIP-like and MCP clusters, have greatly expanded. It remains to be seen if this is the case in chickens, or whether, as for the MHC (Kaufman et al., 1999), the chicken chemokine clusters are simpler and contain fewer genes. MDV has been shown to encode a CXC chemokine, which has been described in the literature as an IL-8 homologue (vIL-8) (Parcells et al., 2001). Although the viral CXC chemokine (vCXC) has high aa identity with human IL-8 and chicken IL-8/CAF, there are several important differences between it and known IL-8s. In mammals the CXC chemokines can be subdivided. Many possess an ELR motif immediately preceding the first cysteine residue, including IL-8. Other CXC chemokines lack the ELR motif. In general, ELR CXC chemokines are involved in angiogenesis, whereas ELR- CXC chemokines are not. The vCXC lacks an ELR motif. In man, the genomic structure of most CXC chemokines consists of four exons and three introns (including IL-8), whereas the genes for PF-4 and NAP2 comprise three exons and two introns. The vCXC

gene also has three exons and two introns. It should not therefore be described as a vIL-8, but rather a vCXC.

Interleukin-15 Birds There is a report in the literature describing the molecular and functional characterization of chicken IL-15 (Choi et al., 1999b). Unfortunately, closer scrutiny reveals that the cytokine described is in fact the previously mentioned chicken IL-2. The true chicken IL-15 homologue was first identified in the EST libraries described earlier (Acc. No. AF152927), and was recently described by Mejri et al. (2001). The predicted protein has 187 aa and contains a 63–66 aa signal peptide, markedly longer than its mammalian homologues. The predicted MW of the mature protein is 14.5 kDa, with four conserved cysteines presumably forming two intrachain disulphide bonds (compare mammalian IL-15 and chicken IL-2). The chicken IL15 gene has six coding exons, as do its mammalian homologues (Kaiser et al., 2001), with similar numbers of aa encoded by each exon with the exception of exon 1, which is longer as it encodes the extended signal sequence. As yet, there is no information on the position, length and number of any 5 UTR exons in the chicken IL-15 gene. The EST libraries contain cDNAs with identity to the mammalian IL-15R alpha chain (Acc. Nos. AI980106 and AI980376).

Interleukin-18 Birds The EST databases contain a cDNA for chicken IL-18. This cDNA was cloned and expressed by Schneider et al. (2000). The predicted protein is 198 aa in length and has approximately 30% aa identity with mammalian IL-18s. In mammals, as with IL-1b, IL-18 is expressed as a propeptide that is cleaved after an aspartic acid by the action of caspase-1. Unlike chicken IL-1b, there is an aspartic acid (Asp29) early in the primary translation product of chicken IL-18, suggesting that the mature form of chicken IL-18 is 169 aa long. This predicted mature protein was expressed

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TUMOR NECROSIS FACTORS

with an N-terminal histidine tag in E. coli, and the recombinant shown to induce IFN-c expression in cultured primary chicken spleen cells, IFN-c activation from primary splenocytes being a classic bioassay for mammalian IL-18. Turkey IL-18 is remarkably similar to chicken IL-18 (Kaiser, 2002), with 96.4% aa identity. Only one IL-18 gene structure has been determined so far in mammals, that of human IL-18. The chicken IL-18 gene is less than one quarter of the length of the human IL-18 gene (Kaiser et al., 2001). Whereas the human IL-18 gene has five exons and four introns, the chicken IL-18 gene has only four exons and three introns. It appears that exons 1 and 2 in man are fused into a single exon (exon 1) in the chicken. Interestingly, the recently sequenced fowlpox genome encodes several predicted proteins with putative immune evasion functions, including an IL-18 binding protein (Afonso et al., 2000).

Other interleukin activities Fish Hybridization with a monkey erythropoietin (EPO) cDNA probe occurs in Northern blots, using rainbow trout kidney, spleen and liver RNA, and antigenic cross-reactivity can be detected in concentrated (four-fold) trout serum using a human EPO radioimmunoassay (Shiels and Wickramasinghe, 1995). The IL-13Ra2 chain has been cloned in rainbow trout (Lockyer et al., 2001). The cDNA contains a 1212 nt ORF coding for a 404 aa protein, with a 69 nt 5 UTR and a 451 nt 3 UTR. The protein has a predicted signal peptide and a single membrane spanning domain and a short intracellular tail. The extracellular domain contains two fibronectin type III modules, a conserved WSXWS motif and four conserved cysteine residues in the amino-terminal part of the domain, typical of class I cytokine receptors. Expression was detectable in the gill, spleen and kidney, but was absent from the brain.

Birds The aforementioned EST libraries contain sequences with identities to the interleukins and interleukin receptors shown in Table 3.2.

TABLE 3.2 Chicken ESTs with identity to mammalian interleukins and interleukin receptors Identity with IL-16 IL-4Ra IL-11Ra IL-13Ra IL-17R IL-21R IL-22R

Accession No.a

EST libraryb

BG713383, BI394090 AF315333, AW061439 BI392763

H H D D D, H D, H D

BG625649

a

Where available b D, University of Delaware EST library; http://www. chickest.udel.edu/; H, University of Hamburg EST library; http://genetics.hpi.uni-hamburg.de/dt40Est. html

TUMOR NECROSIS FACTORS Tumor necrosis factor (TNF) is the principal mediator of the host response to Gram-negative bacteria and is typically released from LPS-stimulated macrophages and monocytes (Abbas et al., 1991). It has been cloned for the first time outwith mammals in fish EST studies (Hirono et al., 2000a), but to date the amphibian, reptilian and avian genes have still to be discovered.

Invertebrates Whilst TNF has not been cloned in any invertebrate to date, a gene for a functionally analogous molecule termed coelomic cytolytic factor-1 (CCF-1) has been sequenced (Acc. No. AF030028) and has been found to have no genetic homology to TNF (Beschin et al., 1999). Thus, these molecules appear to have no common evolutionary origin and whether the invertebrate molecule should be considered a cytokine is a matter of debate.

Fish The Japanese flounder TNF-a cDNA was isolated during EST studies and was the first non-mammalian TNF discovered (Hirono et al., 2000a). The cDNA consists of 1217 nt, encoding a protein of 256 aa. The gene is approximately 2 kb and consists of four exons and three introns. Whilst the molecule has similar homologies to both TNF-a and TNF-b, the lack of a signal peptide, the presence of a transmembrane

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Human TNF-b

Flounder TNF

Human TNF-a 0

1000 2000 Nucleotide

3000

FIGURE 3.1 Gene organization of the flounder TNF gene compared with human TNF-a and TNF-b. The solid boxes represent untranslated regions, while the open boxes represent the coding domains.

domain and the gene organization (Figure 3.1) all suggest this is a TNF-a. The flounder TNF-a gene is expressed at low levels in blood leukocytes, but expression is greatly increased following stimulation with LPS, PMA or ConA. Using the flounder sequence it has been possible to clone the rainbow trout TNF-a gene (Laing et al., 2001b). In this species, the cDNA has an ORF of 738 nt that translates into a 246 aa protein with the same features as for the flounder sequence. The trout gene is also approximately 2 kb and contains three introns. In addition to upregulation of the trout TNF-a gene by stimulation with LPS, treatment of trout macrophages with recombinant trout IL-1b was also very effective. A second trout TNF-a gene has been sequenced more recently (Zou et al., 2002), with 92% aa identity to TNF-a1. This second gene has an ORF of 765 nt, with a 27 nt insertion in the intracellular domain relative to the TNF-a1 gene. Comparison of the 5 flanking regions reveals a CATAAA box 26 nt upstream of the transcription start in both genes, but beyond this many differences in the transcriptional regulatory elements are found. Such differences may account for the differential expression of the genes seen after LPS stimulation, where the TNF-a2 transcript is dominant. A brook trout (Salvelinus fontinalis) TNF-a gene has also been sequenced (Bobe and Goetz, 2001), and has highest homology to the rainbow trout TNF-a2 gene, unlike the Japanese flounder sequence which is more similar to the rainbow trout TNF-a1 gene. A TNFR has also been cloned in the Japanese flounder (Hirono et al., 2000b), similar to the TNFR1 and containing an identifiable death domain. Further members of the TNFR family, with identifiable death

domains, have been cloned more recently in zebra fish (Bobe and Goetz, 2001) and brook trout (Bridgham et al., 2001). The zebra fish ovarian TNF receptor (OTR) consists of a 438 aa protein with a putative transmembrane domain, and as the name suggests is highly expressed in the ovary. TNF decoy receptors have also been cloned in fish (Bobe and Goetz, 2000; Pleguezuelos and Secombes, 2001), where in brook trout they are expressed in the testis and ovary.

Birds Although avian TNF has yet to be cloned, TNF-like activity can be detected in the chicken. After infection with Eimeria (Byrnes et al., 1993a; Zhang et al., 1995) or Marek’s disease virus (Qureshi et al., 1990), release of TNF from chicken macrophages can be detected in cross-reactive mammalian cellular cytotoxicity bioassays. Injection of chickens with such TNF-like factors enhances weight loss due to Eimeria infection, which is partially reversible by treatment with anti-human TNF antisera (Zhang et al., 1995). Human recombinant TNF has been shown to cross-react with chicken cells (Leibovich et al., 1987; Butterwith and Griffin, 1989). Anti-murine TNF-a immunoprecipitates a 50 kDa protein in chickens, as in mammals (Gendron et al., 1991). Although there is a partial gene sequence purporting to be a chicken TNF-b homologue in the databases (Acc. No. M80573), details of how it was obtained are lacking and extensive genome walking to both sides of that sequence failed to identify any TNF-like sequences (P. Kaiser, unpublished results). Rautenschlein et al. (1999) recently analysed supernatants from an LPS-stimulated chicken macrophage cell line, MQ-NCSU, for TNF-like activity. TNF-like activity was purified from the culture supernatants, the peak of activity corresponding to fractions with a molecular weight of 81 kDa or higher. On Western blots, polyclonal anti-human TNF-a antisera crossreacted with a 17 kDa protein under denaturing conditions, suggesting that the chicken TNF-like factor in its active form may be a multimer of 17 kDa monomers (similar to the MW of mammalian TNF-a). The chicken TNF-like factor stimulated macrophages, inducing morphological changes, and enhancing Ia expression and NO production, the latter both on its own and in synergy with chicken IFN-c.

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GROWTH FACTORS

GROWTH FACTORS Transforming growth factor beta TGF-bs belong to a superfamily of structurally related proteins, including activins and inhibins, antiMullerian hormone and bone morphogenetic proteins (BMP) (Burt and Law, 1994). The conserved cysteines give a characteristic ‘cysteine knot’ crystal structure, as seen in other cytokines such as platelet-derived growth factor (PDGF), nerve growth factor and brainderived neurotrophic factor. Within the immune system, TGFbs have both pro- and anti-inflammatory activities, and are particularly well known for their ability to inhibit T and B cell proliferation, macrophage activation and NK cell activity. They exist as three isoforms in fish, birds and mammals, termed TGF-b1/4, TGF-b2 and TGF-b3, with amphibian TGF-b1 termed TGF-b5.

Invertebrates Whilst TGF-b has not been found in invertebrates to date, members of the superfamily are present, such as decapentaplegic protein and Drosophila 60A protein, that participate in dorsal–ventral speciation and development of the embryonic gut in Drosophila (Burt and Law, 1994), and molluscan growth and differentiation factor (mGDF) that displays a unique pattern of expression during development in the oyster Crassostrea gigas (Lelong et al., 2000).

Fish Three isoforms of TGF-b have been sequenced in fish (Laing et al., 2000), corresponding to TGF-b1, TGF-b2 and TGF-b3 of mammals. The homologies to TGF-b genes known in other vertebrate groups are typically higher for the fish TGF-b2 and TGF-b3 genes than for TGF-b1. For example, in plaice (Pleuronectes platessa) the respective genes show 71% aa identity to human TGF-b1, but 84% aa identity to TGF-b2 and TGF-b3. The first teleost TGF-b1 was isolated from rainbow trout (Hardie et al., 1998), and consists of a 1892 nt transcript that translates into a 382 aa precursor molecule. Within this predicted protein can be recognized a signal peptide of 20 aa, three potential glycosylation sites, an integrin binding site and a tetrabasic cleav-

age site (RKKR), giving a typical mature peptide sequence of 112 aa. TGF-b1 has subsequently been sequenced in several teleost species, including carp (Yin and Kwang, 2000b), a hybrid striped bass (Harms et al., 2000), plaice (Laing et al., 2000) and seabream (Acc. No. AF424703). The 5 end of the mature peptide in teleosts differs quite significantly from other known TGF-b1 genes (Figure 3.2). The gene organization of the teleost TGF-b1 gene is also unique, in that whilst it has seven exons as in other vertebrate groups, the exons equivalent to exons 2 and 3 are fused whilst there is an additional intron at the 3 end of the gene within the coding region of exon 7 (Daniels and Secombes, 1999). The gene is expressed in a wide range of lymphoid tissues, including the spleen, kidney, gill and blood. The teleost TGF-b2 gene was first isolated in carp (Sumathy et al., 1997). It shows 81% aa identity to human TGF-b2 and expression is detectable in the heart. Whilst the gene organization is not known, preliminary studies have shown that the extra intron in exon 7 is not present in this isoform in fish (Secombes et al., 2000), and thus may be restricted to the TGF-b1 isoform. Partial sequences for TGF-b2 have also been isolated from plaice and rainbow trout. TGF-b3 was first discovered using genomic DNA from sturgeon (Acipenser baeri), and subsequently partial sequences have been isolated in plaice, eel (Anguilla anguilla) and rainbow trout (Laing et al., 1999, 2000). In both of these isoforms the 5 end of the mature peptide is conserved throughout vertebrates, unlike the situation with TGF-b1. Trout

RKKRQTTTEEICS---DKSESCCVRKLY

Carp

RKKRQTETDQVCT---DKSDGCCVRSLY

Striped bass RKKRSTETKDVCT---AQTETCCVRSLY Plaice

RKKRSTDGTDTCT---AQTETCCVRKLY

Xenopus Chicken Human Mouse Rat

RKKR-GVGQEYCFG--NNGPNCCVKPLY RKKR-DLDTDYCFGPGTDEKNCCVRPLY RKKR-ALDTNYCFS--STEKNCCVRQLY RKKR-ALDTNYCFS--STEKNCCVRQLY RKKR-ALDTNYCFS--STEKNCCVRQLY **** * ***. **

*100% conserved aa; .simular aa.

FIGURE 3.2 Multiple alignment of the 5 end of the mature peptide of TGF-b1 in different vertebrate groups. The tetrabasic cut site is shown in bold immediately upstream of the mature peptide start.

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In addition to TGFb, activins have been cloned in fish (Ge et al., 1993) and are members of the TGFb superfamily.

Amphibians In Xenopus, only two isoforms of TGFb have been sequenced to date, TGFb5 and TGFb2 (Burt and Law, 1994). The mature peptide of Xenopus TGFb5 has 75% aa homology with mammalian TGFb1, whilst Xenopus TGFb2 is 95% homologous to mammalian TGFb2. Recombinant human TGFb1 inhibits T cell growth factor-induced proliferation of splenic blasts (Haynes and Cohen, 1993b), and has mesoderm-inducing activity on amphibian ectoderm (Kimelman and Kirschner, 1987). In addition, recombinant Xenopus TGFb5 inhibits T cell growth factor-induced proliferation of splenic blasts, and anti-TGFb5 serum can inhibit this activity in supernatants from mitogenstimulated Xenopus leukocytes (Haynes and Cohen, 1993b). The organization of the Xenopus TGFb5 gene has been determined and shown to be approximately 20 kb and of the typical 7 exon/6 intron arrangement (Vempati and Kondaiah, 1998a). The 5 flanking region of the TGFb5 gene has also been sequenced and shown to be a functional promoter in LUX reporter gene studies (Vempati and Kondaiah, 1998b). Numerous binding sites for transciption factors were identified, including sites for AP1, AP2, CREBP, GATA, IRF1 and Sp1. BMPs and activins have also been cloned in Xenopus (Thomsen et al., 1990; Dale et al., 1992; Clements et al., 1995).

Birds Three forms of TGF-b have been cloned from chickens: TGF-b4 (equivalent to mammalian TGF-b1) (Burt and Jakowlew, 1992), TGF-b2 (Jakowlew et al., 1990) and TGF-b3 (Jakowlew et al., 1988), which have 80%, 96–99% and 97–99% aa identity, respectively, with their mammalian homologues. All of the chicken TGF-bs have a signal peptide and thus the potential to be secreted. The predicted precursor molecule (minus the signal peptide) is 372, 392 and 389 aa for TGF-b4, TGF-b2 and TGF-b3, respectively. The organization of the genes for TGF-b2 (Burt and Paton, 1991) and TGFb3 (Burt et al., 1995) has been determined. They each

consist of seven exons and six introns, as in their mammalian homologues. The expression of TGF-b in the thymus may regulate the ability of immature thymocytes to progress through the cell cycle and differentiate into mature CD3 thymocytes (Mukamoto and Kodama, 2000). Thymic stromal cells express TGF-b2 and TGF-b3, but not TGF-b4. Thymocytes express all three, but more strongly in CD3 cells than CD3 cells. TGF-b4 mRNA expression has been shown to increase in the caecal tonsils, spleen and duodenum following E. acervulina infection (Choi et al., 1999a), presumably as part of an anti-inflammatory response. Other members of the TGF-b superfamily have been cloned in the chicken, such as the BMPs (Houston et al., 1994) and the inhibins–activins (Chen and Johnson, 1996).

Fibroblast growth factor FGFs are a group of heparin-binding, single chain polypeptides that play a pivotal role in development, cell growth, tissue repair and transformation. Currently over 20 FGF homologues have been described (Boilly et al., 2000) and four FGF receptors of the tyrosine kinase class. Whilst the different FGFs bind to the four FGFR with different affinities, there is no strict specificity between the FGF and FGFR.

Invertebrates In Drosophila melanogaster two FGFRs have been sequenced, DFR1 and DFR2, which are distantly related to vertebrate FGFRs (Shishido et al., 1993). They have two and five extracellular Ig-like domains, respectively, and a highly conserved intracellular kinase domain (60% amino acid homology to vertebrate FGFR kinase domains). The two genes show 79% homology and DFR2 is virtually identical to a previously described DFGF-R (Glazer and Shilo, 1991). They are expressed at all stages of Drosophila development and appear to be particularly important for development of the tracheal system (Klambt et al., 1992). A member of the FGFR family has also been cloned in a sea urchin (McCoon et al., 1996) and in the nematode Caenorhabditis elegans (DeVore et al., 1995). The sea urchin molecule has 47–51% homology to mammalian FGFR and, interestingly,

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GROWTH FACTORS

when expressed in COS cells does not bind FGF-2 or FGF-7.

Fish FGF-2 has been cloned and sequenced in rainbow trout (Hata et al., 1997). It contains 155 aa (approximately 17.3 kDa) and has 70% aa homology with mammalian FGF-2. Trout rFGF-2 binds tightly to heparin-Sepharose and promotes the proliferation of fibroblasts. FGF-3 has been cloned in zebra fish and the rFGF-3 shown to be mitogenic for murine BALB/MK cells known to express FGFR (Kiefer et al., 1996). FGF-6 has also been sequenced in rainbow trout (Rescan, 1998). The gene is composed of three exons, that encode a predicted protein of 206 aa with 64% aa similarity to mammalian FGF-6. Expression was detectable in muscle, heart, testis and brain. An FGF has also been isolated biochemically from the swim-bladder of red seabream (Pagrus major) by heparin-affinity (Suzuki et al., 1994). It has a molecular weight of 22.5 kDa and an isoelectric point of 9.4. It promotes the growth of fibroblasts and mesoderminduction (in a Xenopus assay), and is heat and acidlabile. Antisera to the red seabream FGF have shown that FGF is also produced in the pharynx of developing fish (flounder), where it stimulates cartilage formation (Suzuki and Kurokawa, 1996). Lastly, an EST with homology to FGF-18 is within the zebra fish EST database (Acc. No. BI882039). Four FGFR genes have been sequenced in the Medaka (Oryzias latipes), corresponding to human FGFR-1, FGFR-2, FGFR-3 and FGFR-4 (Emori et al., 1992). The four FGFRs are highly homologous to each other (76%–87%) and to known mammalian FGFRs (approximately 80% homology). Each is a single copy gene that appears to be under different transcriptional control. FGFR-4 has also been sequenced in zebra fish embryos, and uniquely contains four Ig domains in its amino-terminal region (Thisse et al., 1995).

Amphibians It has been known for many years that heparin binding growth factors are present in Xenopus embryos, that have mesoderm-inducing activity inhibitable with anti-FGF sera (Slack and Isaacs, 1989). Six mem-

75

bers of the FGF family have been cloned in Xenopus: FGF-2, FGF-3, FGF-8 (partial sequence), FGF-9, FGF10 and an embryonic (e)FGF closely related to FGF-4 and FGF-6 (Isaacs et al., 1992; Song and Slack, 1996; Christen and Slack, 1997; Yokoyama et al., 2000). Xenopus FGF-2 and FGF-3 have 84% and 71% aa homology to human FGF-2 and FGF-3 respectively, with Xenopus FGF-2 lacking a signal peptide as in mammals. Xenopus FGF-9 shows 93% aa homology to human FGF-9, also lacks a signal peptide but appears to be secreted since it is glycosylated in an in vitro translation system (possibly using its central hydrophobic domain to cross the ER membrane). The Xenopus FGF-10 molecule has 80–83% aa identity to mammalian FGF-10, and 85% aa identity to chicken FGF-10. The regenerative capacity of limb mesenchymal cells has been shown to correlate with FGF-10 expression. Xenopus eFGF has a signal peptide and shows 57–58% aa homology to FGF-4 and 61% homology to FGF-6. FGF-2, FGF-3, FGF-9 and eFGF have mesoderm-inducing activity in isolated animal caps, with FGF-2, FGF-9 and eFGF being expressed maternally (i.e. by persistent maternal transcripts), whereas FGF-3 is not expressed until the early gastrula stage. FGF-9 and eFGF can also influence the anteroposterior axis in Xenopus embryos. FGF-1 has also been cloned in the newt Notophthalmus viridescens, and has aa homologies between 79%–83% with mammalian and avian FGF-1 (Patrie et al., 1997). Lastly, partial Axolotl (Han, 1997) and salamander (Christensen et al., 2001) FGF-8 sequences have been reported. Four FGFR genes has also been cloned in amphibians (Musci et al., 1990; Friesel and Brown, 1992; Shi et al., 1992, 1993; Poulin and Chin, 1994). In newts, the aa homologies to the respective human receptors are 85% for FGFR-1, 73–78% for FGFR-3, 75% for FGFR-3 and 66% for FGFR-4. The variation in FGFR-2 arises from the numerous splice variants of this gene (Shi et al., 1994). Interestingly, the exon IIIb-containing receptors appear to play a role in development of epithelial tissues, whilst the exon IIIc-containing receptors appear to be more important for neural tissue formation. It is clear that there is differential expression and regulation of FGFR during amphibian development (Launay et al., 1994), and that they have distinct roles in amphibian limb regeneration (Poulin et al., 1993).

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Birds At least seven FGFs have been cloned in chickens to date: FGF-1, FGF-2, FGF-3, FGF-4, FGF-8, FGF-18 and FGF-19 (Borja et al., 1993; Niswander et al., 1994; Han, 1995; Mahmood et al., 1995; Vogel et al., 1996; Ohuchi et al., 2000; Ladher et al., 2000), with between 67% and 90% aa identity with their mammalian homologues. As in the other vertebrate classes, four FGFRs have been cloned in birds: FGFR-1 (Cek1), FGFR-2 (Cek3), FGFR-3 (Cek2) and FGFR-like embryonic kinase (FREK) (Pasquale, 1990; Marcelle et al., 1994). FGFR-1, -2 and -3 show 93%, 96% and 97% aa identity with their respective human FGFR homologues. FREK is most closely related to the newt (80% aa identity) and human (72% aa identity) FGFR-4s, but is thought to be distinct as it is expressed strongly in myotomes early in development, followed by expression in all embryonic skeletal muscle and in satellite cells of adult muscle. Splice variants of these FGFRs appear to be under tissue- or area-specific regulation (Sato et al., 1992), as in other vertebrates.

Platelet-derived growth factor Fish A receptor for PDGF, PDGFRb, has been cloned in the puffer fish (How et al., 1996). The gene consists of 21 exons and has 45% aa homology with human PDGFRb, with the kinase domain having much higher homology as with CSF1R (How et al., 1996). The gene is linked tandemly in a head-to-tail array with the CSF1R (see Interleukin-3 and Colony-stimulating factors).

Birds Three cDNA clones were isolated from a chicken hepatoma cell line (LMH) (Horiuchi et al., 2001b), corresponding to short form type 1 (S1), long form (L) and short form type 2 (S2) cDNA clones of the chicken PDGF-A chain. Genomic sequencing and Southern blotting revealed that the three forms of cDNA were generated by alternative splicing. S1 and L mRNAs contained two transcription start sites on one exon. At the aa level, the mature protein encoded by the L clone has 90 and 85% identity with the coding regions

of the long form of human and Xenopus PDGF-A, respectively. The eight cysteine residues conserved in all known forms of PDGF were present in the putative mature peptides of all three forms of chicken PDGF-A. Expression of the three forms of PDGF-A varied among tissues and cells. Chicken thrombocytes, analogous to mammalian platelets, had very low levels of PDGF mRNAs. However, PDGF-A mRNA expression in thrombocytes peaked 4 h after exposure to type 1 collagen or thrombin, and then decreased gradually with continued incubation, suggesting that chicken PDGF in thrombocytes plays an important role in the vascular system and in healing damaged tissue.

CONCLUSIONS The cloning of cytokine genes in non-mammalian vertebrates is rapidly increasing and significant advances have been made in recent years. Representatives of many of the cytokine groups are now known in birds, amphibians or fish. Many cytokine receptor sequences are also now known and show remarkable conservation. Despite these advances in vertebrates, there is still a dearth of information about cytokine genes in invertebrates. Undoubtedly future EST studies and genome programmes will aid the identification of cytokine and cytokine receptor genes in many vertebrate and possibly invertebrate groups. Thus, there remains the possibility that novel cytokine genes have still to be discovered, especially where sequence homology alone may not be sufficient to allow identification.

ACKNOWLEDGEMENTS Thanks are due to Simon Hughes and Nat Bumstead for long and fruitful discussions on chicken chemokines.

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4 Cytokine signaling Brian E. Szente GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA

Words differently arranged have a different meaning, and meanings differently arranged have a different effect. Blaise Pascal (1623–1662)

INTRODUCTION Cytokine signaling and the regulation thereof has undergone something of a renaissance in the past decade, as several families of proteins have come to light. Specifically, these are (1) the JAKs and the Stats which in particular combinations act to ‘get the ball rolling’ as membrane proximal mediators of cellular signaling events; and (2) the SOCS and the PIAS which are among the more potent negative regulators of the JAK-Stat signaling schemes. This review will be dedicated to an examination of the actions of JAKs and Stats, SOCS and PIAS and the deceptively simple, but critical combinatorial interactions between these protein families.

JAKS The Janus kinases, or Jaks, are a family of cytoplasmic protein tyrosine kinases essential for cytokine and The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

growth factor signaling. The mammalian JAK family consists of four members, designated Tyk2, Jak1, Jak2 and Jak3. These are large cytoplasmic kinases ranging in size from roughly 115 to 140 kDa in mass, comprised of approximately 1100 amino acids each (reviewed in Heim, 1999; Leonard, 2001). The expression patterns of three members of the Jak kinase family are fairly ubiquitous, while the expression of Jak3 is primarily restricted to cells of vascular and hematopoietic origin. Furthermore, Jak3 is the only member of the family whose expression appears to be inducible as opposed to constitutive (Verbsky et al., 1996). The domain structure of the Jaks is unique among cytoplasmic protein tyrosine kinases (see Figure 4.1). All have a kinase domain at the C-terminus (Jak Homology domain 1, JH1). The phosphorylation of a conserved tyrosine residue within JH1 stimulates the catalytic activity of the Jak family members. Immediately N-terminal to the kinase domain is a pseudokinase domain (JH2), which lacks many of the residues essential for kinase function, has no Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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JH6

JH5

JH4

JH3

JH2

Pseudokinase Domain Interaction with cytokine receptors

JH1

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Regulation of cell surface expression of receptors

FIGURE 4.1 Domain structure of Jak kinases, with the seven Jak Homology (JH) domains and their respective roles.

functional ATP binding site and instead acts as a regulatory domain (Luo et al., 1997, Leonard and O’Shea, 1998). Other striking structural features of the Jaks are the lack of any Src homology 2 (SH2) and SH3 domains, and a lack of canonical protein–protein interaction domains. Instead, new classifications of functional and protein interaction domains are likely to emerge as the biology of Jaks, related to elements outside of the kinase and pseudokinase domains, is explored. The five additional regions of Jak homology upstream of the pseudokinase domain are designated JH3–JH7, respectively. The interaction of Jak kinases with cytokine receptors is mediated by the N-terminal region, including the JH6 and JH7 domains, of the Jak kinases, as demonstrated in the context of the interactions of the erythropoietin and growth hormone receptors with Jak2 (Argetsinger et al., 1993; Witthuhn et al., 1993, Cacalano et al., 1999), but has subsequently been extended to other cytokine receptors and Jak kinases (reviewed in Leonard, 2001). Further experimental evidence points to discrete regions within the JH7 domain of Jak2 as being essential for cell surface expression of the EPO-R (Huang et al., 2001), and regions of within the JH3–JH4–JH5 domains of Tyk2 serving a similar function for IFNAR1 (Richter et al., 1998). In unstimulated cells, Jaks are generally believed to be present in an inactive form. Some of these proteins are pre-associated with receptor subunits while ligand–induced receptor oligomerization, such as the interaction of a cytokine with its cognate receptor, serves as a trigger to signal the recruitment of Jaks from other intracellular locations into the receptor– ligand complex. Localized clustering of the Jak kinases and their subsequent activation by either auto or trans-phosphorylation is an event that may involve

other members of the Jak kinase family, the Src family of kinases and/or receptor tyrosine kinases. This activation results in an increased Jak kinase activity, after which the activated Jaks then phosphorylate receptors on target tyrosine sites. The phosphotyrosine sites on the receptors can then in turn serve as docking sites for the binding of other SH2-domain containing signaling molecules such as kinases, phosphatases, adapter proteins, and Stats which will be discussed below in more detail. There are a number of cytokines that activate Jak1. These include interferons (both type I and type II interferons), proteins whose receptors contain gp130 (IL-6, IL-11, CNTF, OSM, LIF and CT-1) and proteins whose receptors share the common receptor subunit, cc (IL-2, IL-4, IL-7, IL-9 and IL-15). It was therefore anticipated that the effects of deleting Jak1 would be severe. Consistent with this hypothesis, Jak-1-deficient mice display a profound phenotype which leads to perinatal lethality (Rodig et al., 1998). These animals have severe neurological defects which result in deficient suckling. Cells derived from these mice indicate multiple defects, including those traceable to signaling by cc-dependent cytokines and the interferons. In addition to the neurological deficits in the Jak1deficient animals, immune cell development is also impaired resulting in a severe reduction in the number of lymphoid progenitors (Rodig et al., 1998). Jak2 activation is mediated by a variety of cytokines. All cytokines whose receptors are homodimers of a type I cytokine receptor molecule (Leonard, 2001), such as GH, EPO and TPO, signal in a Jak2-dependent manner. In addition, Jak2 is also used by hematopoietic cytokines and IFNc. Jak2 deficiency results in fetal lethality as a result of defective EPO signaling leading to a severe impact on fetal erythropoiesis (Neubauer et al., 1998; Parganas et al., 1998). The defect in signaling by EPO is so profound that the mice do not develop sufficiently for the ramifications of the other defects to be appreciated in a physiological/developmental context. Cells derived from these animals are further deficient in their responses to IL-3 and IFNc. Jak3 is found solely in association with the common cytokine receptor c chain, cc. Mutations in cc in humans result in X-linked severe combined immunodeficiency (XSCID) (Noguchi et al., 1993). It was hypothesized that mutations in Jak3 might cause an autosomal recessive disease that is both clinically and

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immunologically indistinguishable from XSCID. Further strengthening this hypothesis, children with autosomal recessive SCID were found to have mutations in the Jak3 gene, resulting in either markedly reduced levels of Jak3 or functional inactivation of Jak3 (Russell et al., 1995; Macchi et al., 1995; Candotti et al., 1997). Indeed Jak3 knockout mice suffer from a severe combined immunodeficiency syndrome (SCID) irrespective of sex, thus mirroring the human autosomal recessive disease (Nosaka et al., 1995; Thomis et al., 1995). These animals have small thymuses, and their peripheral T cells do not proliferate after stimulation with PMA, concanavalin A, anti-CD3 antibodies, and the combination of anti-CD3 and anti-CD28 antibodies. They also evince a severe block in the B cell maturation pathway, with most of their B cells arrested at a pre-B maturation step (CD43, CD45R) (Park et al., 1995). This defect, however, is not mirrored in humans, and can be traced to differences in the biological effects of IL-7 in mice and humans (Leonard, 2001). Jak3-deficient mice also demonstrate defects in the NK cell compartment, display splenomegaly at 4 months of age and show significant expansion of the myeloid cell lineage in a T cell-dependent manner (Grossman et al., 1999). Tyk2 is known to be activated primarily by type I IFNs and IL-12, although activation by a multitude of other cytokines including IL-6, IL-10, IL-11, IL-13, TPO, CNTF, CT-1, LIF and OSM has been reported (reviewed in Rane and Reddy, 2000). Consistent with the activation data, signaling by IFNs and IL-12 is diminished in Tyk2-deficient mice (Shimoda et al., 2000; Karaghiosoff et al., 2000), although not completely ablated. Interestingly, Stat3 activation is the primary observable defect in these animals. Signaling by IFNc is also compromised as a consequence of the defect in IL-12 signaling, which may be due in part to the inability of the Tyk2-deficient mice to up-regulate IL-18R expression (Lawless et al., 2000). It appears that Tyk2 is absolutely essential for certain IFNdependent responses, such as the ability to clear vaccinia virus and the capacity of T cells to respond to challenge with LCMV (Karaghiosoff et al., 2000). It is conceivable that other Jak kinases or other signaling pathways can at least partially compensate for the loss of Tyk2 function in the setting of the knockout mouse, given that these animals do not present a lethal phenotype.

STATS There are seven mammalian signal transducers and activators of transcription (Stats) which have been identified in three chromosomal clusters (Copeland et al., 1995). An additional level of complexity is created by the differential splicing (Stat1, Stat3, Stat4 and Stat6) and post-translational, proteolytic processing (Stat5a and Stat5b) of the Stats. Stat proteins are expressed ubiquitously, with one notable exception – Stat4, which is expressed mainly in the thymus and testes. Within Stat proteins, several functional domains have been delineated. These include an N-terminal protein interaction domain – important for positive and negative regulation of Stat activity, a p48 binding domain (in Stats1 and 2), a DNA binding domain, an SH2 domain, and a transactivation domain (see Figure 4.2). The N-terminal region contains the major site of protein–protein interactions of the Stats. In Stat1 and Stat4, the N-terminus is involved in oligomerization of Stat dimers, leading to the formation of Stat tetramers, and perhaps higher-order multimers (Bergad et al., 1995; Guyer et al., 1995; Vinkmeier et al., 1996, 1998). An as yet unidentified phosphatase deactivates Stat1 via a mechanism which is dependent upon the presence of an intact N-terminus (Shuai et al., 1996). Recently the tyrosine phosphatase SHP-2 was demonstrated to dephosphorylate Stat5 in vitro (Yu et al., 2000), although these results await confirmation in a cellular or physiological setting. The N-terminus of Stat1 has also been observed to interact with the nuclear transcriptional coactivators CBP and p300 (Zhang et al., 1996). A site located between amino acids 150 and 250 in both Stat1 and Stat2 interacts with a cofactor known as p48 or ISGF3c (reviewed in Bluyssen et al., 1996). After being activated in response to ligation of the type I IFN receptor,

Dimerization (Stats 1 and 4)

DNA binding domain

SH3

SH2

Transactivation domain

pY pS

FIGURE 4.2 Generic domain structure of Stat proteins, with conserved domain structures delineated.

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Stat1a/Stat2 heterodimers bind to p48. This heterotrimeric complex, designated interferon stimulated gene factor 3 (ISGF3), is translocated to the nucleus where it binds to the interferon-stimulated response element (ISRE or GAS element) in a myriad of interferon-regulated genes (Decker et al., 1997). p48/ISGF3c belongs to the interferon regulatory factor (IRF) and Myb family of transcription factors (Veals et al., 1992). Stat1 and Stat2 are the only members of the Stat family to interact with either p48 or any related proteins. The Stat DNA binding domain is located between amino acid positions 400 and 500 (Horvath et al., 1995). This was defined on the basis of the DNA binding capacities fo Stat1 and Stat3. Mutations in this region impair the DNA binding of the resultant Stat molecules, but do not interfere with their phosphorylation in response to cytokine stimulation. The Stat SH2 domain is localized roughly between amino acid positions 570 and 670, and mediates two distinct sets of functionally important protein–protein interactions. First, this SH2 domain binds with high specificity to phosphotyrosine docking sites on cytokine receptors (Greenlund et al. 1994; Heim et al., 1995), and as a result determines the specific patterns of Stat activation by individual cytokine ligands. Second, the SH2 domain is required for dimerization of Stats, and further determines the specificity of those dimers (Heim et al., 1995). With the sole exception of Stat2, all Stats readily form homodimers, although Stat2 dimers have been observed in Stat1 deficient cell lines (Bluyssen et al., 1995). Heterodimerization of Stats is a more limited phenomenon, but again represents a combinatorial mechanism for generating functional diversity in cytokine signaling pathways. Stat1 is capable of forming heterodimers with Stat2 and Stat3 (Horvath and Darnell, 1997). Stat5 and Stat6 heterodimers on the other hand have been observed in Daudi cells (Fasler-Kan et al., 1998). The Stat transactivation domain is present in the C-terminus of Stat proteins, and contains both tyrosine and serine residues critical to the transcriptional activity of these proteins. The phosphorylation of a single serine residue within this transactivation domain can have dramatic amplifying effects on the transcriptional activity of a given Stat protein (Decker and Kovarik, 2000). In Stat1 and Stat2, the C-terminal domains are capable of binding to the nuclear cofac-

tors CBP and p300 (Bhattacharya et al., 1996; Zhang et al., 1996). Differentially spliced Stats, in which the C-terminal transactivation domains are absent, retain their capacity to bind DNA, but are incapable of activating gene transcription, and consequently serve a dominant negative function. Sequences in the C-termini also play an important role in the dephosphorylation of Stat3 and Stat5. Whereas full length Stat3 and Stat5 are normally dephosphorylated within 3–4 h of activation, the isoforms of these Stats lacking C-termini demonstrate persistent activation due to the retention of phosphotyrosines at or near position 700 (Moriggl et al., 1996; Leonard, 2001). Stat1 is activated by a wide range of cytokines including the interferons, IL-5, IL-6 and IL-10. Other growth factors such as EGF, PDGF and insulin have also been reported to activate Stat1 (Ruff-Jamison et al., 1993; Rane and Reddy, 2000; Leonard, 2001). Given the broad range of activators of Stat1, the phenotype of Stat1-deficient mice was surprisingly selective in terms of defective signaling via the actions of the type I and type II interferons (Durbin et al., 1996; Meraz et al., 1996). IL-6-induced responses, on the other hand, appeared to be unimpaired. These mice exhibit severe defects in responses to microbial antigens (e.g. Listeria monocytogenes) and viruses (murine hepatitis virus and vesicular stomatitis virus) for which host defenses are known to be dependent on interferons. The lack of a defect in signaling in response to other cytokines indicates that there are either redundant pathways in effect or that the in vitro results are simply not reflective of the true situation in vivo. Stat2 activation is observed predominantly in response to stimulation with type I interferons. The situation in Stat2 knockout mice corroborates this, as Stat2-deficient mice exhibit defects related only to signaling by type I interferons (Park et al., 2000). Specifically, cells from these animals were less sensitive to the actions of type I interferons, and consequently more suceptible to viral infection using lower titers of virus. Unexpectedly, however, Stat2-deficient fibroblasts displayed a more severe defect than Stat2deficient macrophages, indicating that different cellular lineages have different levels of dependency on Stat2 for in vivo responses to IFNs. The Stat2-deficient fibroblasts showed a reduced level of Stat1 induction in response to type I interferons, while macrophages from the same animals had levels of Stat1 similar to

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those found in wild-type animals. This indicates that in fibroblasts at least, Stat1 transcription may be dependent on the actions of Stat2. Stat3 is broadly activated by cytokines, and correspondingly, mice lacking Stat3 exhibit perinatal lethality (Takeda et al., 1997). This perinatal lethality has necessitated the study of the effects of Stat3 deletion in a tissue-specific manner (Akira, 2000). The conditional knockout approach has revealed specific defects in Stat3-deficient T cells centering on a specific deficiency in IL-2-induced IL-2Ra expression and an associated defect in IL-2-induced proliferation, similar to that observed in Stat5-deficient mice (Akaishi et al., 1998). The response of these T cells to IL-6 is also impaired. Stat3-deficient neutrophils and macrophages differ from T cells in that they exhibit defective responses to IL-10. Interestingly, the roles of Stat3 have been found to extend well beyond the immune system. Required roles for Stat3 signaling have been defined in the normal involution of mammary epithelium (Chapman et al., 1999) and also for both the normal hair cycle and keratinocyte migration in normal wound healing processes (Sano et al., 1999; Akira, 2000). Stat4 is primarily associated with activation by IL-12, and its expression is restricted to myeloid cells, thymus and testes (Zhong et al., 1994). There are additional reports of Stat4 activation in response to IFNc, IL-2 and IL-17 (Cho et al., 1996; Subramaniam et al., 1999; Wang et al., 1999). Since IL-12 is a major regulator of TH1 cell differentiation, development of the TH1 cell compartment in Stat4-deficient mice was examined. CD4 T cells from these animals were incapable of producing high levels of IFNc in response to IL-12 priming (Kaplan et al., 1996b; Thierfelder et al., 1996). Additionally, IL-12 stimulation did not result in augmented NK cell cytolytic activity in these mice. Stat5a and Stat5b are the most closely related Stat proteins, having greater than 90% homology at the amino acid level. These two Stat proteins are activated in response to the IL-2 family of cytokines (those using cc, excluding IL-4 and IL-13). The major areas of amino acid sequence variability are at the N- and C-termini (Lin et al., 1996). Presently it is not known to what extent the functions of these two proteins are overlapping versus distinct. Individual Stat5a and Stat5b knockout mice have been generated, as have Stat5a/Stat5b double-knockout animals. Stat5a-

deficient mice were first observed to exhibit a major deficit in prolactin signaling (Liu et al., 1995), which is not surprising considering the original discovery of Stat5a as a mammary gland factor (Schmitt-Ney et al., 1992). Stat5b-deficient mice on the other hand exhibit a loss of the normal sexually dimorphic growth rates dependent on pulsatile secretion of growth hormone (Udy et al., 1997). Stat5a expression is not sufficient to compensate for this defect. Both Stat5a- and Stat5bdeficient mice have pronounced defects in the immune system, many of which come as a result of deficiencies in IL-2 induction of IL-2Ra expression (Nakajima et al., 1997; Imada et al., 1998). This in vitro defect is mirrored in vivo in Stat5a knockout animals by the defective expansion of Vb8 T cells in response to superantigen (Staphylococcal enterotoxin B) challenge (Nakajima et al., 1997). Both Stat5a and Stat5bdeficient mice had diminished numbers of NK cells, but the deficiency was more pronounced in the Stat5b-deficient mice. These animals exhibited more severe defects in cytolytic activity and proliferation of NK cells, including drastically reduced perforin gene expression (Imada et al., 1998). Stat5a/Stat5b doubleknockout mice exhibit a more dramatic phenotype than either of the single knockouts. T cells in these animals do not proliferate in response to T cell receptor ligation and IL-2 receptor activation – this is associated with a loss of induction of cyclin D2, cyclin D3 and cdk-6 following stimulation with anti-CD3 antibodies (Moriggl et al., 1999). NK cells in these mice are also conspicuously absent, perhaps due to impaired IL-15 signaling. Additionally, Stat5a and Stat5b are also essential for normal antigen-induced eosinophil and T cell recruitment to the lung in an antigeninduced model of airway eosinophilia (Kagami et al., 1999). The dramatic immune defects in the Stat5a/Stat5b double knockout suggest that while Stat5a and Stat5b may share some properties, they are functionally distinct (reviewed in Lin and Leonard, 2000). Stat6 is activated only in response to IL-4 and IL-13. These cytokines share the IL-4 receptor a chain, which displays multiple docking sites for Stat6 (Hou et al., 1994). Stat6-deficient mice have been generated and exhibit profound defects in the T cell compartment, specifically in IL-4-induced TH2 differentiation and IL-4-stimulated increases in expression of CD23, MHC class II molecules and IL-4Ra (Kaplan et al.,

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1996a; Shimoda et al., 1996). Stat6-deficient B cells were also functionally compromised in terms of immunoglobulin class switching to IgG1 and IgE. B cell and macrophage responses following IL-13 stimulation were also dramatically impaired in the Stat6 knockouts, in terms of both MHC class II expression and the regulation of NO production (Takeda et al., 1996). It is also worth noting that several splice vairants of Stat6 exist, including two (Stat6b and Stat6c) that encode an N-terminal truncation and an SH2 domain deletion respectively (Patel et al., 1998), and one with a deletion in the transactivation domain, which is expressed specifically in mast cells (Sherman et al., 1999).

SOCS The suppressors of cytokine signaling, or SOCS proteins comprise a fairly large and diverse protein family. They are named after the first of their number to be characterized, namely SOCS1, which was independently discovered by three independent laboratories as involved in IL-6 signaling (Starr et al., 1997), interacting with the catalytic domain of JAK2 (Endo et al., 1997), or antigenic similarity to the Stat SH2 domain (Naka et al., 1997). There are in fact five definable SOCS subfamilies, each of which contains the socalled ‘namesake’ motif or ‘SOCS-box’. These are (1) the SH2-SOCS, defined by the presence of a single SH2 domain upstream of the SOCS-box; (2) the ASBSOCS which contain ankyrin repeats; (3) the WSBSOCS which have WD40 repeats; (4) the SSB-SOCS which have SPRY domains; and (5) a pair of small GTPases (RAR and RAR-like) each containing a SOCSbox (Hilton et al., 1998). Those SOCS proteins that have thus far proved to be of major relevance to cytokine signaling have been the SH2-containing SOCS proteins, and it is those which will be reviewed here. Presently eight members of the SH2-containing SOCS subfamily are known, designated as CIS (for cytokine-inducible SH2-containing protein) and SOCS1 to SOCS7. These proteins are characterized by an N-terminal domain of variable length, a central SH2 domain, and a C-terminal domain containing the namesake motif or SOCS box (see Figure 4.3). The N-terminal domains are the most variable regions

SH2

Variable region

Binding to phosphotyrosines on Jaks and activated receptors

SOCS box

Interaction with Elogins B & C (SOCS1 and SOCS3)

FIGURE 4.3 Structure of the SH2-containing SOCS proteins, with the SH2 domain and ‘SOCS box’ namesake motif highlighted. between SOCS family members, and little is known of their precise functions. The N-terminal domains of CIS and SOCS1, SOCS2 and SOCS3 are fairly short, and contain some putative SH3 domain recognition motifs, as well as a roughly 30 amino acid stretch immediately adjacent to the SH2 domain which is required for optimal recognition of other proteins (i.e. Jaks). The central SH2 domain of SOCS proteins is critical for the recognition of target phosphotyrosine moieties (i.e. those found on Jaks and activated cytokine receptors). Finally, the C-terminal domain contains the namesake feature or SOCS box, which may play a role in targeting components of the JakStat signaling pathway for proteasomal degradation. This domain in SOCS1 and SOCS3 has been demonstrated to bind to elongins B and C of the proteasome complex (Kamura et al., 1998; Zhang et al., 1999). Cytokine-inducible SH2 containing protein, or CIS, the first member of this family to be described in detail, was originally reported as an immediate–early gene product induced in hematopoietic cells by EPO and other cytokines (Yoshimura et al., 1995). In Ba/F3 cells stably transfected with either EPO-R or with a chimeric EGF/EPO-R, CIS mRNA was induced within 30 min of cytokine stimulation. This effect was seen to be specific to the signaling via EPO/EPO-R components, as it was not induced in EGF-stimulated Ba/F3 cells stably transfected with the EGF-R. Likewise, GMCSF stimulation of Ba/F3 cells expressing the GM-CSF receptor a-chain and a C-terminally truncated bchain resulted in CIS expression, as did stimulation of the endogenous Ba/F3 IL-3 receptor (Yoshimura et al., 1995). Coimmunoprecipitation experiments suggested that CIS became physically associated with both the tyrosine phosphorylated IL-3 receptor b subunit and the tyrosine phosphorylated EPO-R. Functionally, CIS was characterized as a classic feedback modulator of Stat5-mediated signaling

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(Matsumoto et al., 1997), due to the presence of four Stat5 binding sites identified in the CIS promoter region, which were required for EPO-induced expression of CIS. Furthermore, a mutant IL-2 receptor bchain lacking 147 amino acids at its C-terminus, which could not activate Stat5, was also unable to support the induction of CIS. These results confirmed that Stat5 activation can directly cause an increase in CIS mRNA and protein levels in response to IL-2. It was further demonstrated that overexpression of CIS, under the control of a Dex-inducible promoter, inhibited both the IL-3 and EPO-induced tyrosine phosphorylation of Stat5 and the expression of a Stat5 target gene, namely OSM. Thus, there exists an apparent negative-feedback loop involving CIS and Stat5. Deletion of CIS fails to reveal any phenotypic changes in mice (Marine et al., 1999b), which stands in marked contrast to the striking phenotypes of the SOCS1, SOCS2 or SOCS3-deficient mice (described below). Instead, transgenic models are required to gain an appreciation as to the relevant physiological functions of CIS. Overexpression of CIS induces a phenotype similar to that described for the Stat5a and Stat5b knockout mice (Liu et al., 1997; Udy et al., 1997; Teglund et al., 1998). This phenotype involves failure of terminal differentiation of the mammary glands and consequent failure to lactate following parturition, lower body weight, reduced levels of GHinduced major urinary protein, suppression of Stat5 phosphorylation and a reduction in IL-2Ra expression and Stat5 activation in T cells in response to IL-2 stimulation (Matsumoto et al., 1999). These mice also have significantly lower numbers of cd T cells, NK and NK-T cells. Together these findings strongly suggest that a major physiological function of CIS is the negative regulation of Stat5 responses as pertain to growth, mammary gland function and T cell development, and confirm the roles of CIS in regulating specific physiological responses, including those to GH, PRL and IL-2. SOCS1 inhibits signal transduction by a wide range of cytokines (reviewed in Nicola and Greenhalgh, 2000) including LIF, IL-6, IL-4, GH, prolactin, TPO, the interferons and SCF. The original characterization of SOCS1 suggested that it interacted directly with the kinase domain (JH1) of Jak2 and inhibited kinase activity of all four Jaks, as well as the subsequent

phosphorylation and activation of downstream substrates such as receptors and Stat proteins (Endo et al., 1997). High affinity binding of SOCS1 to Jak2 and complete inhibition of kinase activity required not only the SH2 domain of SOCS1, but also a region of approximately 30 amino acids N-terminal to the SOCS1 SH2 domain (Narazaki et al., 1998; Nicholson et al., 1999; Yasukawa et al., 1999). This N-terminal region has sequence similarity to the pseudosubstrate inhibitory domain of Jak2 (Yasukawa et al., 1999) and could bind independently of the SH2 domain to Jak2 (Nicholson et al., 1999). SOCS-1 has also been shown to interact with other activated kinase domains such as those from Tec (Ohya et al., 1997), c-Kit, Flt3, the CSF-1 receptor (c-Fms) the PDGF receptor (De Sepulveda et al., 1999) and the M-CSF-R (Bourette et al., 2001), all presumably via the SOCS SH2 domain. Further expanding the potential range of actions of SOCS1, it was determined to act as a binding partner for the signaling molecules Vav and Vav2 (guanine nucleotide exchange factors) and Grb2 (an adaptor protein). Finally, isolated SH3 domains of the Fyn, Itk, Nck and the p85 subunit of PI3-kinase were demonstrated to interact with specific proline motifs in the N-terminal domain of SOCS-1 (De Sepulveda et al., 1999). It is interesting to note that although binding of SOCS1 to both Jaks and Tec leads to inhibition of their kinase activities, this does not occur upon binding of SOCS1 to activated kit receptors (De Sepulveda et al., 1999). To date, SOCS1 has not been found to directly regulate the kinase activity of any receptor tyrosine kinases (RTKs), although it has been found to negatively regulate the proliferative signals of several, including the M-CSF-R (Bourette et al., 2001). SOCS1-deficient mice have been generated and characterized by three independent groups (Naka et al., 1998; Starr et al., 1998; Marine et al., 1999b). The phenotype of these knockout animals is severe. By 3 weeks of age, these mice die of a complex disease characterized by (1) fatty degeneration of the liver; (2) monocytic infiltration of the heart, lungs and pancreas; (3) reduced thymic size; and (4) severe T- and Blymphopenia. The spleens of SOCS1 knockout animals are also malformed. Their spleen weights are similar to those of wild-type animals, but they are either entirely devoid of lymphoid follicles, or possess follicles comprised entirely of immature cells. The B lymphocytes of SOCS1 knockout mice have been

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observed to undergo accelerated apoptosis, associated with elevated levels of the pro-apoptotic protein Bax (Naka et al., 1998). It is therefore plausible that SOCS1 may have a role in the negative regulation of pro-apoptotic pathways. The observation that embryonic fibroblasts from SOCS1-deficient animals display increased sensitivity to TNFa-induced cell death would seem to corroborate this hypothesis (Morita et al., 2000). The overall phenotype of the SOCS1deficient animals resembles that seen in neonatal mice with artificially elevated IFNc levels (Gresser et al., 1981). Consequently, mice homozygously null for both the SOCS1 and IFNc genes were bred to determine the role of IFNc in the pathology observed in the SOCS1 knockout animals (Alexander et al., 1999; Marine et al., 1999b). Surprisingly, these mice are healthy, and show little, if any, of the phenotype associated with the SOCS1 knockout animals. The critical role of SOCS1 in negatively regulating IFNc signaling is underscored by the observation that injecting neutralizing anti-IFNc antibodies into SOCS1-deficient mice prevents their disease. SOCS2 has been shown to interact with the activated IGF-1 receptor (Dey et al., 1998). SOCS2deficient mice have been generated (Metcalf et al., 2000), and most organs in these animals appear normal. SOCS2-deficient mice grow and develop normally until weaning, after which they steadily grow larger than their wild-type littermates, becoming ~40% larger by 12 weeks of age. Despite their abnormal size, the growth of long bones and most organs is in proportion to body weight, and there are no defects in the hematopoietic system (Metcalf et al., 2000). There is a signifigant dysregulation of GH and IGF-1 signaling observed in these animals, suggesting a possible role for SOCS2 in regulating signaling by these two cytokines during the postnatal growth period. In point of fact, the SOCS2 knockout phenotype parallels that of mice overexpressing GH or IGF-1, including the characteristic excess collagen deposition in the skin, and the proportional growth of most organs and long bones (Palmiter et al., 1983; Mathews et al., 1988a, 1988b). Interestingly, the molecular basis for the high growth (HG) phenotype has been determined to map to a disrupted SOCS2 locus (Horvat and Medrano, 2001). SOCS3 expression is elevated in response to many of the same cytokine/receptor systems as SOCS-1,

including the gp130 family, IL-4, GH and prolactin. It has also been shown to inhibit signaling by IL-2 and IL-3 (Cohney et al., 1999) and leptin (Bjørbaek et al., 1998, 1999). SOCS3 also appears capable of inhibiting interferon-induced signaling, albeit more weakly than SOCS1 (Sakamoto et al., 1998; Song and Shuai, 1998). At high levels of protein expression, SOCS3 can interact with Jaks, although its affinity for them is decidedly lower than that observed for SOCS1. Numerous lines of investigation have subsequently suggested that the mechanism of action of SOCS3 differs from that of SOCS1. Although both SOCS1 and SOCS3 are capable of binding and co-immunoprecipitating Jak2, only SOCS1 significantly inhibits the in vitro kinase activity of Jak2 (Nicholson et al., 1999). SOCS3 has demonstrated the capacity to interact with Jaks in cells stimulated with either GH or IL-2, but efficient inhibition of Jak kinase activity in these systems requires ligand-bound, phosphorylated GH-R and IL-2Rb, respectively (Cohney et al., 1999; Hansen et al., 1999). It is therefore likely that SOCS3 inhibition of Jak-Stat signaling occurs via an alternative mechanism. Recent studies have determined the comparative binding affinities of SOCS3 for phosphotyrosinecontaining peptides derived from Jaks, Stats and the gp130 subunit of the IL-6 receptor systems. The preferred targets for SOCS3 thus determined were peptides centered around the SHP-2 binding site of gp130, specifically those including the tyrosine at amino acid position 757. In vitro evidence supports this, as mutations of this tyrosine (Y757) on gp130 significantly reduced the capacity of SOCS3, but not SOCS1 to inhibit IL-6 signaling (Nicholson et al., 2000). This observation has been extended to equivalent phosphotyrosines on other cytokine receptors, including, Y401 on the EPO receptor (Sasaki et al., 2000) and Y985 on the leptin receptor (Bjørbaek et al., 2000). One line of thought suggests that the recruitment of SOCS3 to activated receptors may bring it into close apposition to Jaks and allow kinase inhibition by a mechanism similar to that employed by SOCS1. The fact that the binding sites for SOCS3 on the gp130 and Leptin receptors are identical to the SHP-2 binding sites, and given that SHP-2 is a known substrate of the Jaks (Schaper et al., 1998) (Bjørbaek et al., 2000; Nicholson et al., 2000) suggests that SOCS3 may alternatively influence the Ras/MAP kinase

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signaling pathway – perhaps by blocking SHP-2 phosphorylation. Neither SOCS3 transgenic nor SOCS3 knockout mice survive to birth. SOCS3 transgenic mice display a complete failure of fetal liver erythropoiesis, while SOCS3 knockout mice die of widespread erythrocytosis which is most pronounced in the liver (Marine et al., 1999a). The overall suggestion is that of an essential role for SOCS3 as a negative regulator of Jak2mediated EPO signaling. SOCS3 is expressed at low levels in adult tissues, so in order to determine its role in adult hematopoiesis, lethally irradiated adult mice were reconstituted with fetal liver cells from SOCS3deficient animals. An analysis conducted 2 months following reconstitution revealed no differences in the absolute RBC or WBC counts. There was, however, a modest increase (two-fold) in the number of erythroid precursors derived from the bone marrow of these animals (Marine et al., 1999a). These results further indicate a larger role for SOCS3 in the regulation of fetal erythropoiesis than in the regulation of adult erythropoiesis.

PIAS The protein inhibitors of activated stats, or PIAS, family of proteins first came to light when Shuai and colleagues performed yeast two-hybrid screens with the goal of finding proteins capable of interacting with Stat1 outside the C-terminal transcription activation domain (Liu et al., 1998). PIAS1 was the first member of this family to be characterized as it was found to bind to activated/phosphorylated Stat1 homodimers, and inhibit their DNA-binding activity. The binding of PIAS1 however, as demonstrated in the same overexpression systems, was restricted to homodimeric Stat1 while non-phosphorylated Stat1 monomers remained unbound. Furthermore, the binding of PIAS1 was restricted to Stat1, even in the presence of activated Stat2 or Stat3. A search of EST sequence databases using the sequence for PIAS1 and a cDNA library screen revealed four additional family members, PIAS3, PIASxa, PIASxb and PIASy (Chung et al., 1997; Liu et al., 1998). The members of the PIAS family of proteins demonstrate a significant degree of homology (50%), and have a strong conservation of sequence

across species (murine to human). Thus far, several putative functional domains have been defined within the PIAS proteins (Figure 4.4), including a conserved LXXLL (nuclear receptor) motif, a Zn finger domain, a highly acidic region, and a serine/threonine rich region. The sequences of the PIAS proteins are the most divergent at their C-termini, where the acidic and serine/threonine rich regions are located. PIASxa and PIASxb, mentioned above, are likely splice variants, as they differ solely in their C-termini (Liu et al., 1998). Interestingly, PIASy lacks the C-terminal serine/threonine-rich domain, although the functional relevance of this domain remains to be determined. The LXXLL motif contained in PIASy is required for the transrepressional activity of PIASy against Stat1, although this motif does not prevent the DNA-binding activity of Stat1 (Liu et al., 2001). The nine amino acids located at the extreme N-terminus of PIAS1 are also thought to be important for function, as a previously described sequence designated as GBP (for Gu binding protein) is identical to PIAS1 save for a deletion at the N-terminus, and a few additional amino acid differences (Liu et al., 1998; Shuai, 2000). PIAS3 has been reported to be expressed in a broad range of tissues and cell types. It interacts specifically with phosphorylated Stat3 in cells that have been stimulated with cytokines such as IL-6, CNTF and OSM (Chung et al., 1997). Furthermore, in a manner analogous to its counterpart PIAS1, PIAS3 blocks both the DNA-binding activity and subsequent geneactivation mediated by Stat3. Interestingly enough, even within the context of IL-6 stimulation, which results in the phosphorylation of not only Stat3 but Stat1 as well, PIAS3 interacts only with Stat3 and Stat1/3 heterodimers. Among the major drivers of the transcription of

Nuclear receptor motif



Zn finger domain

Acidic region

Serine/Threonine rich region

LXXLL Stat1 interaction (PIAS1)

FIGURE 4.4 Generic domain organization of PIAS proteins, with domains of putative functional importance.

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SOCS genes are the activated Stats, it will be of great interest to see what the effect of PIAS1 and/or PIAS3 will be on the transcription of the SOCS genes – in particular SOCS1 and SOCS3. Presumably, as the mounting experimental evidence indicates, the ability of PIAS proteins to modulate transcriptional activity will be extended beyond the strictly defined JAK-Stat signaling schemes into other signaling cascades. For example, a fragment of PIASxb, designated as Miz1, was demonstrated to interact with the homeobox DNA binding protein Msx2, which incidentally is a modulator of BMP-4 signaling (Gomes and Kessler, 2001). It is therefore likely that the activities of PIAS proteins are more extensive than initially believed.

CONCLUSIONS How interesting and ultimately conservative of nature it is that so much can be accomplished with so little. The majority of interleukin, interferon and growth factor early signal transduction schemes depend heavily upon the actions of the Jak and Stat families of proteins. More striking perhaps, is the fact that so much diversity of response is generated by the specific combinations in which Jaks, Stats and specific receptors interact. On the other hand reside the SOCS and PIAS families of proteins, the majority of functions of which remain to be characterized. Already it is becoming apparent that these proteins are potent negative regulators of Jak-Stat signaling schemes, being highly specific in their protein–protein interactions – regardless of the signaling scheme involved. It will be of intense interest to observe as additional biological functions of these latter families of proteins are elucidated. The purpose of this review has been to emphasize some of the key protein regulators of cytokine signaling, including those responsible for initiating intracellular cascades, as well as those which dampen them. Although a comprehensive review would have included a discussion of the action of specific phosphatases (e.g. SHP-1 and SHP-2), as well as an overview of cytokine receptor turnover, those topics were consciously omitted from this discussion, and the reader is directed to other excellent reviews on those topics (Greenhalgh and Hilton, 2001; Gadina et al., 2001).

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Contribution of Stat SH2 groups to specific interferon signaling by the Jak-Stat pathway. Science 267, 1347–1349. Hilton, D.J., Richardson, R.T., Alexander, W.S. et al. (1998). Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc. Natl Acad. Sci. USA 95, 114–119. Horvat, S. and Medrano, J.F. (2001). Lack of SOCS2 expression causes the high-growth phenotype in mice. Genomics 72, 209–212. Horvath, C.M. and Darnell, J.E. (1997). The state of the STATs: recent developments in the study of signal transduction to the nucleus. Curr. Opin. Cell Biol. 9, 233–239. Horvath, C.M., Wen, Z.L. and Darnell, J.E. (1995). A Stat protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. Genes Dev. 9, 984–994. Hou, J., Schindler, U., Henzel, W.J. et al. (1994). An interleukin-4-induced transcription factor: IL-4 Stat. Science 265, 1701–1706. Huang, L.J., Constantinescu, S.N. and Lodish, H.F. (2001). The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. Mol. Cell 8, 1327–1338. Imada, K., Bloom, E.T., Nakajima, H. et al. (1998). Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity. J. Exp. Med. 188, 2067–2074. Kagami, S.-I., Nakajima, H., Kumano, K. et al. (1999). Both Stat5a and Stat5b are required for antigen-induced eosinophil and T-cell recruitment into the tissue. Blood 95, 1370–1377. Kamura, T., Sato, S., Haque, D. et al. (1998). The Elongin BC complex interacts with the conserved SOCS box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev. 12, 3872–3881. Kaplan, M.H., Schindler,U., Smiley, S.T. and Grusby, M.J. (1996a). Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4, 313–319. Kaplan, M.H., Sun, Y.L., Hoey, T. and Grusby, M.J. (1996b). Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382, 174–177. Karaghiosoff, M., Neubauer, H., Lassnig, C. et al. (2000). Partial impairment of cytokine responses in Tyk2deficient mice. Immunity 13, 549–560. Lawless, V.A., Zhang, S., Ozes, O.N. et al. (2000). Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways. J. Immunol. 165, 6803–6808. Leonard, W.J. (2001). Role of Jak kinases and STATs in cytokine signal transduction. Int. J. Hematol. 73, 271–277. Leonard, W.J. and O’Shea, J.J. (1998). JAKS and STATS: biological implications. Ann. Rev. Immunol. 16, 293–322. Lin, J.-X. and Leonard, W.J. (2000). The role of Stat5a and Stat5b in signaling by IL-2 family cytokines. Oncogene 19, 2566–2576. Lin, J.-X., Mietz, J., Modi, W.S. et al. (1996). Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells. J. Biol. Chem. 271, 10738–10744. Liu, B., Liao, J., Rao, X. et al. (1998). Inhibition of Stat1mediated gene activation by PIAS1. Proc. Natl Acad. Sci. USA 95, 10626–10631. Liu, B., Gross, M., ten Hoeve, J. and Shuai, K. (2001). A transcriptional corepressor of Stat1 with an essential LXXLL signature motif. Proc. Natl Acad. Sci. USA 98, 3203–3207. Liu, X., Robinson, G.W., Gouilleux, F. et al. (1995). Cloning

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Nakajima, H., Liu, X.W., Wynshaw-Boris, A. et al. (1997). An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor a chain induction. Immunity 7, 691–701. Narazaki, M., Fujimoto, M., Matsumoto, T. et al. (1998). Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin-6 signaling. Proc. Natl Acad. Sci. USA 95, 13130. Neubauer, H., Cumano, A., Müller, M. et al. (1998). Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93, 397–409. Nicholson, S.E., Willson, T.A., Farley, A. et al. (1999). Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 18, 375–385. Nicholson, S.E., DeSouza, D., Fabri, L.J. et al. (2000). Suppressor of cytokine signaling-3 preferentially binds to the SHP-2 binding site on the shared cytokine receptor subunit gp130. Proc. Natl Acad. Sci. USA 97, 6493–6498. Nicola, N.A. and Greenhalgh, C.J. (2000). The suppressors of cytokine signaling (SOCS) proteins: Important feedback inhibitors of cytokine action. Exp. Hematol. 28, 1105–1112. Noguchi, M., Yi, H., Rosenblatt, H.M. et al. (1993). Interleukin-2 receptor c chain mutation results in Xlinked severe combined immunodeficiency in humans. Cell 73, 147–157. Nosaka, T., van Deursen, J.M., Tripp, R.A. et al. (1995). Defective lymphoid development in mice lacking Jak3. Science 270, 800–802. Ohya, K., Kajigaya, S., Yamashita, Y. et al. (1997). SOCS-1/ JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase. J. Biol. Chem. 272, 27178–27182. Palmiter, R.D., Norstedt, G., Gelinas, R.E. et al. (1983). Metallothionein-human GH fusion genes stimulate growth of mice. Science 222, 809–814. Parganas, E., Wang, D., Stravopodis, D. et al. (1998). Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93, 385–395. Park, C., Li, S., Cha, E. and Schindler, C. (2000). Immune response in Stat2 knockout mice. Immunity 13, 795–804. Park, S.Y., Saijo, K., Takahashi, T. et al. (1995). Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3, 771–782. Patel, B.K., Pierce, J.H. and LaRochelle, W.J. (1998). Regulation of interleukin-4-mediated signaling by naturally occurring dominant negative and attenuated forms of human Stat6. Proc. Natl Acad. Sci. USA 95, 172–177. Rane, S.G. and Reddy, E.P. (2000). Janus kinases: components of multiple signaling pathways. Oncogene 19, 5662–5679. Richter, M.F., Dumenil, G. Uze, G. et al. (1998). Specific contribution of Tyk2 JH regions to the binding and expression of the interferon a/b receptor component IFNAR1. J. Biol. Chem. 273, 24723–24729. Rodig, S., Meraz, M.A., White, J.M. et al. (1998). Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 93, 373–383. Ruff-Jamison, S., Chen, K. and Cohen, S. (1993). Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science 261, 1733–1736.

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5 Growth hormone Lisbeth A. Welniak and William J. Murphy NCI-Frederick and Intramural Research Support Program, SAIC, NCI-Frederick, Frederick, MD, USA

Science is wonderfully equipped to answer the question ‘How?’ but it gets terribly confused when you ask the question ‘Why?’. Erwin Chargraff

INTRODUCTION

GENE STRUCTURE

Growth hormone (GH), also known as somatotropin, is a molecule with diverse action due in part to the widespread distribution of its receptor. As implied in its name, GH can stimulate the growth and differentiation of muscle, bone and cartilage (Daniels and Martin, 1991). As early as the 1920s pituitary extracts were used to elicit the growth promoting effects in rats and dogs that were later ascribed to GH (Tattersall, 1996). In addition to the pituitary gland, GH is produced in many other tissues. This suggests that in addition to its function as a classic endocrine hormone, GH may also function in an autocrine/ paracrine fashion. In addition to direct receptormediated activity, GH stimulates the production of insulin-like growth factors (IGFs) that mediate many growth promoting actions of the hormone.

GH (GenBank Accession No. J00148 K00612); (Martial et al., 1979) is a member of a family of growth factors with an approximate size of 22 kDa. Other family members include prolactin, placental lactogens, proliferins and somatolactin. GH is also a member of the four helical bundle gene hematopoietic super-family that comprises most of the cytokines and hematopoietic growth factors. In man, the GH gene (GH-N (normal)) is located in a cluster of five closely related genes that spans 47 kb. The other genes in this cluster on chromosome 17 are expressed in the placenta and include human GH-variant (hGHV) and three placental lactogen genes: choriomammosomatotropin A, B and L (CSA, CSB, CSL) (Chen et al., 1989). The genes appear to have arisen from gene duplication as the five genes in the human GH gene cluster have greater similarity among themselves than human GH and non-primate GH (Wallis, 1996). In non-primates, GH

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occurs as a single gene. There has been a large evolutionary change not only in the gene cluster, but also in the primary structure of GH (Ohta, 1993; Wallis, 1994). Between humans and rhesus monkeys, pituitary GH is 95% and 96% identical at the mRNA and protein levels, respectively (Golos et al., 1993). Between mammalian species, bovine GH is more closely related to rat GH than human GH: 83.9% and 83.5% versus 76.5% and 66.8% at the mRNA and protein levels, respectively (Miller et al., 1980).

scription of GH is dependent on a cascade of tissue specific transcription factors (Sornson et al., 1996). Mutations in the pit-1 or prop-1 result in combined pituitary hormone deficiency (Camper et al., 1990; Radovick et al., 1992; Sornson et al., 1996). Pit-1 gene expression is also found in lymphoid, thymic and myeloid cells that express GH (Chen et al., 1997; Delhase et al., 1993; Kooijman et al., 1997a, 1997b) although GH expression in these cells in not always dependent on pit-1 expression (Kooijman et al., 1997b).

GENE REGULATION GH PROTEIN High levels of GH-N gene transcription occur in the somatotroph cells of the anterior pituitary (Wells and de Vos, 1993). The gene is also transcribed in lymphoid and myeloid cells (Binder et al., 1994, Palmetshofer et al., 1995, Rohn and Weigent, 1995). The human GH-N gene is interrupted by four intervening sequences. One of the intervening sequences has two different splice sites. These alternate splice sites account for the production of different peptide sizes (DeNoto et al., 1981). The cloned human GH-N cDNA consists of 29 nucleotides in the 5 untranslated region, 651 nucleotides code for the pre-hormone that includes a signal peptide followed by 108 nucleotides in the 3 untranslated region (Martial et al., 1979). Upstream of the hGH-N gene TATA box are binding sites for NF-1, AP-2, USF and Sp1 (Rousseau, 1992). The human pituitary-specific transcription factor POU1F1 (homolog to murine pit-1/GHF-1) regulates the expression of pituitary GH, prolactin and thyroid stimulating hormone (Li et al., 1990). Mutations in pit-1/GHF-1 lead not only to a loss of expression of the hormones, but also hypoplasia in the pituitary cells responsible for its production (Li et al., 1990). Pit-1/GHF-1 transcription is autoregulated and can be enhanced by downstream events following GHreleasing hormone binding to its receptor. Other hormones can regulate GH gene expression. The GH gene contains binding sites for the thyroid hormone receptor, the glucocorticoid receptor and the retinoic acid receptor (Barlow et al., 1986; Rousseau et al., 1987; Bedo et al., 1989). Mutations resulting in the loss of murine prop-1 (prophet of pit-1) expression demonstrate that tran-

GH is a non-glycoslated polypeptide composed of 191 amino acids (Wallis, 1992). It is a member of the hematopoietic growth factor super-family based on its structural characteristics including an anti-parallel 4-alpha helix bundle fold. The major forms of circulating growth hormone are 20 kDa and 22 kDa monomers and dimers (Soman and Goodman, 1977; Lewis et al., 1977; Baumann et al., 1986). Dimerization is both covalent and non-covalent (Soman and Goodman, 1977; Lewis et al., 1977). Production of GH is controlled by the hypothalamic hormones, growth hormone releasing hormone (GHRH), hypothalamic growth hormone releaseinhibiting factor, and somatostatin (Frohman et al., 1992). GH is controlled by negative feedback regulation of GHRH production (Frohman et al., 1992; Kamegai et al., 1998) and positive feedback regulation of somatostatin production (Zheng et al., 1997). Secretion of the hormone is pulsatile and the highest levels are observed at night. A coordinate response of GHRH, somatostatin and GH control GH pulses (Thorner et al., 1990; Ocampo-Lim et al., 1996). GH secretion is decreased in obesity and rises during starvation. GH levels vary with age. The highest concentrations of circulating GH are detected in the immediate neonatal period. The hormone levels decrease during childhood and then rise again during puberty. In adults, GH secretion falls sharply and continues to decline with age (Figure 5.1). Secretion of GH is also regulated through the GH/IGF-1 axis. IGF-1, also known as somatomedin C, is produced in high concentration in the liver, and at lower concentrations in a variety of other tissues, in

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may occur due to the presence of GH, GH receptor, IGF-1 and IGF-1 receptor in tissues, such as the thymus. Thymic epithelial cells also produce IGF-1 in response to GH that may result in an autocrine loop as IGF-1 stimulates cell growth of the same cells (Timsit et al., 1992; Tsuji et al., 1994; Lin et al., 1997). Rat and human leukocytes have been shown to produce IGF-1 after in vitro stimulation with GH (Geffner et al., 1990; Baxter et al., 1991) and cells of the bone marrow microenvironment express both IGF-1 and its receptor (Landreth et al., 1992; Thomas et al., 1999) (Figure 5.2). The major source of circulating GH is the pituitary. However, GH is produced by other cells including normal lymphocytes and myeloid cells (Weigent et al., 1988; Weigent and Blalock, 1990; Hattori et al., 1990; Varma et al., 1993). GH production has been demonstrated in human peripheral blood lymphocytes (Weigent et al., 1988; Hattori et al., 1990; Varma et al., 1993), rat lymphocytes and macrophages (Weigent and Blalock, 1990). GH mRNA transcripts have been

Somatostatin

GHRH

Hypothalamus

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Liver

FIGURE 5.1 Diagram of pituitary GH control and feedback.

response to GH. Liver-derived IGF-1 participates in the regulation of GH secretion by the pituitary, but it is not necessary for normal growth and development (Yakar et al., 1999). Localized tissue regulation of GH

CD4+ αβTCR

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Exit to periphery

+

GH/IGF-1

Thymulin

FIGURE 5.2 Potential role of GH and IGF-1 in thymopoiesis. Local and pituitary sources of GH may contribute to GH/IGF-1 stimulation of thymopoiesis. Possible targets for the pro-thymopoietic effects are illustrated. THE CYTOKINES AND CHEMOKINES

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detected in human neutrophils (Kooijman et al., 1997a). One of the primary actions of GH is the production of insulin-like growth factor (IGF-1). IGF-1 is responsible for many of the activities attributed to GH, including stimulation of growth. In addition to the activity associated with IGF-1, GH also has direct effects on lipolysis, increased amino acid transport into cells and increased protein synthesis, as well as lactogenic effects through the engagement of the prolactin receptor.

Disease states Deficiency in growth hormone production is associated with dwarfism and a reduction of lean body mass. It is also associated with abnormal glucose homeostasis. Overproduction of GH results in acromegaly and giantism. Administration of GH can result in changes in fluid retention, carbohydrate metabolism and thyroid function (Ho et al., 1989) and may accelerate scolioisis (Clayton and Cowell, 2000). Benign intracranial hypertension is a complication of GH treatment (Malozowski et al., 1993, 1995). Overdose of GH can result in insulin resistance, joint pain, edema, hypertension and acromegalic features. Overexpression of GH is associated with some breast tumors (Tornell et al., 1991). There may be a slight increase in the incidence of leukemia associated with GH treatment in children (Stahnke and Zeisel, 1989).

GH RECEPTORS The GH receptor, also known as the somatogenic receptor, is a single transmembrane glycoprotein. The human protein was cloned from the liver (GenBank Accession No. X06562), the 620 amino acid molecule consists of a signal sequence, an extracellular, transmembrane and intracellular domain. The GH receptor extracellular domain functions as a binding protein and is found in the serum complexed to GH (Leung et al., 1987). The production of the binding protein varies with species. Rodent GH binding protein is derived from alternate splicing and the human GH binding protein arises from proteolysis of the fulllength receptor (Wells and de Vos, 1993).

Similar to the evolutionary changes in GH, a large number of amino acid substitutions (64–65 from the consensus or ‘ancestral’ mammalian GH) have occurred between primate and non-primate GH receptor (Wallis, 1994). Human GH binds the GH receptor from other mammalian species and will stimulate growth in non-primates following administration. However, GH from non-primate mammals is ineffective in humans. Souza and colleagues demonstrated that bovine GH is 3000-fold less potent than human GH in competitive binding assays for the human GH receptor (Souza et al., 1995). The substitution of an arginine for a leucine at position 43 in the primate GH receptor accounts for the specificity along with coordinate changes in the GH protein (Souza et al., 1995). Primate GH binds tightly to and activates both the GH and prolactin receptors (Cunningham et al., 1990; Fu et al., 1992). GH derived from non-primates does not activate PRL receptors. GH and the prolactin (PRL) receptors are members of the hematopoietic receptor family. The family is characterized by overall homology and characteristic motifs including two cysteine pairings and the WSXWS box in the extracellular domain. Limited homology is observed in the intracellular domain among members of the hematopoietic receptor family (Colosi et al., 1993). Similar to other member of the receptor family, engagement of the GH and PRL receptors activates JAK2 (Argetsinger et al., 1993). GH receptor can also signal through the protein kinase C pathway (Doglio et al., 1989). GH has been co-crystallized with either the GH (de Vos et al., 1992) or PRL receptor (Somers et al., 1994). GH binds the GH receptor in a 1:2 complex (de Vos et al., 1992). Binding is sequential as GH binds first one GH receptor to form a dimer and then binds a second GH receptor through a second site (Cunningham et al., 1991). Dimerization of the receptors permits intracellular components to signal (Wells and de Vos, 1993). Likewise, GH binds the PRL receptor in a 1:2 ratio. Binding of primate GH to the PRL receptor is greatly enhanced by zinc (Cunningham et al., 1990). The GH receptor is expressed at high levels in liver and adipose tissues, although it can also be detected in other tissues in rodents, including intestine, brain, testis, heart and skeletal muscle (Kelly et al., 1993).

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GH RECEPTORS

Both GH and IGF-1 receptors are found in the various cell types in the skin (Tavakkol et al., 1992; Oakes et al., 1992). GH and IGF-1 receptors have been demonstrated in the lymphoid tissues of a number of mammalian species (Kiess and Butenandt, 1985; Hull et al., 1996; Clark, 1997; de Mello-Coelho et al., 1997; Chen et al., 1998; Dardenne et al., 1998). In the thymus, the GH receptor has been found on murine CD4CD8 and CD8 thymocytes, as well as thymic epithelial cells (Ban et al., 1991; Timsit et al., 1992; Gagnerault et al., 1996). The receptor is found at variable levels on all hematopoietic lineages in the bone marrow and on subsets of B cells, CD4 and CD8 T cells and macrophages in the secondary lymphoid tissues (Badolato et al., 1994; Rapaport et al., 1995). The PRL receptor is co-expressed on all GH receptor positive peripheral T cells (Gagnerault et al., 1996; Dardenne et al., 1998). Cell surface expression of GH receptor is increased with cell activation of murine or bovine lymphoid cells (Gagnerault et al., 1996; Postel-Vinay et al., 1997; Dardenne et al., 1998). In secondary lymphoid tissue, human B cells constitutively express GH receptors, but the receptor was observed only on activated T cells (Thellin et al., 1998). Studies have shown that NK cell numbers and activity in patients with GH deficiency are depressed suggesting a role for GH in NK cell biology (Kiess et al., 1988; Span et al., 1996). However, the GH receptor has not been demonstrated on NK cells and the activity may be due to engagement of the PRL receptor, which has been demonstrated on rat NK cells (Chambers et al., 1995).

GH and immune function The presence of GH and IGF receptors on many types of immune cells, as well as the production of GH and IGF in bone marrow lymphoid tissues suggest a localized GH/IGF axis and role for GH in immune function. The importance of GH for immune development and function has remained controversial. With the exception of reduced NK cell activity, no consistent alterations in immune development or function have been ascribed to patients with GH deficiency (Kiess et al., 1988; Span et al., 1996). Studies in rodents had suggested that the absence of the anterior pituitary and its hormones

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resulted in T and NK cell immune deficiency that could be corrected with GH administration (Berczi et al., 1981, Saxena et al., 1982, Nagy et al., 1983, Nagy and Berczi, 1989, Murphy et al., 1992a, 1992c). However, studies that are more recent have questioned the reproducibility of these findings (MontecinoRodriguez et al., 1997; Dorshkind and Horseman, 2000). Differences in animal husbandry may account for the discrepancy. While neither GH or IGF-1 are essential for immune cell development or function, there is an extensive literature base demonstrating enhancement of immune function through either direct administration, stimulation of production in vivo, or in vitro immune cell development and functional assays. Administration of IGF-1, GH or GH-stimulating molecules increases thymus size and thymocyte numbers (Beschorner et al., 1991; Murphy et al., 1992c; Montecino-Rodriguez et al., 1998; Koo et al., 2001). IGF-1 administration promotes B cell development in vivo and in vitro (Landreth et al., 1992; Jardieu et al., 1994), although GH administration can result in a transit decrease in B cell numbers in children (Wit et al., 1993; Rapaport and Bozzola, 1997). GH effects are not limited to lymphopoiesis. Either directly or indirectly, GH and or IGF-1 have activity on myeloid (Murphy et al., 1992d; Blazar et al., 1995) and erythroid progenitor (Golde et al., 1977; Merchav et al., 1988; Ratajczak et al., 1998) growth or survival. Both GH and IGF-1 treatment promote myelopoietic recovery following bone marrow transplants in rodents (Tsarfaty et al., 1994; Tian et al., 1998). A summary of the immunomodulatory activities of GH and IGF-1 are presented in Table 5.1 and Figure 5.3. It has been suggested that the primary mechanism by which GH influences immune function is through the promotion of growth and survival of GH receptor and/or IGF-1 receptor bearing cells (Dorshkind and Horseman, 2000). IGF-1 has been shown to prevent apoptosis of myeloid cells (Liu et al., 1997; Kelley et al., 1998). GH may also act as a stress-modulator through the activation of the PRL receptor (Dorshkind and Horseman, 2000). Induction of corticosteroids in stress responses may be countered by GH, but this hypothesis has yet to be proven. GH administration overcomes glucocorticoid suppression of growth and weight gain without reversing the immunosuppressive effects of methylprednisolone

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TABLE 5.1 Effects of GH and IGF-1 on immune cell function Cell or tissue

Activity

Reference

Thymus T Cells

Stimulates thymulin production Increased CTL activity Increased IFN-c production Increased proliferation Promotes adhesion, migration and engraftment Increased proliferation Enhanced Ig production of specific isotypes

Snow et al., 1981 Benfield et al., 1997 Kooijman et al., 1992 Taub et al., 1994; Murphy et al., 1992b Taub et al., 1994; Murphy et al., 1992b Kimata and Yoshida, 1994 Kimata and Yoshida, 1994; Kimata and Fujimoto, 1994 Edwards et al., 1991b; Warwick-Davies et al., 1995 Edwards et al., 1988 Edwards et al., 1991a Wiedermann et al., 1993 Saxena et al., 1982; Crist and Kraner, 1990 Wiedermann et al., 1991 Wiedermann et al., 1991

B Cells Macrophages

Resistance to Salmonella infection Prime for superoxide production Increased TNFa production Chemotaxis Increased number and activity Cell priming Increased adhesion

NK cells Granulocytes

NK Cell GH/IGF-1

Cytotoxicity

GH/IGF-1

CTL Activity IFNγ Production

GH/IGF-1

CD8 + Cell

Antigen Processing

GH/IGF-1

IL-2

CD4 + Cell

TCR

Inflammatory Cascade

Cytokines

Proliferation

APC

CD4 + Cell

Class II + Ag IL 4

GH/IGF-1

B Cell Proliferation

Mϕ IG F-1 GH/IGF-1 PMN

Ig Switch Reactive oxygen intermediates

B Cell

GH/IGF-1

GH Bacteria

Ig Production

(Ortoft et al., 1998). Additional studies are needed to determine if thymic sensitivity to physiological levels of glucocorticoids can be overcome with GH adminstration.

FIGURE 5.3 Potential role of GH and IGF-1 on immune function. GH and/or IGF-1 can act on a wide variety of immune cells to enhance both innate and adaptive immunity.

GH and skin The presence of GH and IGF-1 along with both GH and IGF-1 receptors suggest a role for GH in the skin.

THE CYTOKINES AND CHEMOKINES

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GH overexpression or deficiency is associated with excess or deficiencies in sweating, respectively. Treatment with GH corrects the hypohydrosis in GHdeficient patients (Juul et al., 1995). In vitro, GH increases collagen synthesis and skin strength (Jorgensen et al., 1995; Pierre et al., 1997). GH receptor is expressed in melanocytes (Tavakkol et al., 1992) and is up-regulated in melanoma (Lincoln et al., 1999). Trials have suggested accelerated wound healing in patients receiving GH therapy (Gilpin et al., 1994, Herndon et al., 1995) and healing of skin grafts in animal models (Ghofrani et al., 1999).

SUMMARY The structure and function of growth hormone and its receptors have been intensely studied. These studies have contributed greatly to our understanding of receptor activation and signaling. Over the years, we have learned a great deal of how growth hormone exerts its action. However, much is yet to be learned about why the hormone is produced in a diverse array of tissues and cell types, why the receptors are found on an equally wide variety of cells and what function does this system serve?

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Rapaport, R., Sills, I.N., Green, L. et al. (1995). Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: correlation with growth hormone-binding protein levels. J. Clin. Endocrinol. Metab. 80, 2612–2619. Rapaport, R.G. and Bozzola, M. (1997). Role of B-cells in growth hormone-immune interactions. Acta. Paediatr. Suppl. 423, 82–83. Ratajczak, J., Zhang, Q., Pertusini, E. et al. (1998). The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions – comparison to other cytokines and growth factors. Leukemia 12, 371–381. Rohn, W.M. and Weigent, D.A. (1995). Cloning and nucleotide sequencing of rat lymphocyte growth hormone cDNA. Neuroimmunomodulation 2, 108–114. Rousseau, G.G. (1992). Growth hormone gene regulation by trans-acting factors. Horm. Res. 37, 88–92. Rousseau, G.G., Eliard, P.H., Barlow, J.W. et al. (1987). Approach to the molecular mechanisms of the modulation of growth hormone gene expression by glucocorticoid and thyroid hormones. J. Steroid Biochem. 27, 149–158. Saxena, Q.B., Saxena, R.K. and Adler, W.H. (1982). Regulation of natural killer activity in vivo. III. Effect of hypophysectomy and growth hormone treatment on the natural killer activity of the mouse spleen cell population. Int. Arch. Allergy Appl. Immunol. 67, 169–174. Snow, E.C., Feldbush, T.L. and Oaks, J.A. (1981). The effect of growth hormone and insulin upon MLC responses and the generation of cytotoxic lymphocytes. J. Immunol. 126, 161–164. Soman, V. and Goodman, A.D. (1977). Studies of the composition and radioreceptor activity of ‘big’ and ‘little’ human growth hormone. J. Clin. Endocrinol. Metab. 44, 569–581. Somers, W., Ultsch, M., De Vos, A.M. and Kossiakoff, A.A. (1994). The X-ray structure of a growth hormoneprolactin receptor complex. Nature 372, 478–481. Sornson, M.W., Wu, W., Dasen, J.S. et al. (1996). Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 384, 327–333. Souza, S.C., Frick, G.P., Wang, X. et al. (1995). A single arginine residue determines species specificity of the human growth hormone receptor. Proc. Natl Acad. Sci. USA 92, 959–963. Span, J.P., Pieters, G.F., Smals, A.G. et al. (1996). Number and percentage of NK-cells are decreased in growth hormone-deficient adults. Clin. Immunol. Immunopathol. 78, 90–92. Stahnke, N. and Zeisel, H.J. (1989). Growth hormone therapy and leukaemia. Eur. J. Pediatr. 148, 591–596. Tattersall, R. (1996). A history of growth hormone. Horm. Res. 46, 236–247. Taub, D., Tsarfaty, G., Lloyd, A. et al. (1994). Growth hormone promotes human T cell adhesion and migration to both human and murine matrix proteins in vitro and directly promotes xenogeneic engraftment. J. Clin. Invest. 94, 293–300. Tavakkol, A., Elder, J.T., Griffiths, C.E. et al. (1992). Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin. J. Invest. Dermatol. 99, 343–349.

Thellin, O., Coumans, B., Zorzi, W. et al. (1998). Expression of growth hormone receptors by lymphocyte subpopulations in the human tonsil. Dev. Immunol. 6, 295–304. Thomas, T., Gori, F., Spelsberg, T.C. et al. (1999). Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts and adipocytes. Endocrinology 140, 5036–5044. Thorner, M.O., Vance, M.L., Hartman, M.L. et al. (1990). Physiological role of somatostatin on growth hormone regulation in humans. Metabolism 39, 40–42. Tian, Z.G., Woody, M.A., Sun, R. et al. (1998). Recombinant human growth hormone promotes hematopoietic reconstitution after syngeneic bone marrow transplantation in mice. Stem Cells 16, 193–199. Timsit, J., Savino, W., Safieh, B. et al. (1992). Growth hormone and insulin-like growth factor-I stimulate hormonal function and proliferation of thymic epithelial cells. J. Clin. Endocrinol. Metab. 75, 183–188. Tornell, J., Rymo, L. and Isaksson, O.G. (1991). Induction of mammary adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice. Int. J. Cancer 49, 114–117. Tsarfaty, G., Longo, D. and Murphy, W. (1994). Human insulin-like growth factor I exerts hematopoietic growthpromoting effects after in vivo administration. Exp. Hematol. 22, 1273–1277. Tsuji, Y., Kinoshita, Y., Hato, F. et al. (1994). The in vitro proliferation of thymus epithelial cells stimulated with growth hormone and insulin-like growth factor-I. Cell. Mol. Biol. (Noisy-le-grand) 40, 1135–1142. Varma, S., Sabharwal, P., Sheridan, J.F. and Malarkey, W.B. (1993). Growth hormone secretion by human peripheral blood mononuclear cells detected by an enzyme-linked immunoplaque assay. J. Clin. Endocrinol. Metab. 76, 49–53. Wallis, M. (1992). The expanding growth hormone/prolactin family. J. Mol. Endocrinol. 9, 185–188. Wallis, M. (1994). Variable evolutionary rates in the molecular evolution of mammalian growth hormones. J. Mol. Evol. 38, 619–627. Wallis, M. (1996). The molecular evolution of vertebrate growth hormones: a pattern of near-stasis interrupted by sustained bursts of rapid change. J. Mol. Evol. 43, 93–100. Warwick-Davies, J., Lowrie, D.B. and Cole, P.J. (1995). Growth hormone is a human macrophage activating factor. Priming of human monocytes for enhanced release of H2O2. J. Immunol. 154, 1909–1918. Weigent, D.A., Baxter, J.B., Wear, W.E. et al. (1988). Production of immunoreactive growth hormone by mononuclear leukocytes. Faseb. J. 2, 2812–2818. Weigent, D.A. and Blalock, J.E. (1990). Immunoreactive growth hormone-releasing hormone in rat leukocytes. J. Neuroimmunol. 29, 1–13. Wells, J.A. and de Vos, A.M. (1993). Structure and function of human growth hormone: implications for the hematopoietins. Annu. Rev. Biophys. Biomol. Struct. 22, 329–351. Wiedermann, C.J., Niedermuhlbichler, M., Geissler, D. et al. (1991). Priming of normal human neutrophils by recombinant human growth hormone. Br. J. Haematol. 78, 19–22.

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6 Prolactin Hallgeir Rui and Marja T. Nevalainen Georgetown University, Lombardi Cancer Center, Washington, DC, USA

If you do not expect the unexpected, you will not find it; for it is hard to be sought out, and difficult. Heraclitus, ca. 550–475 BC

INTRODUCTION History Prolactin was originally described as a distinct anterior pituitary hormone with ‘lactogenic’ activity by Stricker and Grueter (Stricker and Grueter, 1928), and the factor was purified and given the name prolactin shortly thereafter (Riddle and Braucher, 1931; Riddle et al., 1932, 1933). Several sources provide reviews and historical references about this landmark discovery and the ensuing research on prolactin actions in mammals, birds, reptiles, amphibians, and fish (Riddle, 1963; Bern and Nicoll, 1968; Nicoll, 1980). Because human growth hormone, in contrast to growth hormone from non-primate mammals, binds to prolactin receptors and has potent lactogenic activity, it was not until the early 1970s that Friesen’s group and others definitively identified human prolactin as a separate entity (Lewis et al., 1971; Hwang et al., 1972).

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

An essentially complete peptide sequence of human prolactin was first derived by Edman degradation of tryptic fragments (Shome and Parlow, 1977), and the entire amino acid sequence, including a 28 residue signal peptide, was deduced from the nucleotide sequence of a human cDNA cloned by Martial’s laboratory (Cooke et al., 1981). Furthermore, extrapituitary production of prolactin was first detected in decidualized endometrial cells (Riddick et al., 1978), and subsequently in many other reproductive tissues, immune cells and brain. Short and long forms of prolactin receptor were cloned by Kelly’s group in the late 1980s (Boutin et al., 1988, 1989). A prolactin receptor-associated tyrosine kinase activity (Rui et al., 1992) was shortly thereafter identified as Jak2 (Rui et al., 1994a; Lebrun et al., 1994). At the same time, Stat5 was identified by Groner’s group to be a Jak2 substrate and a principal signaling protein for prolactin (Wakao et al., 1994; Gouilleux et al., 1994), as further verified by Stat5 knockout mice

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(Liu et al., 1997, Teglund et al., 1998) which show phenotypic changes overlapping with those of prolactin receptor knockout mice (Ormandy et al., 1997) and prolactin knockout mice (Steger et al., 1998). Due to the widespread expression of prolactin, and the realization that prolactin belongs to a family of tetrahelix bundle proteins that activate a corresponding family of cytokine receptors, prolactin is now regarded as a ubiquitous cytokine with autocrine, paracrine, and endocrine roles.

Main activities and pathophysiological roles of prolactin Prolactin is expressed in mammals, birds, reptiles, amfibians and fish, and has a wide spectrum of effects (Nicoll, 1980). In fact, more than 300 distinct biological activities of prolactin have been recorded (Bern and Nicoll, 1968), in large part due to the ubiquitous expression of prolactin receptors (Bole-Feysot et al., 1998). Prolactin has been referred to as luteotropic hormone, luteotropin, mammotropic hormone and mammotropin. To better reflect the diverse and pleiotropic effects of prolactin, the alternative names versatilin and omnipotin also have been suggested (Bern and Nicoll, 1968). In addition to endocrine effects mediated by pituitary prolactin secretion, it has become increasingly evident that prolactin is synthesized at many extrapituitary sites, particularly in reproductive organs, immune cells and brain (for reviews, see BenJonathan et al., 1996; Freeman et al., 2000). It is therefore clear that prolactin can act as a local paracrine and autocrine factor in diverse tissues and cells. Physiological roles of prolactin can be systematized into reproductive and homeostatic effects. Reproductive processes regulated by prolactin include behavioral (libido, parental behavior), sex steroid production (ovaries, placenta, and testes), gestation (blastocyst implantation, placentation, and embryonal development), and lactation. Homeostatic effects have been described to include adaption to a variety of stressors that may involve ensuring appropriate fluid balance, metabolism, and immune function. These and other biological effects of prolactin will be discussed in detail in later sections.

PROLACTIN GENE Chromosome location of the prolactin gene The human prolactin gene is located on chromosome 6 (Owerbach et al., 1981). Evans and colleagues went on to locate prolactin in the interval 6p22.2–p21.3, distal to HLA-C (Evans et al., 1989). The mouse prolactin gene maps to chromosome 13, clustered with genes encoding mouse placental lactogens and other prolactin-like genes (Jackson-Grusby et al., 1988).

Structure and regulation of the prolactin gene The human prolactin gene spans more than 15 kb and contains six exons (Truong et al., 1984; Berwaer et al., 1994). Transcription of the gene is driven by two tissue-specific promoters, a proximal promoter that is used in the pituitary and a very distal promoter that is used in extrapituitary cells and tissues, including decidua, myometrium, and lymphoid cells (DiMattia et al., 1990; Gellersen et al., 1994; Berwaer et al., 1994). A non-coding exon 1a is only expressed in extrapituitary tissues and has a transcriptional start site 5.8 kb upstream of the pituitary start site (Berwaer et al., 1994). In extrapituitary sites, exon 1a is spliced to the first pituitary exon 1b, generating a transcript that is approximately 150 bp larger than the pituitary counterpart (Gellersen et al., 1989), differing only in the 5-untranslated region. The downstream promoter that directs transcription in pituitary lactotrophs is under control of the POU-homeodomain transcription factor Pit-1. There are two clusters of three and eight Pit-1 binding sites within the pituitary promoter of the human prolactin gene. In addition, there is an AP-1 site (Peers et al., 1990) and a degenerate ERE sequence (Gellersen et al., 1995). Transcriptional control of the distal, nonpituitary start site in endometrial stromal cells is linked to decidual differentiation during the secretory phase of the ovulatory cycle (DiMattia et al., 1990; Gellersen et al., 1994). Two consensus binding sites for CCAAT/enhancer-binding proteins (C/EBP) mediate cAMP/PKA-induced activation of this non-pituitary prolactin gene promoter in human decidual cells (Pohnke et al., 1999). Cyclic AMP, alone or in synergy

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with PHA, also stimulated activation of this upstream prolactin gene promoter in Jurkat T-cells, possibly through activation of C/EBP proteins (Reem et al., 1999).

PROLACTIN PROTEIN Structure of prolactin Processing of a 28 residue signal peptide yields a secreted human prolactin polypeptide of 199 amino acid residues, whereas mouse prolactin is two residues shorter (Cooke, 1989). In both species, six cysteines form three intramolecular disulfide bridges. The molecular weight of human prolactin is approximately 23 kDa, but a 26 kDa glycosylated form is also produced. A three-dimensional (3D) structure of prolactin has not been determined, but extensive modeling and structural analysis of prolactin have been provided by Goffin and colleagues (Goffin et al., 1996b). A similar model of the prolactin molecule based on the 3D structure of growth hormone is shown in Figure 6.1. Prolactin is expected to conform to the tetrahelical cytokine fold with antiparallel helices A, B, C and D forming the molecular core, arranged in an up-updown-down organization (Goffin et al., 1996b). Mutational analyses support this 3D structure and have revealed two distinct interaction sites, site 1 and site 2, that mediate binding to prolactin receptors (Goffin et al., 1996b). Cysteine bonds formed by pairwise coupling of residues Cys4–Cys11, Cys58–Cys174, and Cys191–Cys199 are not shown in Figure 6.1. In addition, a 3D structure of the related polypeptide, ovine placental lactogen, complexed to rat prolactin receptors has been solved (Elkins et al., 2000).

FIGURE 6.1 Ribbon model of the three-dimensional structure of prolactin. Four alpha helices organized in an up-up-down-down configuration make up the core of the molecule (helices A–D). Cysteine bonds are not marked. Prolactin was modeled by Dr Amy Swain (Macromolecular Structure Laboratory, National Cancer Institute, Frederick, MD, USA) based on the crystal structure of human growth hormone (de Vos et al., 1992). For detailed discussion of regions and amino acid residues that represent putative interaction sites with prolactin receptors, see analyses by Goffin et al., 1996a and Bole-Feysot et al., 1998).

Prolactin variants Prolactin circulates in blood as monomers of 23–26 kDa (Lewis et al., 1985). In addition, larger ‘macroprolactins’ (big-prolactin, big-big prolactin) represent both homo-oligomeric aggregates and immunoglobulin-complexed prolactin (Sinha, 1995). In certain asymptomatic subjects with hyperprolactinemia stable circulating complexes between immunoglobulin and prolactin have been identified

(Bonhoff et al., 1995; Hattori and Inagaki, 1998), suggesting that anti-prolactin autoantibodies may occasionally neutralize the activity of hormone. Furthermore, physiological proteolysis of prolactin yields an N-terminal 16 kDa variant with distinct biological activities, and that activates a unique, yet-to-be identified receptor (Mittra, 1980; Clapp and Weiner, 1992; Clapp et al., 1993; D’Angelo et al., 1999).

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Important homologies

Proteolysis

Human prolactin is most homologous to growth hormone (16% amino acid identity) and placental lactogen (13% amino acid identity). In rodents, prolactin is closely related to a series of prolactin-like genes that are expressed in placenta in temporally defined patterns during the course of pregnancy (Table 6.1; for reviews, see Forsyth, 1994; Soares et al., 1998).

Mature prolactin, formed by proteolytic removal of a 28 residue signal peptide, can be further modified by proteases (see review by Sinha, 1995). Cathepsin-D proteolysis at position 133 generates two fragments of 16 and 8 kDa, which may exist both as disulfide-linked heterodimers and as monomers (Mittra, 1980; Cole et al., 1991; Baldocchi et al., 1993). In addition, kallikrein, a trypsin-like protease, cleaves prolactin at position 173 to remove the C-terminal disulfide loop and give rise to a 22 kDa fragment (Anthony et al., 1995). Particular interest has been linked to the antiangiogenic effect of the 16 kDa prolactin fragment (Clapp and Weiner, 1992; Clapp et al., 1993; D’Angelo et al., 1999). The new concept that prolactin, GH and PL each have proangiogenic effects that are reversed to antiangiogenic effects by proteolytic conversion into the corresponding N-terminal 16 kDa fragments is especially intriguing (Struman et al., 1999).

Posttranslational modifications Glycosylation A proportion of pituitary and circulating prolactin is glycosylated in most species. Approximately 20% of circulating human prolactin is glycosylated through N-linkage at position 31 (Lewis et al., 1985; Champier et al., 1987). Mouse prolactin is also glycosylated, but the extent and linkage site remains to be determined (Sinha, 1995). The physiological function of glycosylation of prolactin may be to reduce biological potency, while extending the half-life of the molecule (Hoffmann et al., 1993). TABLE 6.1 Prolactin-like proteins produced by placenta and decidua in rodents Prolactin Placental lactogen I Placental lactogen II Prolactin-like protein-A Prolactin-like protein-B Prolactin-like protein-C Prolactin-like protein-C variant Prolactin-like protein-C alfa Prolactin-like protein-C beta Prolactin-like protein-D Prolactin-like protein-E Prolactin-like protein-F Prolactin-like protein-G Prolactin-like protein-H Prolactin-like protein-I Prolactin-like protein-J Prolactin-like protein-K Prolactin-like protein-L Prolactin-like protein-M Prolactin-related protein Proliferin (1–3) Proliferin-related protein Decidual/trophoblast PRL-related protein (d/t PRP) Adapted from Soares et al., 1998.

Phosphorylation A significant proportion of prolactin molecules are phosphorylated on serine and threonine residues (Oetting et al., 1986). In bovine pituitaries, between 20 and 80% of prolactin was phosphorylated, particularly on Ser90, with Ser26 and Ser34 constituting minor sites (Kim and Brooks, 1993). In rat prolactin, Ser177 was found to be the major site of phosphorylation (Wang et al., 1996), and this site is positionally conserved in prolactin from all species. In general, phosphorylation of prolactin is associated with reduced bioactivity (Wang and Walker, 1993; Brooks and Saiduddin, 1998), but does not appear to affect biological half-life (Brooks and Saiduddin, 1998). The extent of phosphorylation of pituitary prolactin also varied during the estrus cycle in rodents (Ho et al., 1993). Because prolactin is phosphorylated in secretory granules during release from pituitary lactotrophs, it is possible that phosphorylation serves to reduce local bioactivity during secretion. Substitution of the positionally conserved serine residue Ser179 in human prolactin with either Asp or Glu, which are structural mimics of phosphoserine, was associated with reduced bioactivity in both mutants. Indeed, in some assays the Ser179Aspmutant was reported to act as a prolactin antagonist

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(Chen et al., 1998). However, antagonist activity was not detected in phosphorylated bovine prolactin (Brooks and Saiduddin, 1998), and a recent report suggested that the Ser179Asp-mutant rather was an agonist (Bernichtein et al., 2001). Further work is needed to resolve the physiological and pharmacological effects of prolactin phosphorylation.

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TABLE 6.2 Cells and tissues that express prolactin Nervous system Brain Pituitary: Lactotrophs Pituitary: Somatolactotrophs Hypothalamus Pons-medulla Other regions Endocrine system Adrenal Pancreas: Cells of Langerhans islands Urinary system Kidney: Parietal cells

Cellular sources that produce prolactin

Circulatory system Blood vessels: endothelial cells

Prolactin is produced by lactotrophs and somatolactotrophs of the anterior pituitary. These cells constitute between 20 and 50% of cells in the anterior pituitary. In addition, local production of prolactin occurs in a broad range of cell types, particularly in uterus, mammary gland, prostate, cells of the immune system and certain brain regions (Table 6.2). Immortalized pituitary cell lines that secrete prolactin include the widely studied rodent GH3, GH4C1 and MMQ cell lines (Gautvik et al., 1983; Kineman and Frawley, 1994). In addition, the human B-lymphoblastoid IM-9-P3 cell line secretes considerable amounts of prolactin (DiMattia et al., 1988).

Immune system T lymphocytes B lymphocytes NK cells Mononuclear cells Thymic epithelial cells

Stimulatory and inhibitory stimuli A multitude of inhibitors and stimulators of pituitary prolactin secretion have been identified (for review, see Thorner et al., 1998; Freeman et al., 2000). Secretion of prolactin, unlike that of other pituitary hormones, is under tonic inhibition by hypothalamic dopamine. Thyrotropin releasing hormone, vasocative intestinal peptide, and prolactin-releasing peptide are potent stimulatory peptides (Thorner et al., 1998; Hinuma et al., 1998). In addition, estrogens are strong stimulators of pituitary prolactin secretion (Day et al., 1990; Murdoch et al., 1995). Leptin also appears to stimulate pituitary prolactin secretion, an observation with important implications for the role of prolactin in metabolism (Watanobe et al., 2000). In addition, ergot-derivatives (e.g. bromocriptine) act as dopamine agonists and inhibit pituitary prolactin release, whereas many psycopharmaceutical drugs

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Reproductive system (female) Uterus: Decidualized endometrial stromal cells Uterus: Myometrial cells Ovary Mammary: Epithelial cells Reproductive system (male) Testis: Leydig cells Prostate: Epithelial cells Integumentary system: Skin: Dermal fibroblasts Sweat glands Adapted from Ben-Jonathan et al., 1996.

stimulate prolactin secretion. Table 6.3 summarizes the most established regulators of pituitary prolactin secretion (adapted from Thorner et al., 1998; Freeman et al., 2000).

Exogenous and endogenous modulators Steroid hormones frequently modulate the effects of prolactin. For instance, estrogen and progesterone from placenta suppresses the lactogenic effect of pituitary prolactin in the mammary gland, whereas glucocorticoids exert a synergistic effect. Thus, for milk production to begin, the reduction in estrogen and progesterone levels associated with shedding of

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TABLE 6.3 Stimuli that regulate pituitary prolactin secretion Enhancers

Inhibitors

Physiological Pregnancy (estrogen effect) Nursing/Suckling Neurogenic Nipple stimulation/Chest wall trauma/ Breast manipulation/Spinal cord lesions Sleep Stress (hypoglycemia) Exercise Pharmacologic Dopamine antagonists (phenothiazines, haloperidol, metoclopramide, reserpine, methyldopa, amoxapine, opiates) Estrogen Thyrotropin releasing hormone (TRH) Vasoactive intestinal peptide (VIP) Vasopressin Prolactin releasing hormone/peptide Histamine Endogenous opioid peptides Opioids Monamine oxidase inhibitors Cimetidine (intravenous) Verapamil Leptin Pathologic Pituitary tumors Hypothalamic/pituitary stalk lesions Neuraxis irraditation Chest wall lesions Spinal cord lesions Hypothyroidism Chronic renal failure Severe liver disease Seizures

Dopamine agonists (bromocriptine, levodopa) GABA Acetylcholine Somatostatin Prolactin

Pseudohypoparathyroidism Pituitary destruction/removal Lymphocytic hypophysitis

Adapted from Thorner et al., 1998; Freeman et al., 2000.

placenta at parturition is needed. However, interactions between prolactin and steroids are highly dependent on cell type and hormonal milieu. For instance, whereas prolactin is anti-apoptotic in lymphoid cells, and glucocorticoids are proapoptotic, prolactin and glucocorticoids appear to have a synergistic anti-apoptotic effect in differentiated mammary gland.

Disease states In humans, prolactin is important for both physiological and psychological aspects of reproductive func-

tion, and the hormone also affects certain aspects of immune cell function. Evidence for these roles of prolactin in humans has to a large extent been disclosed by symptoms and signs associated with hyperprolactinemia, the most frequent endocrine disturbance of the pituitary. Galactorrhea, anovulation and consequent amenorrhea, decreased libido and impotence are typical effects of chronic hyperprolactinemia (Thorner et al., 1998). On the other hand, as illustrated by hypophysectomy or isolated idiopathic prolactin deficiency, lack of pituitary prolactin in adults is not associated with vital deficiencies beyond fertility problems (Turkington, 1972; Kauppila et al., 1987).

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PROLACTIN RECEPTORS The intact, 22 kDa prolactin molecule binds selectively to high-affinity prolactin receptors. The first prolactin receptor to be cloned was the short isoform from rat liver (Boutin et al., 1988), and long rat and human isoforms were identified shortly thereafter from breast and ovarian cells (Boutin et al., 1989).

Long

C-C

C-C

C-C

D1

C-C

Intermediate "Nb2-like" C-C

D1

C-C

Short "S1b" C-C

D1

PRLbp (soluble)

C-C

C-C

D1

C-C

D1

D2

D2

D2

D2

D2

WSXWS

WSXWS

WSXWS

WSXWS

WSXWS

TM

TM Box 1

Y237

TM Box 1

Y237

Box 2 Y283

Box 2 Y283

Y351

Y346 Y352

Y290 Y318

Y290 Y318

Box 1

206

TM Y237

Box 2 Y283

Y290

Box 1

288

Y237

Y283

325

Y381

Human prolactin receptor gene – Chromosome location and splice variants The human prolactin receptor gene is located on chromosome 5 (p13–14) and contains at least 11 exons for an overall length 100 kb (Arden et al., 1990; Hu et al., 2001). Several splice variants of human prolactin receptors have been identified and the resulting proteins are presented in Figure 6.2. A long form of human prolactin receptor contains 622 amino acid residues (Boutin et al., 1989). One of the human intermediate receptor forms contains 325 residues and its cytoplasmic domains results from deletion of codons corresponding to approximately 200 cytoplasmic amino acids and an associated frameshift that yields a unique C-terminal fragment of 13 residues (Kline et al., 1999). This first intermediate isoform structurally resembles the prolactin receptor characterized in rat Nb2 cells (Ali et al., 1991), and is able to activate tyrosine kinase Jak2 (Kline et al., 1999). A second intermediate human prolactin receptor isoform, S1a, and a short isoform S1b that both include skipping of exon 10 and incorporate sequence encoding 39 and three amino acid residues, respectively, from a newly described exon 11 (Hu et al., 2001). The S1a isoform appears to be less stable and perhaps of lesser biological importance than the S1b isoform. Both S1a and S1b are able to suppress signal transduction to the b-casein promoter by the long receptor form in cotransfection assays (Hu et al., 2001). In addition to several transmembrane prolactin receptor isoforms, an alternatively spliced mRNA encoding a soluble human prolactin-binding protein (PRLbp) has been reported (Fuh and Wells, 1995). Soluble prolactin binding proteins may also arise by proteolysis of membrane-anchored prolactin receptor, but no specific protease has been characterized (Kline and Clevenger, 2001). It is usually

Intermediate "S1a"

Y406

376

Y485

Y522

Y587

622

FIGURE 6.2 Overview of splice variants of human prolactin receptors. The extracellular domain of prolactin receptor variants presented is comprised of two fibronectin III-like subdomains D1 and D2. D1 contains two pairs of disulfide-bonded cysteines (C–C) and subdomain D2 contains the WS-motif, a hallmark of the cytokine receptor superfamily. Homology Box1 is another feature of cytokine receptors and is found in the cytoplasmic domain of all transmembrane proactin receptor isoforms. The long human prolactin receptor (Boutin et al., 1989), the intermediate ‘Nb2–like’ form (Kline et al., 1999), the intermediate S1a and short S1b (Hu et al., 2001), as well as a soluble prolactin binding protein, PRLbp (Fuh and Wells, 1995), are presented. The intermediate form described by Clevenger’s laboratory has been tentatively presented as ‘Nb2-like’ isoform, due to its structural resemblance to the rat intermediate Nb2 cell prolactin receptor isoform (Ali et al., 1991), in order to distinguish it from the S1a intermediate form. Tyrosine residues within the cytoplasmic domains are indicated.

assumed that the formation of prolactin/PRLbp complexes enhances the half-life of circulating prolactin. Moreover, since activation of the prolactin receptor occurs by ligand-induced dimerization, such complexes may interfere with signaling through transmembrane prolactin receptor by forming nonproductive heterodimers (Lesueur et al., 1993). Finally, preliminary work has identified a transcript that may encode a human prolactin receptor ΔS1 that lacks a significant portion of the extracellular domain (Kline et al., 1999). This is of significance due

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to description of constitutive activation of prolactin receptors following genetic removal of portions of the extracellular domain, particularly the D2 subdomain (Gourdou et al., 1996; Lee et al., 1999).

Rodent prolactin receptor genes – splice variants The mouse prolactin receptor gene (Davis and Linzer, 1989) spans 120 kb and is located within a cluster of cytokine receptor loci on chromosome 15 (p12–13) (Gearing et al., 1993). At least seven transcripts that encode four different prolactin receptor isoforms have been described (Buck et al., 1992). The mouse prolactin receptor gene contains 13 exons. The first nine exons are common to all mouse prolactin receptor isoforms. Exon 10 encodes the cytoplasmic tail of the long isoform (exon 10), and exons 11, 12 and 13 encode the cytoplasmic tails of three short isoforms named prolactin receptor S1 (exon 12), S2 (exon 11) and S3 (exon 13). Five different promoter regions have been identified in the mouse prolactin receptor gene (Ormandy et al., 1998). The rat prolactin receptor gene gives rise to three isoforms, a long, an intermediate, and a short form (Bole-Feysot et al., 1998). In the rat prolactin receptor gene, three promoters have been identified and tissue-specific usage has been demonstrated for two of them (Moldrup et al., 1996; Hu et al., 1998).

Biological importance of prolactin receptor splice variants The demonstration of prolactin receptor isoforms with long or short cytoplasmic domains in man, rodents and ruminants (Bignon et al., 1997; Hu et al., 2001), indicates a conserved function of splice variants across species. Their biological relevance is further supported by regulated splicing and tissuespecific expression patterns of the various isoforms (Bole-Feysot et al., 1998). The main functional difference between isoforms appears to be their ability to interact with intracellular effector proteins, and therefore their signaling capacity. In general, short forms may work as negative regulators of signaling by long prolactin receptor isoforms. However, more complex, independent or interactive functions of short forms may exist.

Prolactin receptor protein structure The single-pass, transmembrane prolactin receptor belongs to the class 1 cytokine receptor family (Bazan, 1990). Prolactin receptor molecules are glycosylated and are synthesized as precursors that include a signal peptide of 19 to 24 amino acids. While most other members of the cytokine receptor class form bipartite receptor complexes that involve two distinct gene family members, the prolactin receptor is simpler in that it only depends on a single gene.

Extracellular domain The extracellular domain of the prolactin receptor conforms structurally to the typical cytokine receptor extracellular domain. It contains a sequence of 210 amino acids referred to as the cytokine receptor homology (CRH) region (Bazan, 1990). The prolactin receptor extracellular domain is divided into two subdomains D1 and D2 of 100 amino acid residues each. Both subdomains show analogy with the fibronectin type III module (Kelly et al., 1991). Each D1 and D2 subdomain folds into seven beta-strands that form a sandwich of two antiparallel b-sheets (Somers et al., 1994; Elkins et al., 2000). In contrast to many other cytokine receptors, prolactin receptors do not contain additional extracellular subdomains. A highly conserved feature within the extracellular domains of prolactin receptor are two pairs of disulfide-linked cysteines in the N-terminal subdomain D1 (Cys12–Cys22 and Cys51–Cys62 in human prolactin receptor). A second characteristic feature is the WS-motif located in the membrane-proximal region of subdomain D2. The disulfide bonds and the WS-motif appear to be required for proper folding and trafficking of cytokine receptors to the cell membrane, although neither feature is directly involved in ligand binding (Hilton et al., 1995; Bole-Feysot et al., 1998). A crystal structure of ovine placental lactogen bound to the extracellular domains of two prolactin receptors has been solved (Christinger et al., 1998; Elkins et al., 2000). Prolactin is expected to bind to its receptor in a similar way, but relatively weaker interaction forces between prolactin and the second receptor molecule have been postulated (Elkins et al., 2000).

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Ligand specificity of prolactin receptors Within the cytokine receptor family, the prolactin receptor is most closely related to the growth hormone receptor. In fact, growth hormone from man and other primates, as well as placental lactogen, also bind and activate prolactin receptors (Kossiakoff et al., 1994). In contrast, in subprimate species growth hormone does not activate prolactin receptors. Placental lactogen is synthesized by mammals only and is therefore not found in lower vertebrates. While there is currently no specific receptor identified for placental lactogens (Gertler, 1997; Gertler et al., 1998), placental lactogens are able, perhaps in a speciesdependent manner, to heterodimerize prolactin and growth hormone receptors (Herman et al., 2000). This is especially intriguing since it raises the possibility for signal specificity between prolactin, growth hormone and placental lactogens, even if each hormone alone can activate prolactin receptors in primates.

Transmembrane domain The single-pass transmembrane domain of the human prolactin receptor contains 24 amino acid residues (aa 211–234) that are predicted to form a stable a-helix. The functional importance of individual amino acids within the transmembrane domain has not yet been examined. It would be of specific interest to determine whether specific side chains facilitate dimerization or higher order oligomerization, as has been suggested for erythropoietin receptors (Constantinescu et al., 2001b).

Cytoplasmic domain Prolactin receptor isoforms differ primarily, if not exclusively, in their cytoplasmic domains. Among the transmembrane forms of the human prolactin receptor, the size of the cytoplasmic domain varies from 54 amino acid residues in the S1b short isoform (Hu et al., 2001), to 388 amino acid residues in the long isoform (Boutin et al., 1989). Different cytoplasmic domains imply unique interaction capacities with primary and secondary effector proteins. There are no known enzymatic motifs intrinsic to the cytoplasmic domain of prolactin receptors. Two

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conserved structural features are the membraneproximal Box1 and Box2 homology regions (Yu-Lee et al., 1998b; Bole-Feysot et al., 1998). Box1 is an eight amino acid residue, proline-rich and hydrophobic motif (aa 243–250). The second consensus region, Box2, is less conserved between cytokine receptors than Box1 and is comprised of hydrophobic and acidic residues (aa 288–298). Whereas Box1 and Box2 are present in long and intermediate transmembrane prolactin receptor isoforms, Box2 is absent in the short isoforms. Mutational analysis has demonstrated that Box1 prolines are required for activation of the Jak2 tyrosine kinase (Pezet et al., 1997a). Truncated receptor forms that lack Box2, but have an intact Box1 may also be unable to activate Jak2 (DaSilva et al., 1994).

Cell types and tissues expressing prolactin receptor Prolactin receptors are expressed in a wide range of cells and tissues. In addition to prolactin target tissues with well-characterized biological responsiveness to prolactin, such as mammary gland, ovaries and prostate, many other organs have been found to express prolactin receptor. An extensive list is provided in Table 6.4 (Bole-Feysot et al., 1998). Studies of genetic mouse models lacking either prolactin or prolactin receptor have revealed significant phenotypes that reflect several indispensable functions of prolactin receptors in the mouse, including mammary gland differentiation, ovarian steroid production and uterine blastocyst implantation and placentation (Ormandy et al., 1997; Steger et al., 1998). While blastocyst implantation and early placentation to some extent may be overcome by progesterone replacement in prolactin receptor null mice, prolactin receptors are nonetheless needed for full-term pregnancies in mice (Reese et al., 2000; Binart et al., 2000). Furthermore, disruption of prolactin receptors may also critically affect brain function and turnover of bone, fat and hair follicles (Lucas et al., 1998; Clement-Lacroix et al., 1999; Craven et al., 2001; Freemark et al., 2001; Kelly et al., 2001). In contrast, the absence of anticipated phenotypes related to immune cell function in prolactin or prolactin receptor null mice (Horseman et al., 1997; Bouchard et al., 1999), as well as in other prolactin target tissues, may reflect more redundant or

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TABLE 6.4 Distribution of prolactin receptors in vertebrates Immune system Lymphoid tissue: Spleen Lymphocytes: T, B Macrophages Ganglia Thymus: Nurse cells Thymus: Epithelial cells Thymus: Intestinal cells

Nervous system Brain Cortex Hippocampus Choroid plexus Striatum Cochlear duct Corpus callosum Hypothalamus Astrocytes Glial cells Retina Olfactory system Ganglia

Urinary system Kidney: Cortex Bladder (fish, reptiles, amphibians) Reproductive system (female) Ovary: Granulosa cells Ovary: Thecal cells Ovary: Corpus luteum (luteal cells) Oviduct Mammary gland: Epithelial cells Milk Tumors Uterus: Endometrium Uterus: Placenta Uterus: Amnion

Endocrine system Pituitary: Anterior lobe Pituitary: Intermediate lobe Adrenal cortex Pancreas: Cells of Langerhans Islands Circulatory system Heart: Cardiac muscle Heart: Atria

Reproductive system (male) Testis: Germ cells Tetsis: Spermatozoa Testis: Leydig cells Testis: Sertoli cells Epididymis Seminal vesicle Prostate

Respiratory system Lung Gills (fish and larval amphibians) Musculoskeletal system Bone tissue: Chondrocytes Bone tissue: Cartilage Bone tissue: Osteoblasts Skeletal muscle

Integumentary system Skin: Epidermis Hair follicle Sweat glands

Digestive system Liver: Hepatocytes Liver: Kupffer cells Submandicular gland Submaxillary gland Esophagus Stomach Intestine Duodenum Jejunum Ileum Colon Crop sac (birds)

Adipocytes (birds) Brown adipose tissue

Adapted from Bole-Feysot et al., 1998.

compensable roles of prolactin. Arguably, vertebrates have probably evolved backup mechanisms for the most ancient and fundamental functions of prolactin, e.g. osmoregulation (Bern and Nicoll, 1968). Observed changes in the severity of autoimmune diseases by

hyperprolactinemia also support redundant roles of prolactin in certain cells and tissues. Likewise, overexpression of prolactin leads to marked prostate hyperplasia in transgenic mice (Wennbo et al., 1997b), although the male accessory organs of prolactin

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receptor null mice appear normal (Ormandy et al., 1997).

Cell lines expressing prolactin receptors The Nb2 lymphoma line was originally established by Peter Gout from an estrogen-treated rat (Gout et al., 1980). This cell model is highly responsive to prolactin and was instrumental for initial detection of prolactin induced tyrosine kinase activity (Rui et al., 1992; Rillema et al., 1992), which led to the identification of Jak2 tyrosine kinase as the initial effector of prolactin receptors (Rui et al., 1994a; Lebrun et al., 1994). Nb2 cells have on average 12,000 prolactin receptors and proliferate in response to very low concentrations of prolactin (Gout et al., 1980; Ali et al., 1991). This feature has led to the extensive use of Nb2 cells as a bioassay for prolactin, although Nb2 cells also proliferates in response to interleukin-2 and interleukin-7 (Sadeghi and Wang, 1992; Kirken et al., 1994). Whereas the Nb2 cell line is valuable for studying mitogenic and anti-apoptotic effects of prolactin, mouse mammary epithelial cells such as COMMA-D or HC11 are particularly useful to study prolactininduced cell differentiation in vitro (Medina et al., 1987; Hynes et al., 1990). In addition, several human breast cancer cell lines express high levels of prolactin receptors, e.g. T47D, MCF-7 or BT-20 (Shiu, 1979). The biological effects of prolactin in human breast cancer cells are still being determined, but reported effects include differentiation (Ackland et al., 2001), proliferation (Das and Vonderhaar, 1997), and induction of motility (Maus et al., 1999).

Regulation of receptor expression Long versus short isoforms Many details of how prolactin receptor expression is regulated remain to be established. Several tissuespecific promoter regions are involved and presumably controlled by different transcription factors (Moldrup et al., 1996; Ormandy et al., 1998; Galsgaard et al., 1999; Hu et al., 1999). Furthermore, the relative expression of alternatively spliced isoforms is highly regulated, leading to a variable pattern of isoform expression in different tissues. For instance, the long

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prolactin isoform is the predominant form in the mammary gland, whereas the short isoform predominates in the liver (Nagano and Kelly, 1994; Ouhtit et al., 1994).

Up-regulation and down-regulation Depending on cell type and experimental conditions, prolactin may either up-regulate (Rui et al., 1986) or down-regulate its receptor (Suard et al., 1979). Downregulation is typically attributed to increased rate of internalization of bound receptor complexes. Upregulation may include unmasking of cryptic binding sites (Rui et al., 1987) or result from direct transcriptional upregulation by Stat5 activation (Galsgaard et al., 1999). The effect of prolactin on its receptor levels is also a function of hormone concentration and duration of exposure. Thus, both positive and negative feedback loops may exist that involve prolactin and its receptor. In many organs, steroid receptors are involved in regulating prolactin receptor expression. Prolactin receptors are for instance up-regulated by estrogens in both mammary gland and liver (Jolicoeur et al., 1989). In prostate, prolactin receptor levels are increased by testosterone (Nevalainen et al., 1996).

TRANSMEMBRANE SIGNAL TRANSDUCTION Prolactin-induced receptor dimerization and Jak2 activation Based on mutagenesis and homology analyses with growth hormone, for which the crystal structure of the liganded receptor complex has been solved (de Vos et al., 1992), a similar two-site model has been developed for prolactin (Goffin et al., 1996b). Consistent with this model, a crystal structure of placental lactogen bound to prolactin receptors revealed the anticipated complex of one ligand molecule bound to two prolactin receptors (Elkins et al., 2000). Initial prolactin receptor activation, therefore, is postulated to involve dimerization of two receptor molecules per prolactin molecule. By oligomerizing extracellular receptor domains, prolactin binding also brings cytoplasmic receptor

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domains together. This, in turn, leads to activation of the intracellular, receptor-associated tyrosine kinase Jak2 (Rui et al., 1994a). Jak2 activation is specifically thought to occur by transphosphorylation of catalytic domains following close juxtaposition of two Jak2 molecules upon prolactin binding and receptor aggregation. Functional data supporting this model of homodimerization of prolactin receptors include the ability of bivalent monoclonal anti-receptor antibodies to mimic the effect of prolactin on Jak2 activation and proliferation of Nb2 lymphocytes (Rui et al., 1994b). In contrast, monovalent anti-receptor Fab-fragments require religation with bivalent anti-Fab antibodies to become active. Furthermore, the mitogenic potency of five mAbs correlated with their ability to induce tyrosine phosphorylation of Jak2 (Rui et al., 1994b). Additional experimental support for receptor dimerization include the demonstration that hormone analogs without functional binding Site 2 are unable to induce prolactin receptor homodimerization, and act instead as competitive antagonists by binding receptors through site 1 (Bernichtein et al., 2001).

Receptor dimerization may not be sufficient Although prolactin receptor activation appears to require dimerization, it may not be sufficient. Of five anti-prolactin receptor antibodies with comparable affinities and ability to bind to prolactin receptors on Nb2 cells, only three acted as receptor agonists (Rui et al., 1994b). Inactive prolactin receptor dimers may therefore exist, due to inappropriate relative orientation or intermolecular distances of the dimerized receptors molecules. In fact, recent crystallographic data obtained with the related erythropoietin receptor suggest that inactive receptors are present on the cell surface as preformed dimers with an unproductive distance between the transmembrane, and hence, cytoplasmic domains. Crystallographic data suggest that binding of erythropoietin will bring the cytoplasmic domains together by pivoting together the extracellular domains of the preformed dimer in a scissor-like movement (Livnah et al., 1999). Evidence for preformed erythropoietin receptor aggregates were also supported by antibody-mediated immunofluorescence copatching of epitope-tagged

receptors at the surface of live cells, an effect that was at least to some extent dependent on the transmembrane domain (Constantinescu et al., 2001b). A similar paradigm of ‘dimerization of dimers’ or aggregation of higher order oligomers now needs to be considered for transmembrane signaling by prolactin receptor.

Receptor interaction with Jak2 Current data suggest that the Jak2 tyrosine kinase is pre-associated with the inactive prolactin receptor. Consistent with this view, Jak2 is activated within 20 s of prolactin receptor triggering and can be selectively coprecipitated with the receptor (Rui et al., 1992, 1994a). The interaction requires an intact receptor Box1 homology domain (Pezet et al., 1997a), but Jak2 interaction may also depend on additional contact points. Studies of the erythropoietin receptor have identified several membrane-proximal residues required for proper Jak2 activation, and suggested that proper rotational orientation of Jak2 was also required (Constantinescu et al., 2001a). Furthermore, the Box2 region may also be important for Jak2 activation, since prolactin receptors lacking Box2 may not activate Jak2 even if Box1 is intact (DaSilva et al., 1994). This notion is consistent with the apparent absence of Jak2 activation in short prolactin receptor isoforms that lack Box2.

Intracellular signaling – Substrates of Jak2 Jak2 becomes autophosphorylated on tyrosine residues following prolactin receptor activation (Rui et al., 1994a). Furthermore, cytoplasmic domains of long and intermediate prolactin receptor isoforms become phosphorylated upon Jak2 activation, and at least some of this phosphorylation is attributed to Jak2 (Bole-Feysot et al., 1998). Prolactin receptor-associated proteins, either preassociated or associated following initial phosphorylation of critical tyrosine residues, constitute probable substrates for Jak2. Such substrates are most likely found among proteins with src homology domain 2 (SH2) or phosphotyrosyl binding (PTB) domains, which are known to associate specifically with tyrosine phosphorylated protein motifs (Pawson and

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PRLR PRL PRLR

Cell membrane

Sos1 Ras

SHP2

Grb2 Shc

Raf1

Src P-

-P

P-

PI3K IRS1 Akt

-P P- Jak2 Jak2 -P P-P

MEK

?

ERK1/2 Stat5

Stat3

Stat1

Fos Jun

Nuclear membrane

Jun Fos

Stat5

Stat3

Stat1

FIGURE 6.3 Overview of PRL receptor receptor signaling pathways. PRL causes oligomerization of prolactin receptors and activate the receptor-activated Jak2 tyrosine kinase. Main signaling pathways include Stat5, Stat1, and Stat3, transcription factors that are activated by tyrosine phosphorylation, dimerization and translocation to the cell nucleus. A second main pathway leads to activation of MAP kinases, involving the Shc, Grb2, Sos, Ras, Raf cascade, but also possibly other signaling routes. Interactions between prolactin receptors and Src kinases (e.g. Src, Fyn), tyrosine phosphatase SHP2, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and other transducing molecules remain to be more firmly defined. The extent of positive and negative signaling by short prolactin receptor isoforms is also under active investigation.

Scott, 1997). Whether Jak2 substrates are strictly confined to proteins permanently or transiently anchored to the receptor complex has not yet been determined, but colocalization with Jak2 is certainly required. In general, prolactin signaling in a given cell will depend on availability of cellular mediators (signaling proteins, adaptor proteins, co-regulators) and on the expression ratio of prolactin receptor splice variants. Furthermore, prolactin signaling will be influenced by crosstalk from alternate, concurrently active pathways. A single signaling scheme is therefore not expected to fit all prolactin target cells. Downstream of signal transduction, the diversity of biological effects may also to a large extent depend on how cellular chromatin structure determines which genes are available for transcriptional regulation. Nonetheless, the transcription factor Stat5 appears to be a very

general mediator of prolactin effects in a variety of cells. A general outline of prolactin receptor signal transduction is presented in Figure 6.3.

Prolactin signaling through the Jak2-Stat5 pathway The most direct signaling route from the transmembrane prolactin receptor to the cell nucleus involves Signal transducers and activators of transcription (Stats). There are seven mammalian genes encoding Stat transcription factors (Darnell et al., 1994). Stat5 was originally identified as a prolactin-activated nuclear factor that binds to the b-casein gene promoter in sheep mammary gland (Wakao et al., 1994). Subsequent work has revealed the existence of two highly homologous isoforms, Stat5a and Stat5b, with

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overlapping functions (Liu et al., 1995; Grimley et al., 1999). The initial activation of Stat5 involves Jak2mediated phosphorylation of a positionally conserved tyrosine residue that causes dimerization of Stat5 molecules (Gouilleux et al., 1994). Transient juxtaposition of Stat5 and Jak2 is needed for this phosphorylation to occur, and involves docking of Stat5 via its SH2 domain to specific phosphotyrosine residues within the activated receptor complex (Pezet et al., 1997b). Interestingly, tyrosine phosphorylation of Stat5 alone is not sufficient for translocation (Kazansky et al., 1999). A second coordinated activation event involves translocation of dimerized Stat5 from the cytoplasm into the cell nucleus, which permits Stat5 to bind to promoter elements and regulate transcription of specific genes (Gouilleux et al., 1994; Kazansky et al., 1999). The details of nuclear translocation are yet to be clarified, and regulated nuclear export may be a key element of nuclear accumulation of Stat transcription factors (Mowen and David, 2000). Regions of the prolactin receptor that are important for Stat5 activation have been identified. The molecular mechanism involves transient recruitment of Stats to select tyrosine phosphorylated sites on the prolactin receptor. Studies of mouse and rat prolactin receptors have implicated the most C-terminal tyrosine (corresponding to Y587 of human prolactin receptor) as a substrate of Jak2 and as a major docking site for Stat5 (Pezet et al., 1997b; Mayr et al., 1998). In addition, the mouse prolactin receptor residue corresponding to Y485 of human prolactin receptor is also a Stat5 docking site (Pezet et al., 1997b; Mayr et al., 1998). However, other sites may also be involved in prolactin-induced Stat5 activation, since a highly truncated receptor variant lacking both of these prolactin receptor tyrosine residues remained capable of activating Stat5, albeit to a lesser extent (DaSilva et al., 1996).

Stat5 serine phosphorylation In addition to Stat5 tyrosine phosphorylation by Jak2, prolactin can regulate phosphorylation of serine residues within the transactivation domains of Stat5a and Sta5b by a proline-directed serine kinase (Kirken et al., 1997a, 1997b). Specifically, a PSP-motif of Stat5a (S726) and Stat5b (S731) are major shared phosphorylation sites (Yamashita et al., 1998), whereas Stat5a

has a second major serine phosphorylation site, serine 780 within a SPP-motif (Pircher et al., 1999; Beuvink et al., 2000; Yamashita et al., 2001). In Nb2 cells, two pathways appear to regulate phosphorylation of the PSP-motif, one that is prolactin-activated and PD98059-resistant, and one that is constitutively active, PD98059-sensitive and which preferentially targets Stat5a over Stat5b (Yamashita et al., 1998). The biological roles of Stat5 serine phoshorylation have remained elusive (Yamashita et al., 1998; Beuvink et al., 2000), but emerging evidence suggests a modulatory role that may be positive or negative in a coregulator- and promoter-dependent manner (Yamashita et al., 2001, Park et al., 2001).

Other Stats Whereas Stat5 appears to be the central Stat activated by prolactin, prolactin can also activate Stat1 and Stat3 (David et al., 1994; DaSilva et al., 1996; Schaber et al., 1998). The region(s) of the prolactin receptor required for activation of these Stats are still poorly defined. In the rat intermediate prolactin receptor (Nb2 form), tyrosine residues Y382 and Y309 have been proposed to bind STAT 1 (Wang et al., 1997). Furthermore, a truncated prolactin receptor that lacked Y382, but contained Y309, was still capable of activating Stat1 and Stat3 more effectively than the long receptor form (DaSilva et al., 1996). Tyrosine residues within the Jak2 molecule itself or in other proteins of the receptor complex may also mediate Stat1 and Stat3 activation (DaSilva et al., 1996).

Src and other non-Jak2 tyrosine kinases Cytoplasmic tyrosine kinases of the Src-family can also be regulated by prolactin. Prolactin-induced Fyn activation in rat T lymphoma Nb2 cell line (Clevenger and Medaglia, 1994) has been proposed to mediate prolactin-induced activation of phosphatidyl-inositol 3-kinase (al-Sakkaf et al., 1997). Similarly, a role for Src-kinases in mediating prolactin-induced cell proliferation in transfected mouse BaF3 pro-B cells has been suggested (Vara et al., 2001). Prolactin-induced activation of Src was detected in hepatocytes from lactating rats (Berlanga et al., 1995). Interestingly, in a chick embryo fibroblast reconstitution model of pro-

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lactin signaling, a prolactin receptor with a mutated Box1 that lacked the ability to activate Jak2 was still able to mediate prolactin-induced activation of c-Src (Fresno Vara et al., 2000). Thus, Jak2–independent, prolactin-induced activation of Src tyrosine kinases may occur. Additional non-Jak, non-Src tyrosine kinases may also be activated by prolactin in a cell-dependent manner, including focal adhesion kinase (Canbay et al., 1997), and the TEC tyrosine kinase (Kline et al., 2001). Their biological significance for prolactin signaling remains to be established.

Mitogen-activated protein kinase (MAPK) family The MAPK family of proline-directed serine/threonine kinases includes extracellular regulated kinases (ERKs), p38Hog, and Jun N-terminal Kinase (JNK) (Davis, 1995). Prolactin, like many other cytokines and growth factors, is capable of activating several ERKs, including ERKs 1, 2, 3 and 4 in Nb2 cells (Buckley et al., 1994; Camarillo et al., 1997) and at least ERKs 1 and 2 in breast cancer cells (Das and Vonderhaar, 1996b; Llovera et al., 2000a). One probable pathway involves the MAP kinase kinase, MEK1, and the MAP kinase kinase kinase, Raf-1 (Clevenger et al., 1994; Das and Vonderhaar, 1996a), although the existence of an alternative PI3K-dependent pathway has also been proposed (Goupille et al., 2000). The Ras activator, Raf-1, may in turn be activated by the prolactin receptor through recruitment of the adaptor protein SHC and the Grb2–SOS complex (Erwin et al., 1995; Das and Vonderhaar, 1996b). An alternative route to Ras activation could involve the Vav protein (Clevenger et al., 1995b). Several recent reports also demonstrate that prolactin can activate JNK in various cell types (Schwertfeger et al., 2000; Olazabal et al., 2000; Cheng et al., 2000). Whether Jak2 is required for prolactin activation of MAP kinase cascades is currently unknown, although Jak2 has been demonstrated to be essential for GH induced MAPK activation (Winston and Hunter, 1995).

Phosphatidyl-inositol 3-kinase (PI3K) In Nb2 cells, prolactin may induce rapid tyrosine phosphorylation of the 85 kDa subunit of the PI3K,

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which, along with insulin receptor substrate-1, appears to associate with the prolactin receptor in a prolactin-dependent manner (Berlanga et al., 1997). Furthermore, PI3K is reported to associate with the adaptor protein Cbl in Nb2 cells, which itself undergoes inducible tyrosine phosphorylation in response to prolactin (Hunter et al., 1997, 1999). The possible role of prolactin-activation of PI3K in regulating protein kinase B (Akt) and the mTOR/S6 kinase pathway deserves further examination (Vara et al., 2001), especially in light of the anti-apoptotic effects of prolactin in Nb2 cells (Leff et al., 1996; Clevenger et al., 1997; Al-Sakkaf et al., 2000; Tessier et al., 2001). However, prolactin-induced survival of prostate epithelial cells did not appear to involve the Akt pathway (Ahonen et al., 1999).

Other signaling molecules Accumulating evidence demonstrates a positive role for the tyrosine phosphatase SHP-2 in prolactin signal transduction (Ali et al., 1996; Berchtold et al., 1998; Ali, 2000). Other signaling proteins that are activated by prolactin and that will require further investigation as cell-dependent mediators of prolactin effects are adaptor proteins, such as insulin receptor substrates, Cbl and Nck (Pawson and Scott, 1997). Furthermore, prolactin-induced calcium entry in hamster ovary cells (Ratovondrahona et al., 1998; Sorin et al., 2000) and chloride transport in mouse mammary cells (Selvaraj et al., 2000) may represent novel prolactin effector pathways. These and other signaling components may mediate prolactin effects to a variable extent in a diverse set of target cells.

Negative signals – SOCS proteins Activation of Jak-Stat pathways by prolactin or other cytokines generally leads to induced expression of a family of negative regulators that help return Jak-Stat pathways to steady-state after activation. These inhibitory proteins are named SOCS (suppressor of cytokine signaling, or CIS, cytokine-inducible SH2 protein) and include at least eight members (CIS, SOCS-1 to 7) (Chen et al., 2000). At least some SOCS genes are direct targets of Stat transcription factors, and provide an efficient negative feedback loop for Jak-Stat signaling. CIS, SOCS-1 and SOCS-3 appear to

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have particular importance for regulating cytokine receptors and Jak tyrosine kinases (Chen et al., 2000). In the case of prolactin, inducible up-regulation of CIS, SOCS-1, -2 and -3 has been observed, although with variable kinetics (Pezet et al., 1999). In particular, SOCS-1 and SOCS-3 exerted negative effects on prolactin signaling (Pezet et al., 1999; Tomic et al., 1999), whereas SOCS-2 appeared to bind to prolactin receptors and counteract the inhibitory effect of SOCS-1, but not of SOCS-3 (Pezet et al., 1999). SOCS proteins may both act locally by directly inhibiting Jak kinase activity, block recruitment of Stats to the receptor complex, or facilitate targeting of associated proteins for degradation in the proteasome (Chen et al., 2000).

Signaling roles of individual prolactin receptor splice variants No simple classification of prolactin-receptor isoforms (see Figure 6.2) into signaling and non-signaling forms has yet emerged. While it is clear that long and intermediate isoform can mediate positive prolactin signals, short isoforms have been reported to act as signal disruptors (Perrot-Applanat et al., 1997b; BoleFeysot et al., 1998; Hu et al., 2001), but also to mediate positive signals (Das and Vonderhaar, 1995; Bignon et al., 1999). In general, the ability of individual prolactin receptor splice variants that differ in their cytoplasmic domains to positively mediate prolactin effects is expected to depend on their ability to activate intracellular effector molecules. First, ability to activate Jak2 following receptor dimerization is anticipated to be a key feature of signaling prolactin receptor isoforms. However, Jak2–independent signaling may occur in some cases (Fresno Vara et al., 2000). Second, the signaling repertoire of a prolactin receptor isoform will depend on the ability of its cytoplasmic domain to attract and interact with signaling intermediaries. Phosphotyrosyl docking sites represent key modules, but other regulated or constitutive association sites may exist that differ between isoforms. Third, a non-signaling isoform is expected to disrupt signaling by a signalcompetent partner by forming non-productive heterodimers (Bole-Feysot et al., 1998). There are several reports of such inhibition by short isoforms. In transfected cells, short prolactin receptor variants that lack Box2 function as dominant-negative iso-

forms, inhibiting inducible cell proliferation (Chang et al., 1998) or activation of milk protein gene transcription (Perrot-Applanat et al., 1997b; Hu et al., 2001). An additional consideration involves cooperative effectsofpairedreceptortyrosineresidues. Specifically, heterodimerization of two engineered prolactin receptor mutants that each lacked a separate tyrosine residue in the cytoplasmic domains led to a loss of inducible Jak2 tyrosine phosphorylation, despite marked inducible Jak2 tyrosine phosphorylation upon homodimerization of each mutant (Chang et al., 1998). Thus, tyrosine residues within receptor dimers may act in trans to induce full Jak2 tyrosine phosphorylation. This phenomenon may become helpful for studies of interactions between ‘natural’ prolactin receptor heterodimers.

Transcription factors activated When activated, STAT factors translocate to the nucleus, where they transactivate target gene promoters by binding to consensus DNA sequences. Specifically, Stat5 binds as a dimer to promoters containing the optimal GAS (interferon-gamma activated site) elements of the consensus TTCNNNGAA (Wakao et al., 1994; Darnell, 1997). In addition, Stat5 may bind physiologically to less optimal sites as tetramers, provided two sites are located in tandem with proper spacing (John et al., 1999). An increasing number of genes are identified that are responsive to Stat5 (for reviews, see Grimley et al., 1999, Lin and Leonard, 2000). Stats cooperate with a variety of other transcription factors within the context of individual gene promoters. However, Stat5 may also interact directly with other transcription factors in a manner that at least partly is DNA independent. For example, Stat5 interacts with the glucocorticoid receptor to stimulate b-casein gene transactivation, even in the absence of glucocorticoid receptor DNA-binding domain (Stocklin et al., 1996, 1997). However, DNA binding by GR appears to be essential for Stat5 cooperative stimulation of b-casein gene expression at low GR levels (Doppler et al., 2001). Interaction between prolactin-activated Stat5 and the progesterone receptor may also occur (Richer et al., 1998), and cooperative interaction between NF1 and STAT5

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on WAP gene expression has been reported (Li and Rosen, 1995). Furthermore, while prolactin-induced Stat1 stimulates IRF-1 gene transcription, Stat5a and Stat5b inhibit prolactin-inducible IRF-1 promoter activity, possibly through squelching by Stat5 of a coregulatory factor required for Stat1-activation of the IRF-1 promoter (Luo and Yu-Lee, 1997; Book McAlexander and Yu-Lee, 2001).

Genes regulated Prolactin has a wide spectrum of biological effects. The repertoire of genes regulated by prolactin, either directly or indirectly, is correspondingly broad. A multi-approach analysis in Nb2 lymphocytes revealed approximately 70 genes that were either up- or downregulated by prolactin (Bole-Feysot et al., 2000). Genes induced include early growth related genes such as c-fos, c-jun, c-myc, c-src, IGF-1 and mid- and late-G1 genes such as ornithine decarboxylase (ODC), heat shock proteins (hsp), or gfi-1. In addition transcription factor IRF-1, Bax, Bcl-2 and many cyclins are up-regulated (Yu-Lee, 1990; Crowe et al., 1991; Leff et al., 1996; Bole-Feysot et al., 1998). In the mammary gland, genes involved in epithelial cell differentiation and milk protein genes are stimulated by prolactin, including caseins, lactoglobulin and whey acidic protein (Shiu and Iwasiow, 1985; Hennighausen et al., 1997).

BIOLOGICAL EFFECTS OF PROLACTIN In vitro activities Because prolactin receptors are expressed ubiquitously, and couple to several parallel signal transduction pathways, prolactin affects the function of a variety of cells. The effects are cell- and contextdependent, and include regulation of growth, survival, differentiation and cellular activation state. Particularly pronounced effects have been described on cells of the mammary gland and other reproductive organs, but also on cells of the immune system (see reviews Topper et al., 1986; Groner and Gouilleux, 1995; Ben-Jonathan et al., 1996; Yu-Lee, 1997; Ferrag

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et al., 1997; Yu-Lee et al., 1998a; Clevenger et al., 1998)).

In vivo biological activities – normal physiological roles The physiological effects of prolactin include reproductive and homeostatic effects. Reproductive effects include mammary gland differentiation, fertility, sex steroid production and behavior. Homeostatic effects may be categorized into stress adaption responses, water/electrolyte balance (osmoregulation), metabolism, angiogenesis and skin function.

Reproductive effects Mammary gland In mammals, prolactin plays a critical role in stimulating mammary gland differentition, and is important for initiation and maintenance of milk production (Topper et al., 1986; Rosen et al., 1994; Groner and Gouilleux, 1995). An interesting comparative function of prolactin in a submammalian species is the induction of cropmilk in the cropsac of nursing pigeons (Horseman and Buntin, 1995). In mammals, prolactin-induced mammary gland differentiation occurs in synergy with insulin and corticosteroids, and only after circulating estrogens and progesterone levels fall at parturition will prolactin induce milk secretion (for extensive reviews, see Hennighausen et al., 1991; Rosen et al., 1994; Groner and Gouilleux, 1995). Gene knockout studies in mice have verified the critical roles that prolactin and prolactin receptors play for mammary gland differentiation and lactation (Horseman et al., 1997; Ormandy et al., 1997; Steger et al., 1998; Goffin et al., 1999a). Specifically, prolactin was found to affect mammary gland morphogenesis by controlling ductal side branching and terminal end bud regression in virgin mice through indirect mechanisms, but acted directly on the mammary epithelium to induce lobuloalveolar development during pregnancy (Brisken et al., 1999). Furthermore, gene knockout studies have demonstrated that transcription factor Stat5a is a central downstream mediator of prolactin action in mammary glands (Liu et al., 1997; Hennighausen et al., 1997; Groner and Hennighausen, 2000).

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Gonads Prolactin regulates ovarian and testicular function both directly through gonadal prolactin receptors (Russell and Richards, 1999; Huang et al., 2001) and indirectly by modulating gonadotropin secretion (Smith, 1980; Ben-Jonathan et al., 1996). Prolactin stimulates dopamine release in the hypothalamus and inhibits gonadotropin secretion, and physiological hyperprolactinemia during pregnancy and lactation and pathological hyperprolactinemia are associated with suppression of the hypothalamicpituitary-gonadal axis. Inhibition of pulsatile secretion of GnRH from the hypothalamus by prolactin results in impaired gonadotropin secretion and inhibition of gonadal function (Thorner et al., 1998). However, fertility and circulating testosterone levels were normal in male prolactin knockout mice, whereas ovarian steroid production was insufficient for fertility in females (Steger et al., 1998). The direct effect of prolactin on testicular function appears to be particularly pronounced in seasonally breeding animals, in which prolactin may act directly on the testes as a gonadotropin (Jabbour et al., 1998). Prolactin receptors are expressed in Leydig cells as well as in steroidproducing cells in the ovary (Rolland and Hammond, 1975; Charreau et al., 1977; Shirota et al., 1990). Furthermore, evidence suggests direct effects of prolactin on rodent ovarian function, particularly in the regulation of progesterone metabolism (Ouhtit et al., 1993a; Martel et al., 1994; Zhong et al., 1997; Russell and Richards, 1999).

Uterus/placenta Prolactin is produced in decidualized endometrial cells and myometrial cells (Riddick et al., 1978; DiMattia et al., 1990; Gellersen et al., 1991; Gellersen et al., 1994; Stewart et al., 1995). As a local uterine factor prolactin is thought to be important for normal progression of pregnancy (for review, see Soares et al., 1998), although the exact roles of decidual prolactin and prolactin-like proteins/placental lactogens in this process remain to be firmly established. Several lines of evidence indicate that decidual prolactin diffuses into the amniotic fluid (Rosenberg et al., 1980; Riddick and Maslar, 1981; Riddick and Daly, 1982; Riddick et al., 1983). Two proposed functions for decidual prolactin involve regulation of

water-electrolyte balance of the amnion (Tyson, 1982) and local immune reactions to prevent rejection of the implant (Handwerger et al., 1992). A role for amniotic fluid prolactin in lung maturation has also been proposed (Johnson et al., 1985a). Studies based on prolactin receptor null mice suggest that prolactin is directly involved in the maintenance of full-term pregnancy via effects on uterus, rather than a predominant role in preimplantatory egg development, implantation and decidualization, which seem to depend more on ovarian prolactin receptor expression and progesterone synthesis (Reese et al., 2000). Furthermore, another specific role of prolactin may be to induce extensive angiogenesis of placenta associated with trophoblast invasion, based on the observation that prolactin markedly stimulated local expression of the angiogenic factor bFGF (Srivastava et al., 1998), and observed direct proangiogenic effects of prolactin (Struman et al., 1999).

Prostate Besides androgens, prolactin is involved in regulation of growth and differentiation of prostate. Prolactin receptors are expressed at high levels in fetal human prostate (Leav et al., 1999). During development of prostate in rats, prolactin induces growth of the gland (Negro-Vilar et al., 1977; Hostetter and Piacsek, 1977; Perez-Villamil et al., 1992). Furthermore, targeted disruption of the prolactin gene in mice caused reduction in prostate size (Steger et al., 1998). No specific prostate phenotype was detected in prolactin receptor null mice (Ormandy et al., 1997), whereas absence of the Stat5a gene, which is one of the components of prolactin signaling in prostate, was associated with disorganization of the epithelium and cystic changes in prostate acini (Nevalainen et al., 2000). In contrast to prolactin knockout mice, transgenic overexpression of prolactin caused massive growth of prostates of older mice (Wennbo et al., 1997b). This effect is supported by a number of in vivo studies that have demonstrated a trophic effect of prolactin on mature rodent prostate (Prins and Lee, 1983; Johnson et al., 1985b; Rui and Purvis, 1987; Sissom et al., 1988; Schacht et al., 1992; Tabar et al., 2000; McPherson et al., 2001). Demonstration of prolactin-induced hyperplastic changes in rat and human prostate epithelium,

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coupled with stimulated proliferation (Nevalainen et al., 1991, 1997b) and inhibition of prostate epithelial cell apoptosis (Ahonen et al., 1999) based on longterm tissue explant organ cultures as an in vitro model, has been important for establishing the direct and androgen-independent effects of prolactin on prostate epithelium. In addition to stimulating growth of prostate, prolactin regulates metabolic events related to citrate production in differentiated prostate epithelium (Costello and Franklin, 1994; Costello et al., 2000). Detection of local production of prolactin within the epithelial compartment of rodent and human prostate has suggested regulation by prolactin of growth and differentiation of normal (Nevalainen et al., 1997a, 1997b) and, possibly, malignant prostate (Nakamura et al., 1990; Rana et al., 1995; Janssen et al., 1996; Leav et al., 1999; Melck et al., 2000, Xu et al., 2001).

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receptors in the kidney (Sakai et al., 1999), in intestinum (Krishnamra et al., 2001) and in secretory organs, such as mammary, prostate and lacrimal glands (Clevenger and Plank, 1997; Nevalainen et al., 1997b; Cady et al., 2000). In mammary epithelial cells, prolactin decreases the transport of sodium and increases the transport of potassium across the cell membranes (Falconer and Rowe, 1975; Falconer et al., 1983). Also, prolactin stimulates the uptake of amino acids by mammary epithelial cells (Vina et al., 1981; Valve et al., 2001). In kidney, prolactin acts on the proximal convoluted tubule of the renal nephron to promote sodium, potassium, and water retention (Stier et al., 1984). Furthermore, decidual prolactin present in amniotic fluid affects water transport across amniotic membranes (Handwerger and Freemark, 1987).

Prolactin and stress Behavior and psychological effects The behavioral effects of prolactin in vertebrates are primarily linked to reproduction. Prolactin stimulates parental behavior, such as nesting and egg hatching in birds, and nest building and nursing of pups in mice, rats and rabbits (for reviews, see Scapagnini et al., 1985; Dutt et al., 1994; Horseman and Buntin, 1995). Confirming this long-standing notion of behavioral effects of prolactin, which was first observed by Riddle and colleagues (Riddle et al., 1935), elegant studies of prolactin-receptor knockout mice demonstrated a reduction in maternal behavior in these animals (Lucas et al., 1998). Furthermore, recent studies have identified prolactin as one of the hormonal factors associated with anxiety-related behaviors (Landgraf et al., 1999; Gerra et al., 2000; Torner et al., 2001).

Effects on osmoregulation Regulation of water-electrolyte balance by promoting solute transport across cell membranes seems to be the most prominent role of prolactin in fish and reptiles, and might represent the most ancient function of prolactin (Bern and Nicoll, 1968; Eckert et al., 2001; Sandra et al., 2000). In mammals, the role of prolactin in osmoregulation is perhaps reflected in local production of prolactin and expression of prolactin

Prolactin is a stress hormone (Drago et al., 1989). Circulating prolactin levels rise rapidly in response to emotional and physical stress in both humans and other mammals (Noel et al., 1972; Meites and Clemens, 1972; Siegel et al., 1980; Fujikawa et al., 1995; Servatius et al., 2000). In soldiers undergoing an exhausting combat course, prolactin levels were elevated at the start of the course, presumeably in anticipation of stress, but underwent a decrease over the 3 days of continuous physical strain (Aakvaag et al., 1978). Consistent with these and other observations (Jones and Hallworth, 1999; Dave et al., 2000), it has been postulated that stress situations associated with passive coping are accompanied by increased plasma prolactin levels, whereas stress situations associated with active coping are associated with unchanged or even lowered levels (Lacey et al., 2000). Furthermore, data indicate that prolactin affects neuroendocrine, behavioral and autonomic responses to stress, such as changes in the motility of gastric musculature and gastric acid secretion, and in thermoregulation (Drago and Amir, 1984; Drago et al., 1990; Drago et al., 1993). In rats, hyperprolactinemia has been demonstrated to protect against stress-induced ulcers (Drago et al., 1985). These and other data suggest that stress-induced hyperprolactinemia is not a mere consequence of

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stress activation, but may play a role in physiological mechanisms leading to restoration of body homeostasis (Dorshkind and Horseman, 2001). Decreased libido in stressed and hyperprolactinemic subjects is perhaps an endurance-preserving stress adaption response. Future studies of stress in mouse prolactin and prolactin receptor knockout models should provide new insight into the role of prolactin in stress adaption.

Metabolism The liver is involved in clearance of circulating prolactin, and expression of dysproportionally high levels of the putative ‘non-signaling’ short prolactin isoform by liver cells may facilitate this function (Jahn et al., 1997; Perrot-Applanat et al., 1997a). However, prolactin has also some growth hormone-like effects on the liver, and stimulates IGF-1 production (Murphy et al., 1988; Strain and Ingleton, 1990). Independently, prolactin and growth hormone stimulate beta-cell proliferation and insulin production in Langerhans islets (Billestrup and Nielsen, 1991; Sorenson and Brelje, 1997; Nielsen et al., 1999). Prolactin-induced proliferation of beta cells and insulin production by Langerhans islets are both mediated by Stat5 (Friedrichsen et al., 2001). Furthermore, decreased bone formation and reduction of bone mineral density in prolactin-receptor null mice, combined with detection of prolactin receptors in osteoblasts but not osteoclasts, suggested an involvement of prolactin in bone metabolism (Clement-Lacroix et al., 1999). In light of previously reported effects of prolactin on cholecalciferol secretion by the kidney (MacIntyre et al., 1978; Brown et al., 1980), an indirect effect of prolactin on calcium metabolism might be involved in the development of the phenotype of bones in prolactin receptor deficient mice. Recently, increased expression of receptors for prolactin was reported in adipose tissue of transgenic mice overexpressing prolactin (Ling et al., 2000). Conversely, older prolactin receptor knockout mice have reduction in body weight, which was specifically associated with reduced abdominal fat mass (Freemark et al., 2001). These findings suggest that prolactin has a role in modulating adipose tissue development and distribution.

Effects on immune function Extensive literature has documented effects of prolactin on various functions of immune cells, including lymphoid cells (T, B, NK), monocytes, macrophages, and thymic epithelial cells (for extensive reviews, see Berczi, 1994; Murphy et al., 1995; Leite De Moraes et al., 1995; Weigent, 1996; Ferrag et al., 1997; Yu-Lee, 1997; Velkeniers et al., 1998; De Mello-Coelho et al., 1998; Yu-Lee et al., 1998a; Clevenger et al., 1998; Matera et al., 2000; Dorshkind and Horseman, 2000; Dorshkind and Horseman, 2001). However, recent work involving gene targeting in mice has suggested that the ancestral and fundamental roles of prolactin as a regulator of hematopoiesis and immune function have been effectively shared by other and more specialized tetrahelical cytokines. In knockout mice lacking either the gene for prolactin or the prolactin receptor, a redundancy of prolaction function in the immune system was revealed by the finding of normal immunity and hematopoietic parameters (Horseman et al., 1997; Bouchard et al., 1999). Although prolactin was not found to be critical for immune function in mice, redundant functions of prolactin on immune cells will be more difficult to uncover and may require combinatorial knockout strategies. Nonetheless, a series of studies document that hyperactivity of the prolactin– prolactin receptor axis affects immune function, particularly autoimmunity, hematopoietic cell growth promotion and leukemogenesis (for reviews, see Neidhart, 1998; Hooghe et al., 1998). Furthermore, elevated prolactin levels during secondary infections in HIV-patients may have diagnostic and therapeutic implications (Montero et al., 2001).

Skin In seasonal animals, such as blue fox and red deer, evidence indicates that prolactin stimulates the transition from winter to summer coat (for review, see Curlewis, 1992). Also, prolactin receptors are expressed in ovine skin wool follicles (Choy et al., 1997) and in rat skin (Ouhtit et al., 1993b). Prolactin may promote proliferation of skin epithelial cells and support hair growth (Paus, 1991; Stenn and Paus, 1999). These findings are relevant for the occasional

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finding of hirsutism in hyperprolactinemic patients (Thorner et al., 1998).

PATHOPHYSIOLOGICAL ROLES AND DIAGNOSTIC UTILITY Normal levels and effects Basal levels of circulating prolactin in adults vary considerably, with levels of 4.0–25 μg l in nonpregnant women (median 10.0 μg l), and levels of 0.5–19 μg l in men (median 8.5 μg l) (Le Moli et al., 1999). Serum prolactin, which increases throughout pregnancy, falls with the onset of labor and then exhibits variable patterns of secretion depending on whether breast-feeding occurs.

Role of prolactin in disease Reproduction Among pituitary tumors, 60% secrete prolactin and cause a state of chronic hyperprolactinemia. Pituitary adenomas reveal pathophysiological effects of prolactin in men by inducing decreased libido, impotence, gynecomastia, galactorrhea, hypospermia, and occasionally reduced beard growth (Thorner et al., 1998). In premenopausal women, the cardinal effects of hyperprolactinemia are cessation of normal cyclic ovarian function, amenorrhea, galactorrhea, decreased libido, occasional hirsutism and increased long-term risk of osteoporosis (Palermo et al., 1994; Thorner et al., 1998). Specifically, pathological milk discharge, galactorrhea, is a result of hyperprolactinemia in both men and women and attests to the importance of prolactin in lactation.

Autoimmune diseases Accumulating data suggest that elevated prolactin levels represent a risk factor for certain autoimmune diseases in humans and rodents. These include adjuvant arthritis in rats, collagen type II-induced arthritis in rats and mice, type I diabetes in mice, and systemic lupus erythematosus (SLE) in mice and humans (for reviews, see Walker et al., 1995; McMurray, 1996;

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Neidhart, 1998; Ostensen, 1999). Furthermore, a connection between elevated prolactin levels and rheumatoid arthritis (RA) also has been suggested, but this correlation is less clear. Interestingly, there is a genetic association between RA and genes encoded in the HLA complex, particularly HLA DR4, and the human prolactin gene is located in close proximity to the HLA region on the short arm of chromosome 6. The hypothesis has been put forth that associations between HLA DR4 and reproductive risk factors in RA are due to linkage disequilibrium between DR4 and an abnormally regulated prolactin gene polymorphism (Brennan et al., 1996).

Hematopoietic cancer An involvement of prolactin in the development and progression of leukemia and lymphoma has also been suggested (for review, see Hooghe et al., 1998). For instance, a mitogenic prolactin receptor mutant has been described in the prolactin-dependent Nb2 lymphoma (Ali et al., 1991). Furthermore, autocrine production of prolactin is detectable in several transformed human lymphocytic lines, including IM-9-P3, Jurkat, Hut-78, U937 and YT (DiMattia et al., 1988; Pellegrini et al., 1992). The fact that tyrosine kinase Jak2, the principal downstream mediator of prolactin action (Rui et al., 1994a; Lebrun et al., 1994), is oncogenic in cells of both lymphoid and myeloid origin (Peeters et al., 1997), lends support to the notion that prolactin may promote growth of hematopoietic cancers. In fact, elevated serum prolactin was detected in more that 50% of patients with acute myeloid leukemia (Hatfill et al., 1990), although this observation might be due to an associated stress response.

Osmoregulation Excessive levels of amniotic fluid, polyhydramnion, is often associated with diabetes mellitus, multiple pregnancies, or fetal malformation. Interestingly, amniotic fluid in polyhydramnion is characterized by selectively reduced levels of prolactin, and not of other hormones (Luciano and Varner, 1984; Sarandakou et al., 1992). At the same time, patients with chronic polyhydramnion showed reduced numbers of prolactin receptors within the chorion leave (Healy et al., 1985).

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AREAS OF ACTIVE RESEARCH Several areas of prolactin biology are the subject of particularly active research. Efforts continue to further understand signal transduction pathways used by prolactin in various cell and tissue-specific contexts. Related to these efforts, characterizing and understanding the roles of alternatively spliced receptor variants for the biology of prolactin is one area that now needs to be further explored. Furthermore, understanding biological effects of prolactin in terms of gene expression programs using large scale gene expression analyses is another area of focus. Biological effects that continue to attract scientific focus are roles of prolactin in stress and autoimmune disease. Increased attention is being given to the role of prolactin in angiogenesis (D’Angelo et al., 1999; Struman et al., 1999; Martini et al., 2000), as well as the involvement of prolactin in development and progression of cancer. Particularly, ongoing work on the role of prolactin in breast and prostate cancer is summarized below.

Prolactin and breast cancer The involvement of prolactin in initiation and formation of mammary tumors in rodents is well established. Specifically, the incidence of spontaneous mammary tumors is significantly increased in mice that have pituitary isografts secreting large amounts of prolactin (Muhlbock and Boot, 1959). There is also a direct correlation between serum prolactin levels and susceptibility of various rat strains to induction of mammary tumors by chemical carcinogens (Boyns et al., 1973). Furthermore, hyperprolactinemia stimulates mammary tumor growth in rodents, whereas hypoprolactinemia reduces the tumor volume (Welsch and Gribler, 1973, 1977). Recently, Wennbo and colleagues demonstrated that transgenic female mice overexpressing the prolactin gene spontaneously developed mammary tumors (Wennbo et al., 1997b). Importantly, the same group also showed that activation of prolactin receptors, but not GH receptors, induced mammary tumors in mice overexpressing human GH (Wennbo et al., 1997a). In contrast, correlations between plasma prolactin levels and breast cancer risk in women have been controversial (for reviews, see

Clevenger and Plank, 1997; Llovera et al., 2000b). However, a recent well-designed study demonstrated that high prolactin levels were associated with increased risk of breast cancer in postmenopausal women (Hankinson et al., 1999). It is therefore possible that the role of prolactin in breast cancer progression is dependent on additional hormonal factors. The involvement of locally produced prolactin in promoting breast cancer growth in an autocrine or paracrine manner has been widely discussed (Clevenger and Plank, 1997; Vonderhaar, 1999) and supported by a number of studies (Clevenger et al., 1995a; Wennbo and Tornell, 2000). Coupled with the large number of prolactin receptor positive breast cancer specimens, a positive role of prolactin as a breast tumor promoter has stimulated development of prolactin receptor antagonists (Goffin et al., 1999b; Kinet et al., 1999; Llovera et al., 2000a; Bernichtein et al., 2001). However, it should be taken into account that prolactin may activate both tumor suppressive differentiation pathways and tumor promoting growth pathways. Therefore, prolactin receptor antagonists may be more effective in a subfraction of breast cancers. Success of new anti-prolactin therapies may depend on further knowledge of intracellular targets and pathways of prolactin in breast cancer.

Prolactin and prostate cancer An involvement of prolactin in prostate neoplasia has been established and this role of prolactin continues to be the subject of active research. In rats, induction of preneoplastic lesions in prostate epithelium by chemical carcinogens was enhanced by hyperprolactinemia (Nakamura et al., 1990). Transgenic mice overexpressing prolactin developed hyperplastic tumors of prostate, although malignant tumors were not reported (Wennbo et al., 1997b). Receptors for prolactin are expressed in normal human prostate (Nevalainen et al., 1997b), and in dysplastic and neoplastic lesions of human prostate epithelium (Leav et al., 1999). Prolactin has been shown to directly stimulate proliferation of prostate epithelial cells (Nevalainen et al., 1991, 1997b), but also to inhibit apoptosis (Ahonen et al., 1999). These observations suggest two growth-promoting mechanisms that may mediate prolactin-induced progression of prostate cancer. Growth of human prostate

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cancer cells is stimulated by prolactin (Janssen et al., 1996) and inhibited by suppression of prolactin receptors (Melck et al., 2000). Since the growth of prostate cancer can initially be inhibited by androgen withdrawal, one of the key elements of progression of prostate cancer is the achievement of the ability by prostate cancer cells to grow independently of androgens. Prolactin could be one of the factors that provides prostate cancer cells with the ability to survive in the growth environment lacking androgens and to overcome the growth arrest induced by androgen ablation. Local production of prolactin by prostate epithelium (Nevalainen et al., 1997a, 1997b) supports this notion, and may also explain the limited success of adjuvant therapies of prostate cancer with inhibitors of pituitary prolactin secretion (Rana et al., 1995; Horti et al., 1998).

SUMMARY Prolactin is a tetrahelix bundle cytokine most closely related to growth hormone and placental lactogen, and binds to specific prolactin receptors that belong to the WS-motif cytokine receptor family. Prolactin is secreted in a highly regulated manner into the circulation by the anterior pituitary, and acts on peripheral target tissues as a hormone. In addition, prolactin is expressed at many extrapituitary sites, particulary within female and male reproductive organs and cells of the immune system, acting locally as an autocrine or paracrine cytokine. Due to the ubiquitous expression of prolactin receptors, prolactin has a wide range of cellular and physiological effects. In mammals, prolactin is particularly critical for differentiation of the mammary gland and for lactation. Hyperprolactinemia, the most common pituitary disorder, causes infertility and decreased libido in men and women. Finally, prolactin has been implicated as a promoter of neoplastic growth, and may also influence the activity of certain autoimmune diseases.

ACKNOWLEDGEMENTS The work has been supported by National Institutes of Health grants DK52013 and CA83813.

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transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na-K-2Cl- cotransporter. Mol. Endocrinol. 14, 2054–2065. Servatius, R.J., Natelson, B.H., Moldow, R. et al. (2000). Persistent neuroendocrine changes in multiple hormonal axes after a single or repeated stressor exposures. Stress 3, 263–274. Shirota, M., Banville, D., Ali, S. et al. (1990). Expression of two forms of prolactin receptor in rat ovary and liver. Mol. Endocrinol. 4, 1136–1143. Shiu, R.P. (1979). Prolactin receptors in human breast cancer cells in long-term tissue culture. Cancer Res. 39, 4381–4386. Shiu, R.P. and Iwasiow, B.M. (1985). Prolactin-inducible proteins in human breast cancer cells. J. Biol. Chem. 260, 11307–11313. Shome, B. and Parlow, A.F. (1977). Human pituitary prolactin (hPRL): the entire linear amino acid sequence. J. Clin. Endocrinol. Metab. 45, 1112–1115. Siegel, R.A., Conforti, N. and Chowers, I. (1980). Neural pathways mediating the prolactin secretory response to acute neurogenic stress in the male rat. Brain Res. 198, 43–53. Sinha, Y.N. (1995). Structural variants of prolactin: occurrence and physiological significance. Endocr. Rev. 16, 354–369. Sissom, J.F., Eigenbrodt, M.L. and Porter, J.C. (1988). Antigrowth action on mouse mammary and prostate glands of a monoclonal antibody to prolactin receptor. Am. J. Pathol. 133, 589–595. Smith, M.S. (1980). Role of prolactin in regulating gonadotropin secretion and gonad function in female rats. Fed. Proc. 39, 2571–2576. Soares, M.J., Muller, H., Orwig, K.E. et al. (1998). The uteroplacental prolactin family and pregnancy. Biol. Reprod. 58, 273–284. Somers, W., Ultsch, M., De Vos, A.M. and Kossiakoff, A.A. (1994). The X-ray structure of a growth hormoneprolactin receptor complex. Nature 372, 478–481. Sorenson, R.L. and Brelje, T.C. (1997). Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm. Metab. Res. 29, 301–307. Sorin, B., Vacher, A.M., Djiane, J. and Vacher, P. (2000). Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor. J. Neuroendocrinol. 12, 910–918. Srivastava, R.K., Gu, Y., Ayloo, S. et al. (1998). Developmental expression and regulation of basic fibroblast growth factor and vascular endothelial growth factor in rat decidua and in a decidual cell line. J. Mol. Endocrinol. 21, 355–362. Steger, R.W., Chandrashekar, V., Zhao, W. et al. (1998). Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 139, 3691–3695. Stenn, K.S. and Paus, R. (1999). What controls hair follicle cycling? Exp. Dermatol. 8, 229–233; discussion 233–236. Stewart, E.A., Jain, P., Penglase, M.D. et al. (1995). The myometrium of postmenopausal women produces prolactin in response to human chorionic gonadotropin and alpha-subunit in vitro. Fertil. Steril. 64, 972–976. Stier, C.T., Jr., Cowden, E.A., Friesen, H.G. and Allison, M.E. (1984). Prolactin and the rat kidney: a clearance and micropuncture study. Endocrinology 115, 362–367.

Stocklin, E., Wissler, M., Gouilleux, F. and Groner, B. (1996). Functional interactions between Stat5 and the glucocorticoid receptor. Nature 383, 726–728. Stoecklin, E., Wissler, M., Moriggl, R. and Groner, B. (1997). Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription. Mol. Cell. Biol. 17, 6708–6716. Strain, A.J. and Ingleton, P.M. (1990). Growth hormone- and prolactin-induced release of insulin-like growth factor by isolated rat hepatocytes. Biochem. Soc. Trans. 18, 1206. Stricker, P. and Grueter, R. (1928). Action du lobe anterieur de l’hypophyse sur la montée laiteuse. Compt. Rend. Soc. Biol. (Paris) 99, 1978–1980. Struman, I., Bentzien, F., Lee, H. et al. (1999). Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: an efficient mechanism for the regulation of angiogenesis. Proc. Natl. Acad. Sci. USA 96, 1246–1251. Suard, Y.M., Kraehenbuhl, J.P. and Aubert, M.L. (1979). Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits. J. Biol. Chem. 254, 10466–10475. Tabar, L., Dean, P.B., Duffy, S.W. and Chen, H.H. (2000). A new era in the diagnosis of breast cancer. Surg. Oncol. Clin. N. Am. 9, 233–277. Teglund, S., McKay, C., Schuetz, E. et al. (1998). Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell. 93, 841–850. Tessier, C., Prigent-Tessier, A., Ferguson-Gottschall, S. et al. (2001). PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity. Endocrinology 142, 4086–4094. Thorner, M.O., Vance, M.L., Laws, E.R.J. et al. (1998). The Anterior Pituitary. Philadelphia: WB Saunders. Tomic, S., Chughtai, N. and Ali, S. (1999). SOCS-1, -2, -3: selective targets and functions downstream of the prolactin receptor. Mol. Cell. Endocrinol. 158, 45–54. Topper, Y.J., Sankaran, L., Chomczynski, P. et al. (1986). Three stages of responsiveness to hormones in the mammary cell. Ann. NY Acad. Sci. 464, 1–10. Torner, L., Toschi, N., Pohlinger, A. et al. (2001). Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of antisense targeting of the prolactin receptor by molecular modeling. J. Neurosci. 21, 3207–3214. Truong, A.T., Duez, C., Belayew, A. et al. (1984). Isolation and characterization of the human prolactin gene. Embo. J. 3, 429–437. Turkington, R.W. (1972). Phenothiazine stimulation test for prolactin reserve: the syndrome of isolated prolactin deficiency. J. Clin. Endocrinol. Metab. 34, 246–249. Tyson, J.E. (1982). The evolutionary role of prolactin in mammalian osmoregulation: effects on fetoplacental hydromineral transport. Semin. Perinatol. 6, 216–228. Valve, E.M., Nevalainen, M.T., Nurmi, M.J. et al. (2001). Increased expression of FGF-8 isoforms and FGF receptors in human premalignant prostatic intraepithelial neoplasia lesions and prostate cancer. Lab. Invest. 81, 815–826. Vara, J.A., Caceres, M.A., Silva, A. and Martin-Perez, J. (2001). Src family kinases are required for prolactin induction of cell proliferation. Mol. Biol. Cell. 12, 2171–2183.

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7 Erythropoietin Christoph Kasper Klinik für Innere Medizin II, Friedrich-Schiller-Universität, Jena, Germany

To teach a man how he may learn to grow independently, and for himself, is perhaps the greatest service that one man can do for another. Benjamin Jowett

INTRODUCTION In 1906, Carnot and DeFlandre demonstrated the occurrence of a retuculocytosis in normal rabbits injected with plasma from rabbits made anemic by bleeding and thus postulated a humoral factor they called hemopoietin. The reports by Hjort in 1936, Krumdieck in 1943, and Erslev in 1953 confirmed the existence of a humoral factor that controls erythropoiesis, and Bonsdorff and Jalavisto named it erythropoietin (1948). In 1950, Reissman demonstrated that hypoxia stimulated the production of erythropoietin. In the following years the kidney was found to be the site of production of erythropoietin (Jacobson et al., 1957; Fisher and Birdwell, 1961; Kuratowaska et al., 1961; Nathan et al., 1964). Erythropoietin was the first hematopoietic growth hormone to be cloned. In 1977, Miyake et al. isolated and purified erythropoietin from the urine of severely anemic patients. The identification of the amino acid sequence of the pure urinary protein enabled it to The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

synthesize erythropoietin DNA probes for isolation and cloning of the erythropoietin gene (Jacobs et al., 1985; Lin et al., 1985; Powell et al., 1986). Chinese hamster ovary (CHO) cells transfected with the erythropoietin gene linked to an expression vector were shown to produce biologically active recombinant human erythropoietin, and hence, recombinant human erythropoietin became available for clinical use. Erythropoietin is the primary regulator of erythropoiesis, and promotes the survival, proliferation and differentiation of erythroid progenitor cells.

STRUCTURE OF ERYTHROPOIETIN The erythropoietin gene is highly conserved between species (Table 7.1). There is a 80–82% amino acid identity between the human sequence and that of pig, sheep, mouse and rat erythropoietin genes (Wen

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TABLE 7.1 Properties of human erythropoietin and the human erythropoietin receptor Erythropoietin Protein Precursor protein: amino acids Mature protein: amino acids Molecular weight (kDa) N-Glycosylation sites Disulphide bonds Intracytoplasmatic tyrosine Gene Gene size (kb) Gene exons Gene introns Gene location: chromosome Cell sources

Erythropoietin receptor

193 165 30.4 3 2

507 66 0 2 8

5.4 5 4 7q11–q22

8 7 19p

interstitial kidney cells, hepatocytes (brain, placenta, testes, spleen, lung)

CFU-E, BFU-E, megacaryocytes, placental tissue, endothelial cells, neural cells

et al., 1993). The gene encoding human erythropoietin contained a single-copy in a 5.4 kb region (Jacobs et al., 1985; Lin et al., 1985) and is located on chromosome 7q11–q22 (Law et al., 1986; Powell et al., 1986; Watkins et al., 1986). It contains five exons and four introns coding for the prohormone of 193 amino acids (Jelkmann et al., 1992). Intron I, the 5 and 3 flanking regions are also highly conserved between species (Galson et al., 1993) and are involved in the regulation of the erythropoietin gene expression (see below). The 3.9 kb of the 5 flanking sequence contains consensus recognition sites for various cytokines, metal-response elements, glucocorticoidresponse elements, and adenosine 3,5-cyclic monophosphatase-response elements, as well as binding sites for AP1, nuclear factor (NF)jb, and Sp1 transcription factors (Huang et al., 1997). The 3 flanking region includes nitrogen-regulatory/oxygensensing consensus sequences, tissue-specific regulatory elements, and binding sites for AP and Sp1 (Lee-Huang et al., 1993). Erythropoietin is a glycoprotein with a molecular weight of 34 kDa, and the prohormone is synthesized as a 193 amino acid polypeptide. A leader sequence of 27 amino acids from the NH2 terminus and the arginyl residue from the carboxy-terminus are cleaved intracellularly from the primary translation product (Lai et al., 1986; Recny et al., 1987). The mature glycoprotein hormone consists of 165 amino acids, and the erythropoietin peptide has a molecular mass of 18 kDa

(Lai et al., 1986). It is extensively glycosylated with a carbohydrate moiety of approximately 40%, resulting in a total mass of 30.4 kDa (Davis et al., 1987). There are three tetraantennary N-linked (Asp 24, 38 and 83) and one small O-linked (Ser 126) acidic oligosaccharide side chains (Egrie et al., 1986; Lai et al., 1986; Sasaki et al., 1987; Broudy et al., 1988), required for the correct processing and export of the hormone (Delorme et al., 1992; Dube et al., 1988), molecular stability (Narhi et al., 1991), and for its full in vivo activity (Higuchi et al., 1992). The carbohydrate contains fructose, mannose, N-acetylglucosamine, galactose and N-acetyl neuraminic acid (Dordal et al., 1985). Four cysteines residues linked by disulfide bonds between cysteine 7–161 and 29–33 (Sytkowski, 1980; Wang et al., 1985) are essential for full in vivo biological activity (Nielsen et al., 1987). Despite little similarity in the amino acid sequence erythropoietin has a tertiary conformation analogous to that of growth hormone, prolactin and interleukin-6 (Bazan, 1990a). Recombinant human erythropoietin expressed in CHO cells has been compared with human urinary erythropoietin by circular dichroism, UV light absorbance, and fluorescence spectroscopy, without identifying any significant difference in amino acid sequence, position of their two disulfide bridges, glycosylation position, and secondary structure (Davis et al., 1987). However, recombinant human erythropoietin has a higher in vivo activity than urinary

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erythropoietin (Browne et al., 1986). Initially, a lyophilized human urinary erythropoietin with a specific activity of 1 international unit (IU) was defined as the activity which elicits the same erythropoiesisstimulating effect as 5 lg cobalt (Annable et al., 1972), but has been replaced now by a DNA-derived human erythropoietin standard with a specific activity of 130 000 IU per milligram fully glycosylated protein (Storring and Gaines Das, 1992). There are two forms of erythropoietin available for clinical use without any difference in the pharmacokinetics: the alpha-form, which contains 9% carbohydrate, and the beta-form, which contains 24% carbohydrate.

PRODUCTION Early studies in rats indicated that nephrectomy almost completely abolished the increase in erythropoietin plasma levels following bleeding (Jacobson et al., 1957), and cobalt (Fisher et al., 1961) or hypoxic stimulation (Kuratowaska et al., 1961) significantly increased the erythropoietin titers in perfusates of isolated kidneys. Furthermore, the degree of erythropoiesis in the bone marrow of anephric patientsis low (Nathan et al., 1964), and in patients with end-stage renal failure with severe anemia the serum erythropoietin level is extremely low, but will revert to normal after successful renal transplantation (Denny et al., 1966). Histochemistry studies in the mouse using in situ hybridization to localize erythropoietin mRNA confirmed that the kidney is a primary site of erythropoietin synthesis (Koury et al., 1988; Lacombe et al., 1988). In the kidney the mRNA is expressedininterstitialcellsoftheperitubularcapillary bed with fibroblast-like characteristics in various species (Eckard et al., 1989; Suzuki and Sasaki, 1990; Bachmann et al., 1993; Darby et al., 1995; Liapis et al., 1995; Fisher et al., 1996). There are no intracellular stores of erythropoietin, and anemia leads to an exponentialincreaseinthenumberofinterstitialcellsinparallel with the exponential increase in erythropoietin mRNA and serum erythropoietin (Koury et al., 1989). In patients with renal adenocarcinoma and polycythemia, their tumoral cells produce erythropoietin (Da Silva et al., 1990). The other major site of erythropoietin production in adults is the liver, contributing to approximately

10–15% to circulating plasma levels (Erslev et al., 1980). However, hepatic production cannot compensate for loss of the kidneys in case of chronic renal failure. The hepatocytes are the major cell type producing erythropoietin mRNA in the liver (Schuster et al., 1992). Using in situ hybridization in transgenic mice, epithelial cells surrounding the central veins have been identified to express erythropoietin mRNA (Koury et al., 1991). Those cells are supposed to share some characteristics with the erythropoietin producing cells in the kidney (Maxwell et al., 1994). On the other hand, the liver appears to be the major site of erythropoietin production during fetal life with a shift from the liver to the kidney during gestation (Zanjani et al., 1977, 1981), although more recent studies also demonstrated an early production in the kidneys (Lim et al., 1994; Wintour et al., 1996). To a minimal degree expression of erythropoietin mRNA has also been found in the brain (Masuda et al., 1994; Digicaylioglu et al., 1995; Marti et al., 1996; Yamaji et al., 1996), placenta (Conrad et al., 1996), testes, spleen and lung (Tan et al., 1991; Fandrey and Bunn, 1993).

REGULATION OF PRODUCTION Erythropoietin production is regulated by hypoxia that leads to an increase in the level of gene transcription (Figure 7.1) (Schuster et al., 1989). There are no intracellular stores of the hormone. The erythropoietin gene expression is controlled by complex interactions between DNA and nuclear proteins. Using transgenic mice expressing constructs of the human erythropoietin gene cis-regulatory regions have been identified (Semenza et al., 1989). Sequences required for the expression in the kidney, kidney inducible elements (KIE), have been located in a region 9.5 to 14 kb (Semenza et al., 1991a), and a negative regulatory element (NRE) in a region 0.4 to 6 kb from the 5 end of the human erythropoietin transcription start site (Semenza et al., 1990). Liver inducible elements (LIE) in a region are located within 0.7 kb to the 3-, and 0.5 kb to the 5-flanking regions. Transgenic mice lacking KIE and LIE, but still comprise NRE do not express erythropoietin mRNA in their kidneys (Semenza et al., 1991a).

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KIE kbp 14 9.5

NRE 6 0.4

I

II

III

IV

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5 Promoter

3 Enhancer p300 ARNT HIF-1a

HNF-4 HNF-4

FIGURE 7.1 Erythropoietin gene regulation. Downstream the 5 transcriptional site a 117 bp encompassing erythropoietin promoter does not have consensus TATA or CAAT elements, and acts synergistically with a 3 enhancer (Blanchard et al., 1992). Approximately 120 bp upstream the 3 end of the polyadenylation site the 50 bp enhancer responsible for hypoxia-inducible erythropoietin gene expression has been identified (Beck et al., 1991; Semenza et al., 1991b; Blanchard et al., 1992). The hypoxia-inducible enhancer contains three different segments (Semenza and Wang, 1992). Near the 5 end of the enhancer is a conserved sequence, the binding site for a transcription factor designated hypoxia-induced factor-1 (HIF-1) (Beck et al., 1993; Wang and Semenza, 1993). The C-terminal portion of HIF-1 binds to the adenovirus E1A binding p300 (Arany et al., 1996). In the middle a functional domain conveying oxygenregulates response has been identified (Pugh et al., 1994). The 3 end is a binding site for the hepatocyte nuclear factor 4 (HNF-4) (Galson et al., 1995). The formation of a complex of these proteins induced by hypoxia binds to the enhancer and thus signals to the promoter of the erythropoietin gene (Huang et al., 1997). HIF-1 has been purified from Hep3B and HeLa cells and characterized as a heterodimer transcription factor containing two basic helix–loop–helix proteins, a 120–kDa HIF-a and a 90-kDa HIF-b subunit (Wang et al., 1995; Wang and Semenza, 1995). HIF-a previously has been identified as the aryl hydrocarbon nuclear translocator sequence (ARNT) (Hoffman et al., 1991). Hypoxia does not alter the level of the mRNA encoding either HIF-a or HIF-b, suggesting a posttranscriptional mechanism of regulation. In addition to erythropoietin, HIF-1 interacts with vascular endothelial growth factor (VEGF), several glycolytic enzymes, glucose-transporter 1, inducible nitric oxide synthase, haem oxygenase and transferrin (Wenger

and Gassman, 1997). The HIF-1 induced activation of VEFG is also involved in angiogenesis and may play a role in tumorigenesis. However, oxygen sensing mechanisms are still not completely understood. It is proposed that the oxygen sensor is a heme protein that changes its conformation depending on the binding of oxygen to its heme moiety. The iron atom of heme can be replaced by cobalt, thereby mimicking the hypoxic state (Goldberg et al., 1988). Other studies suggested the involvement of a cytochrome P-450 protein (Fandrey, 1990), or of the hydrogen peroxide system (Fandrey et al., 1994). Cytokines such as interferon-gamma (IFN-c), tumor necrosis factor (TNF), and interleukin 1 (IL-1) have been reported to inhibit erythropoiesis in vitro as well as in vivo (Broxmeyer et al., 1986; Means and Krantz, 1991; Means et al., 1992), and these cytokines are able to inhibit the production of erythropoietin (Jelkmann et al., 1991; Faquin et al., 1992; Vannucchi et al., 1994). This provides a rationale for the clinical use of erythropoietin.

SITE OF ACTION Erythropoietin is essential for the viability, proliferation and differentiation in the erythrocytic lineage. The suppression of apoptoses is supposed to be the primary mechanism by which erythropoietin maintains the erythropoiesis (Koury and Bondwant, 1992). Following multipotent stem cells, the most primitive erythrocyte progenitor is the burst-forming unit-erythrocyte (BFU-E) which gives rise to colonyforming unit-erythroid (CFU-E). The BFU-E is mostly unresponsive to erythropoietin and requires other growth factors such as interleukin-3 (IL-3) and stem cell factor (SCF). Only low numbers of erythropoietin receptors are seen on more mature BFU-E. The density of the erythropoietin receptor on the cell surface increases with maturation to the CFU-E as the main target cell of erythropoietin (Sawada et al., 1990). When the cell reaches the orthochromatic normoblast stage during erythroid cell development the erythropoietin receptors almost completely disappear, and reticulocytes and mature erythrocytes do not have any receptors. However, the CFU-E contain only a low density of approximately 1000 erythropoietin receptor molecules per cell (Sawyer et al., 1989;

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D’Andrea and Zon, 1990). When erythropoietin binds to its receptor it is rapidly internalized, undergoing endocytoses and degradation (Sawyer et al., 1987). Mice homozygous for deletions of erythropoietin or its receptor die at embryonic day 12.5 owing to severe anemia (Wu et al., 1995b), though primitive yolk sacderived erythrocytes are still produced and their fetal livers contain normal numbers of BFU-E and CFU-E, indicating that erythropoietin signaling is not required for lineage determination, but for proliferation and differentiation in erythrocytes. Thus, erythropoietin is not involved in commitment of the erythroid lineage and seems to act mainly as a survival factor. It has been suggested that the erythropoietin receptor mainly transduces anti-apotopic signals. After infection with the prolactin receptors and further stimulation with prolactin rather than erythropoietin erythroid progenitors from murine fetal liver differentiate into erythroblasts (Socolovsky et al., 1997). While both, the prolactin and erythropoietin receptor belong to the same cytokine receptor family they trigger lineage differentiation according to the stochastic model. Furthermore, HCD57 cells infected with retroviral vectors encoding Bcl-2 or Bcl-xL remain viable in the absence of erythropoietin (Silva et al., 1996). However, erythropoietin may promote more than CFU-E survival. Although the overexpression of Bcl-2 or Bcl-xL in transgenic mice or fetal liver cells attenuates the programmed cell death no red cells development occurs (Lacronique et al., 1997; Chida et al., 1999). The function of erythropoietin is not restricted to erythroid committed cells. Erythropoietin receptors were also detected in megakaryocytic cells (Fraser et al., 1989; Yoshida et al., 1992), in placental tissue (Sawyer et al., 1989), in endothelial cells (Anagnostou et al., 1990, 1994), in neural cells (Masuda et al., 1993) (see below) and in human bronchial tumors (Kayser and Gabius, 1992).

RECEPTOR The erythropoietin receptor has been cloned by an expression strategy from a cDNA library of murine erythroleukemic cells (D’Andrea et al., 1989). The human erythropoietin receptor gene is localized on chromosome 19p (Winkelman et al., 1990). There are

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eight exons and seven introns, encoding a 507 amino acid peptide with a molecular weight of 66 kDa. In the human gene, exons 1–5 encode the 251-amino acid extracellular domain, exon 6 a 20-amino acid membrane-spanning a-helical region, and exons 7–8 the 236-amino acid cytoplasmic domain (Youssoufian et al., 1993). The human erythropoietin receptor has no N-linked glycosylation sites, but a high frequency of serine and threonine residues (Jones et al., 1990). There is 82% identity between the human and murine erythropoietin. Cross-linking of erythropoietin to the cell surface of erythroid cells has revealed two accessory molecules of 85 and 100 kDa that are not recognized by anti-p66 antibodies (D’Andrea and Zon, 1990; Mayeux et al., 1991). However, their function has to be determined. The erythropoietin receptor belongs to the type 1 family of single-transmembrane cytokine receptors. This family shares a conserved extracellular domain composed of fibronectin type III (FNIII) subdomains, as well as a conserved a-chain cytoplasmatic box 1 motif which binds selectively to Janus kinases (JAK) (Bazan, 1990b). The extracellular region of the erythropoietin receptor contains two FNIII-subdomains (D1 and D2), which form an L-shape, with the long axis of each domain aligned at approximately 90 to the other axis. The NH2-terminal D1 domain consist of four-on-four a-strands, and the D2 domain of seven aniparallel a-strands (Livnah et al., 1996). The D1 domain forms an h-type fold with a hybrid FNIII/immunoglobin-like topology, and two distal pairs of cysteine residues form disulfide bridges. The membrane proximal D2 domain folds with standard s-type FNIII topology, and contains a conserved WSXWS motif (i.e. tryptophan–serine–any amino acid–tryptophan–serine), which is important for the erythropoietin receptor folding (Quelle et al., 1992). D1 and D2 domains contribute together six loops for erythropoietin interactions. The cytoplasmatic region which is rich in the amino acids proline, glutamine, and aspartase contains a box 1 domain (residues 257–264) that is specific to JAK2 (Zhuang et al., 1994; Jiang et al., 1996), a box 2 domain (residues 303–313), and eight phosphotyrosine sites (Tyr 343, 401, 429, 431, 443, 460, 464, 479) that mediate the recruitment of Src homology-2 (SH2) domain-encoding effectors. An extended box 2 (residues 329–372) is essential for binding the tyrosine kinase receptor KIT after activation by

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its ligand and causes tyrosine phosphorylation of the erythropoietin receptor indicating a functional interaction between both receptors (Wu et al., 1995a). Erythropoietin activates the erythropoietin receptor by dimerization (Philo et al., 1996). One p66 molecule binds erythropoietin with high affinity (Kd around 1 nM), the other with a lower affinity (Kd around 2 lM). Using mutations and deletions, the active sites of erythropoietin have been mapped (Boissel et al., 1993; Wen et al., 1994; Elliott et al., 1997). Bivalent, but not monovalent monoclonal antibodies directed to the extracellular domain of the erythropoietin receptor induce proliferation of erythropoietin-dependent cell lines and formation of BFU-E suggesting a receptor activation through its dimerization (Elliot et al., 1996). The same effect can be obtained using small erythropoietin mimetic peptides (EMPs) (Livnah et al., 1996; Wrighton et al., 1996). Although EMP do not share any sequence homology with erythropoietin they bind specifically to the erythropoietin receptor. Point mutations in the extracellular domain (R129C, E132C or E133C) form disulfide bonds and constitutively also activate the receptor (Watowich et al., 1994). In particular, the mutation of the arginine 129 residue into a cysteine is oncogenic and induces erythroleukemia (Longmore and Lodish, 1991). On the contrary, EMP33 is able to dimerizate, but not to activate, the receptor indicating an essential role of the conformational change in the dimerized receptor for its signaling (Livnah et al., 1998; Remy et al., 1999). The existence of preformed inactive receptor dimers on the cell surface has been proposed, mediated by the D1–D2 intervening regions and providing a separation of 79 Å at the base of their extracellular domains (Livnah et al., 1996). After binding an agonist the extracellular receptor domains change their structure in a defined orientation with a spacing of 39 Å providing an adjustment of their cytoplasmatic components and leading to signaling (Wilson and Jolliffe, 1999). Besides binding erythropoietin, the erythropoietin receptor can be activated by other mechanisms. The gp55 envelope protein encoded by the murine Friend virus induces erythroleukemia in mice after binding to and activating the murine erythropoietin receptor (Wolff and Ruscetti, 1985; Li et al., 1990). Furthermore, synthetic peptides that do not share any sequence homology with erythropoietin are able to stimulate the erythropoietin receptor (see below).

SIGNALING The hemopoietin receptor family including the erythropoietin receptor does not have endogenous tyrosine kinase activity. Erythropoietin induces dimerization of its receptor, triggering activation of JAK2 tyrosine kinase which is constitutively associated with the erythropoietin receptor (Figure 7.2) (Witthuhn et al., 1993). Successively, the erythropoietin receptor is phosphorylated after erythropoietin stimulation and contains docking sites for various intracellular transcription factors. Subsequently these proteins can be phosphorylated and activated leading to a stimulation of several early response genes and effectors of mitogenesis (e.g. c-myc, c-jun, c-fos). The JAK2 molecule is constitutively associated to the box 1 domain of the erythropoietin receptor close to the transmembrane, and dimerization with conformational changes brings both JAK2 molecules into close proximity leading to transphosphorylation and activation of JAK2. Tyrosine 1007 corresponds to the activation loop in the other tyrosine kinase and mutation of Y1007 inactivates JAK2 (Feng et al., 1997). The activation of JAK2 leads to the phosphorylaion of the

EPO D1

D2

JAK2

JAK2 Y343 - P: Y401 - P: Y429 - P: Y431 - P: Y443 - P: Y460 - P: Y464 - P: Y479 - P:

STAT5, Pim1 STAT5, SHP-2, CIS1 (STAT5), SHP-1 (STAT5)

Lyn Lyn, PI-3

FIGURE 7.2 After binding of erythropoietin and homodimerization JAK2 is activated and this results in phosphorylation of the eight tyrosine residues of the erythropoietin receptor.

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eight tyrosines of the intracellular erythropoietin receptor domain. These phosphorylated tyrosines are docking sites for various intracellular proteins containing SH2 domains. Mice deficient for JAK2 die as embryos by day 13 from severe anemia and have an impaired number of erythroid progenitors, although the erythropoiesis in the yolk sac proceeds (Neubauer et al., 1998; Parganas et al., 1998). Thus JAK2 is required at an earlier stage of erythropoiesis than the erythropoietin receptor itself. The signal transducer and activator of transcription (STAT) pathway plays a major role in cytokineinduced signaling. Both, STAT5A and STAT5B are fully activated by the tyrosine residues Y343 and Y401, while Y429 and Y431 only partially activate them (Damen et al., 1995; Pallard et al., 1995; Wakao et al., 1995; Chin et al., 1996; Gobert et al., 1996; Klingmüller et al., 1996; Quelle et al., 1996). JAK2-mediated STAT5 phosphorylation results in the formation of stable STAT5 homodimers. These translocate into the nucleus and bind to specific regulatory sequences. Adult mice defective in both STAT5 isoforms have a normal erythroid development, indicating a redundant role in erythropoieses (Teglund et al., 1998). However, embryonic STAT5 / mice evolve severe anemia and this may result from a dysregulated erythropoietin/JAK2/STAT5/Bcl-X response pathway (Socolovsky et al., 1999). Upon its phosphorylation by JAK2, STAT5 stimulates several transcription factors, e.g. cytokineinducible SH2 protein-1 (CIS1), Pim1 and Bcl-XL. CIS1 is a member of the CIS/SOCS/JAB protein factors family containing a C-terminal 47-AA to 50-AA CIS/SOCS homology domain and a central SH2 domain (Yoshimura, 1998). The divergent aminoterminal domains may inhibit phosphorylated tyrosine kinase. CIS1 gene expression is induced by erythropoietin and its transcription mediated by STAT5 (Yoshimura et al., 1995). Binding at Y401 CIS1 inhibits STAT5 signaling and may promote erythropoietin receptor degradation (Matsumoto et al., 1997; Verdier et al., 1998). After phosphorylation of Y343 the Pim1 kinase is activated (Miura et al., 1994a), and this may attenuate apoptosis after cytokine withdrawal (Lilly and Kraft, 1997). In particular, erythropoietin increases Bcl-XL and Bcl-2 expression (Silva et al., 1996; Quelle et al., 1998; Gregory et al., 1999). During the terminal differentiation stages of

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human and mouse erythroblasts Bcl-XL expression strongly increases with little effect on other members of the Bcl-2 family (Gregoli and Bondurant, 1997), proposing Bcl-XL as the primary effector of erythropoietin-dependent erythroid progenitor cell survival. STAT5A- and B-deficient mice bear severe anemia at day 13.5 during embryotic development, and derived erythroid progenitor cells undergo programmed cell death. Presumably, this may result from a dysregulated erythropoietin/JAK2/STAT5/ Bcl-X response pathway (Socolovsky et al., 1999). Recently, it has been shown that STAT5A- and Bdeficient mice either had near-normal steady-state hematocrit values, but could not efficiently increase erythropoietic rate in response to stress, or had chronic anemia, suggesting a severe deficit in steadystate erythropoiesis (Socolovsky et al., 2001). In these mice a correlation between decreased Bcl-XL expression and increased apoptosis of early erythroblasts with the severity of anemia has been found. Alternatively, an activation of STAT1 and STAT3 may become a compensatory mechanism. Both, STAT1 and STAT3 can bind the promoter of the Bcl-XL gene and up-regulate the gene expression. In murine erythroleukemia cell lines STAT1 and STAT3 were activated by erythropoietin (Ohashi et al., 1995; Penta and Sawer, 1995). Recently, it has been shown that Y432 is critical for the erythropoietin-induced activation of STAT1 and STAT3 and that JAK2 and c-Fes tyrosine kinases are directly involved in their activation (Kirito et al., 2002). Apart from JAK2 other signaling factors may also be involved in erythropoietin signaling. The Src tyrosine kinase Lyn binds with its SH2 domain to phosphorylated Y464 and Y479 and activates STAT5 (Chin et al., 1998). Although Lyn homozygous knockout mice do not exhibit obvious hematopoietic deficiencies, a defect in Lyn expression blocks the ability of erythropoietin to induce differentiation in erythroleukemic J2E cells (Tilbrook et al., 1997). However, antisense oligonucleotides against Raf-1 and against Src, but not against Lyn inhibit cell proliferation (Caroll et al., 1991; Kubota et al., 2001). At least the two other phospho-tyrosine kinases Syk (Duprez et al., 1998) and Tec (Machide et al., 1995; Yamashita et al., 1998) are known to be involved in erythropoietin signaling. The second signaling pathway activated through the erythropoietin receptor is the mitogen-activated

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protein kinase (MAPK) pathway. The phosphorylated tyrosine 479 recruits the SH2 domain of the p85 subunit of the phophatidylinositol-3 (PI3) kinase and this may result in activation of the extracellular-signalregulated (ERK)-2 MAPK through a Ras-independent pathway (Damen et al., 1993b; Klingmüller et al., 1997). Phosphorylated Y479 does not activate STAT5 or Ras, but supports the formation of normal numbers of CFU-E and BFU-E. The p110 subunit of PI3 kinase phosphorylates phosphatidylinositol 4,5-biphosphate at the 3 position to yield phosphatidylinositol 3,4,5triphosphate. An alternative pathway for the activation of PI3-kinase involves tyrosine phosphorylation of the adaptor protein insulin receptor substrate-2 (IRS2) and its subsequent association with PI3-kinase (Verdier et al., 1997). This may account for the activation of PI3-kinase by erythropoietin receptor mutants lacking Y479, and for the PI3-kinase-linked activation of Ras/MAPK. PI3-kinase is also connected to the MAPK-pathway via kinase C-epsilon (PKC-e) (Klingmüller et al., 1997). Downstream of PI3-kinase SHIP and AKT, a regulator of Bad, are activated (Damen et al., 1996; Lioubin et al., 1996; Franke et al., 1997). SHIP associates with the erythropoietin receptor via SHC and Grb2, and catalyzes the hydrolysis of phosphatidylinositol 3,4,5-triphosphate and inositol 1,3,4,5-tetraphosphate to phosphatidylinositol 3,4biphosphate and inositol 1,3,4-triphosphate, respectively (Damen et al., 1993a). Grb2 associates with SHC at phosphorylated Y464 and subsequently binds to mSOS/Ras/Raf (Ravichandran et al., 1995; Gram et al., 1997). Following erythropoietin stimulation Gab2 provides docking sites for both SHP2 and PI3-kinase (Ravichandran et al., 1995). Activation of phospholipase C-gamma (PLC-c) via phosphorylation induces diacylglycerol production, 1,4,5-triphosphate formation, and Ca2 mobilization (Ren et al., 1994b). Furthermore, PLC-c stimulates PKC-e leading to transcription of c-myc (Li et al., 1996). Although the MAPK pathway is sufficient for normal erythroid differentiation, it does not appear to be essential (Miura et al., 1994). Mutant receptors lacking Y479 still support erythroid differentiation. Through activation of several anti-apoptotic proteins such as Bcl-2, Bcl-XL and protein kinase B erythropoietin maintains erythroid differentiation. Similarly, the activation of AP-1 transcription factors mediated by Y343 and Y464 leads to an inhibition of programmed

cell death (Bergelson et al., 1998). This indicates a redundancy in the erythropoietin receptor-mediated signaling pathway. Two tyrosine phosphatases, SHP-1 and SHP-2, are also involved in erythropoietin-induced signaling. SHP-2 plays a positive role in stimulating cell proliferation. It is phosphorylated after binding to Y401, and mutation of Y401 blunts proliferative activity (Tauchi et al., 1996). SHP2 activation depends on tyrosine phosphorylation of its two SH2 domains (Pluskey et al., 1995). Specific substrates for SH2 are not established, although Grb2, Gab1, Shc, SHIP and PI3kinase may be involved. In contrast, the phosphatase SHP1 (or hematopoietic cell phosphatase) acts as a negative regulator subsequently leading to dephosphorylation of JAK2 by binding to Y429 (Klingmüller et al., 1997). Cells with a truncated erythropoietin receptor lacking this portion of the intracellular domain are not able to bind SHP1 and are hypersensitive to erythropoietin. An autosomal dominant benign form of polycythemia in family members lacking Y429 has been described (De la Chapelle et al., 1993). While the erythropoietin receptor is essential for terminal differentiation of erythroid cells, it does not induce any unique intracellular signal. For example, the prolactin receptor can rescue EPO / erythroid progenitors (Socolovsky et al., 1998), and using the same signaling proteins the product of the bcr-abl oncogene can also induce erythroid differentiation. Bcr-abl effectively supports normal erythroid proliferation, differentiation and maturation in JAK2deficient fetal liver cells (Ghaffari et al., 2001). Recently, it has been shown in mouse embryos that human granulocyte–macrophage colony-stimulating factor (hGM-CSF) can stimulate the proliferation of primitive and definitive erythroid cells independently of erythropoietin receptor signal if they express the hGM-CSF receptor, but not erythropoietin receptors, and the activity is comparable to that of erythropoietin in definitive, but not primitive, erythropoiesis (Hisakawa et al., 2001). Interestingly, the hGM-CSF receptor activates signaling molecules similar to the erythropoietin receptor, such as JAK2 and STAT5. The transcription factor GATA-1 is essential for normal erythropoieses. The family of GATA transcription factors bind to the DNA of promoter or enhancer of many genes comprising two zinc fingers. GATA1 and

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SP1 appear to cooperate with other transcriptional factors to activate or inhibit transcription (Fisher et al., 1996). Mice deficient for GATA1 die of anemia at 9.5–10.5 days of embryonic development (Fujiwara et al., 1996), and similarly, GATA2 and GATA3 have been shown to be essential for erythropoiesis (Ohyagi et al., 1994). There is a negative regulatory element at 30 bp in the 5 promoter region of the erythropoietin gene containing a GATA site (Imagawa et al., 1994), and the gene encoding the erythropoietin receptor contains functional GATA-binding sites in its promoter and enhancer (Zon et al., 1991; Heberlein et al. 1992). It has been shown that both GATA-1 and erythropoietin, cooperate to promote erythroid cell survival by regulating Bcl-X, thus preventing programmed cell death, while more immature erythroid cells require c-kit ligand (Gregory et al., 1999; Kapur and Zhang, 2001). Activation of protein kinase C (PKC) by erythropoietin maintains GATA-2 and Bcl-XL expression (Tsushima et al., 1997).

CLINICAL STUDIES In patients with chronic renal failure and low endogenous serum erythropoietin level, rhEPO – epoietin alfa and beta – has been successfully used to correct anemia since 1985 (Winearl et al., 1986; Eschbach et al., 1987). Between 90 and 95% of the patients treated with recombinant human erythropoietin have a positive response, become transfusion-independent, and improvement of anemia is associated with an improved quality of life, increased exercise capacity, as well as an improved cardiac function. Initially, rhEPO was administered intravenously three times weekly, but more recent studies have confirmed the efficacy and tolerability of subcutaneous administration of rhEPO once to three times weekly (Weiss, 2001). The US National Kidney FoundationsDialysis Outcome Quality Initiative (NKF-DOQI) guidelines recommend a target hematocrit of between 33 and 26% or a hemoglobin concentration between 11 and 12 g/dl (NKF-DOQI Work Group, 1997; Eknoyan et al., 2001), and the European Best Practive Guidelines (EBPG) a target hemoglobin greater than 11 g/dl with no upper limit specified (European Best Practive Guidelines, 1999). However, the optimum target hemoglobin concentration remains to be determined.

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While leading to an increased cardiac output anemia is a risk factor for left ventricular hypertrophy and dilation (Eckard, 2001). Although clinical trials indicate no cardiovascular benefit in correcting the anemia in patients with symptomatic cardiac disease or severe left ventricular dilation (Besarab et al., 1998; Foley et al., 2000), there is evidence that earlier intervention can prevent cardiovascular disease (Foley et al., 2000; McMahon et al., 2000). This however, is the subject of the CREATE (cardiovascular reduction early anemia treatment with epoietin beta) study (Macdougall, 2001). A continued need for more than 300 IU kg1 rhEPO subcutaneously (or more than 450 IU kg1 intravenously), seen in approximately 5–10% of the patients, has been defined as an inadequate response to rhEPO (NKF-DOQI Work Group, 1997). The most common cause is an absolute or functional iron deficiency. According to the EBPG (1999), ferritin levels should be kept between 200 and 500 ng ml1, the transferrin saturation between 20–40%, and the percentage of hypochromic red blood cells below 10%. In case of iron deficiency intravenous iron should be supplemented. Other causes for hyporesponsiveness to rhEPO include infection and inflammation, severe hyperparathyroidism, inadequate dialysis and deficiencies of vitamin B12, folic acid and possibly vitamin C (Drüeke, 2001). About one-third of end-stage renal disease patients maintained on hemodialysis and rhEPO therapy develop arterial hypertension (Winearl et al., 1986; Eschbach et al., 1987; NKF-DOQI Work Group, 1997; EBPG, 1999). Other side effects include thrombotic events and seizures. Recently, a novel erythropoieses-stimulating protein (NESP) with a prolonged half-life has been proven efficient (e.g. darbepoetin alfa) (Egrie et al., 1997; Macdougall et al., 1999; Glaspy et al., 2001). The amino acid sequence of NESP differs from human erythropoietin at 5 positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn and Pro90Thr), allowing additional N-linked carbohydrate chains at positions 30 and 88. It has a molecular weight of 37.1 kDa. Outside the setting of uremia, recombinant human erythropoietin has been proven effective in several indications (Cazzola et al., 1997). Independently from other causes the anemia of chronic disease (ACD) is characterized by a normochromic/normocytic

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anemia with reticulocytopenia, a shortened survival of erythrocytes, hypoferremia in the presence of adequate iron stores, and an inappropriate low erythropoietin serum level (Cartwright, 1966; Cash and Sears, 1989; Miller et al., 1990; Means et al., 1992). Macrophages may play a central role in the pathogenesis, not only by the secretion of inhibitory cytokines but also by phagocytosis of erythrocytes. Certain cytokines such as IFNs, TNF and IL-1 are increased in patients with ACD (Denz et al., 1990; Faquin et al., 1992). Macrophages down-regulate their transferrinreceptor (TFR) expression and ferritin content when exposed to IFN-c (Byrd and Horwitz, 1993). This may result in a reduced availability of iron. Furthermore, erythroblasts of patients with ACD have a decreased number of TFRs and a lower affinity of the receptor to transferrin (Feelders et al., 1993). These cytokines are also able to inhibit the production of erythropoietin (Jelkmann et al., 1991; Faquin et al., 1992; Vannucchi et al., 1994). Interestingly, the inhibitory effect of those cytokines can be overcome by pharmacological concentrations of erythropoietin (Schooley et al., 1987; Johnson et al., 1989, 1990). In the early 1990s, Ludwig et al., (1990) successfully tested the efficacy and safety of recombinant erythropoietin in 13 anemic patients with multiple myeloma. Since then, several studies demonstrated the positive effect of rhEPO in the treatment of anemia in patients with cancer using different dose schedules and different response criteria (review in Kasper, 2001). Erythropoietin therapy corrected anemia in approximately 50–60% of patients with hematological malignancies, and in two-thirds of patients with solid tumors. A statistically significant correlation between the increase in hemoglobin level and improvement in overall quality of life (QOL) was demonstrated with the greatest improvement when the hemoglobin level increased from 11 to 12 g/dl (range, 11 to 13) (Glaspy et al., 1997; Demetri et al., 1998; Cleeland et al., 1999). While tumor oxygenation is an independent prognostic factor influencing overall survival (Vaupel et al., 1996), preliminary data suggest that combined antitumor treatment and therapy with recombinant erythropoietin may improve overall survival (Silver and Piver, 1998; Glaser et al., 1999; Littlewood et al., 2001). In the heterogeneous group of myelodysplastic syn-

dromes (MDS), only few patients show a significant response to treatment with erythropoietin (HellströmLindberg, 1994). However, it is becoming evident that the generally rather low response rate of erythropoietin in patients with MDS will be dramatically improved by the combination of erythropoietin with other cytokines such as granulocyte colony-stimulating factor (G-CSF) or granulocyte– macrophage colony-stimulating factor (GM-CSF) (Negrin et al., 1996; Hellström-Lindberg et al., 1998; Economopoulos et al., 1999; Remacha et al., 1999; Stasi et al., 1999) especially in prolonged administration (Mantovani et al., 2000). Besides in cancer patients, ACD occurs in different diseases and can be treated successfully with recombinant erythropoietin, e.g. rheumatoid arthritis (Baer et al., 1987; Kaltwasser et al., 2001) and AIDS (Fischl et al., 1990). Furthermore, recombinant human erythropoietin has been used in autologous blood donation (Goodnough et al., 1989) and the anemia of prematurity (Maier et al., 1994; Bader et al., 2001). Until recently, no important side effects have been associated with the administration of recombinant human erythropoietin in diseases other than renal failure (Ehmer et al., 1996). In uremia, however, severe hypertension is observed with occasionally encephalopathy and seizure. However, a French group identified 13 (plus nine more added in proof) patients in whom pure red-cell aplasia developed during treatment with recombinant human erythropoietin, in whom neutralizing antierythropoietin antibodies occurred (Casadevall et al., 2002). The patients became transfusion-dependent, but some of them responded to immunosuppressive therapy. These antibodies were not directed against the carbohydrate moiety. It has been suggested that a subtle difference in the carbohydrate structure of epoietin and endogenous erythropoietin creates an epitope on the epoietin polypeptide to which an antibody binds, thereby inactivating not only epoietin, but the endogenous hormone as well (Bunn, 2002). Functional iron deficiency, defined as an imbalance between iron needs in the erythroid marrow and iron supply, is a major factor limiting the efficiency of erythropoietin therapy (Beguin, 1998). Iron supplements should be given when the percentage of hypochromic red blood cells is greater than 10%, or the transferrin saturation below 15%.

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REFERENCES

ERYTHROPOIETIN BEYOND ERYTHROPOIESIS Erythropoietin is not just an ‘erythropoietin’ but may play a broader biologic role (Cerami, 2001). The presence of erythropoietin receptors in neural cells (Masuda et al., 1993) and the ability of neurons and astrocytes to produce erythropoietin under hypoxic conditions has been detected (Masuda et al., 1994; Digicaylioglu et al., 1995; Marti et al., 1996;Yamaji et al., 1996). Furthermore, erythropoietin might play a role in brain development (Liu et al., 1994). Pretreatment with erythropoietin attenuated neural damage in animal models, but administration of soluble erythropoietin receptor exacerbated tissue damage (Sakanaka et al., 1998; Brines et al., 2000). It has been suggested that erythropoietin is able to cross the blood–brain barrier via a specific receptor-mediated transport mechanism (Brines et al., 2000). Furthermore, systemic administration of erythropoietin after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction (Sirén et al., 2001). The erythropoietininduced neuropotection may be a result of the inhibition of programmed cell death via activation of specific transcription factors (Digicaylioglu and Lipton, 2001; Sirén et al., 2001). Thus, erythropoietin might provide new stroke treatment.

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8 Interleukin-2 Jian-Xin Lin and Warren J. Leonard Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Things should be made as simple as possible, but not any simpler. Albert Einstein

INTRODUCTION Interleukin-2 (IL-2) is the prototype member of a family of cytokines that have pleiotropic actions in the immune system. It was discovered in 1976 as a growth factor present in conditioned medium from phytohemagglutinin (PHA)-stimulated normal human lymphocytes that was able to specifically support the growth of activated normal T lymphocytes in vitro and was therefore denoted as T cell growth factor (TCGF) (Morgan et al., 1976). The early biochemical characterization of natural IL-2, generation of purified human IL-2 from Jurkat T cells, the subsequent molecular cloning of its cDNA from various mammalian species, and the eventual production and purification of large quantities of recombinant IL-2 greatly facilitated investigations of this molecule, its signaling pathways and its actions. Especially during the past decade, our knowledge of

the molecular mechanisms by which IL-2 exerts its effects has tremendously advanced, in part as the result of studies using mice in which IL-2, each chain of the IL-2 receptor, as well as a number of IL-2activated signaling molecules have been individually deleted by homologous recombination. Moreover, in humans, mutations in the genes encoding IL-2 and IL-2Ra cause autoimmunity, while defective expression of IL-2Rb, IL-2Rc, or Jak3 has each been found in patients with severe combined immunodeficiency syndromes. Defective expression of these proteins in these patients provides the molecular bases for developing clinical protocols for gene therapy eventually to correct these immunodeficiency disorders. In addition, with the discovery that both IL-2Rb and IL-2Rc are shared by receptors for other IL-2 family cytokines, we have acquired an enhanced appreciation of the contributions of these molecules towards cytokine specificity, pleiotropy and redundancy.

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IL-2 cDNA cloning, genomic structure and chromosomal localization Human IL-2 has a molecular weight of about 15 kDa as estimated by gel filtration, and its heterogeneity in mobility on SDS-PAGE is most likely due to the posttranslational modifications of the molecule, including differential glycosylation (Gillis et al., 1980; Robb et al., 1981). Human and murine IL-2 cDNAs predict open reading frames of 153 amino acids and 169 amino acids, respectively. The main difference between human and murine IL-2 is a unique repeat of 12 consecutive glutamine residues at the N-terminus of mature murine IL-2 (Kashima et al., 1985). The significance of this glutamine segment is unclear. Both the human and murine IL-2 pro-proteins contain a 20 amino acid long signal peptide sequence (Devos et al., 1983; Taniguchi et al., 1983; Kashima et al., 1985; Yokota et al., 1985; Degrave et al., 1986). The homology in the coding regions between human and murine IL-2 is 72% at the nucleotide level and 65% at the amino acid level (Degrave et al., 1986). IL-2 cDNAs from gibbon (Chen et al., 1985), bovine (Cerretti et al., 1986; Reeves et al., 1986), rat (McKnight et al., 1989), ovine (Seow et al., 1990), and porcine (Goodall et al., 1991) sources have also been isolated. Among these species, the overall homology for IL-2 at the amino acid level is approximately 50%. The IL-2 gene is located on human chromosome 4q (Seigel et al., 1984), mouse chromosome 3 (Webb et al., 1990), and feline chromosome B1 (Seigel et al., 1984). The human and murine IL-2 genes are similarly organized: both contain a TATA box, a transcription initiation site, four exons, three introns, and two potential polyadenylation signals (Fujita et al., 1983; Fuse et al., 1984; Degrave et al., 1986).

function relationships. Data from X-ray crystallographic studies (Brandhuber et al., 1987a, 1987b) that were later refined by computer modeling methodology (Bazan, 1992) (Plate 8.1) (see Plate section), and which also incorporated the data from mutagenesis studies, have revealed that the human IL-2 molecule contains four core a-helices B, C, A and D, which are connected by a long downward A-B loop that also contains a short helical segment initially identified by X-ray crystallography, a B-C loop and a long C-D crossover loop. Proline 65 (Pro 65) disrupts the B helix into B and B helices. The B helix is connected to the C-D loop via the formation of a disulfide bond between amino acids Cys 58 and Cys 105. Both of these two cysteine residues are important for biological activity of IL-2, whereas Cys 125 is not (Wang et al., 1984). Analyses in which various regions of IL-2 were deleted revealed that the amino terminus (residues 1–20), the carboxyl terminus (residues 121–133) and two of the three cysteine residues (Cys 58 and Cys 105) (Ju et al., 1987; Collins et al., 1988) are vital for biological activity. There are three forms of IL-2 receptors, binding IL-2 with low-, intermediate-, or high-affinity. The low-affinity receptor contains IL-2Ra, intermediatedaffinity receptor contains IL-2Rb and IL-2Rc (now denoted as the common cytokine receptor c chain, cc; discussed below), and high affinity receptor contains all three chains (Figure 8.2). Site-directed mutagenesis analyses have demonstrated that four amino acids (Lys 35, Arg 38, Phe 42 and Lys 43) in the A-B loop of human IL-2 appear to be essential for its interaction with the IL-2Ra chain, but not with IL-2Rb (Weigel et al., 1989; Sauve et al., 1991). Conversely, Asp 20 of Affinity Low-

IL-2

IL-2

Structural and functional relationships for IL-2 As an important step towards understanding the functions of IL-2 and thereby towards designing therapeutic approaches to modulate immune responses mediated by this protein, many efforts were initiated in the late 1980s to early 1990s to resolve its tertiary structure and understand its structure/

Intermediate-

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IL-2

IL-2Rα γc

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FIGURE 8.2 Different combinations of three IL-2 receptor subunits form low-affinity, intermediateaffinity, and high-affinity IL-2 receptors.

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human IL-2 (Collins et al., 1988; Weigel et al., 1989) and Leu 31, Asp 34, Leu 35 and Leu 38 of murine (Zurawski and Zurawski, 1989; Zurawski et al., 1990) IL-2 in the A helix are required for its interaction with IL-2Rb but not with IL-2Ra. Interestingly, Leu 17 in the A helix and Trp 121 in the D helix of human IL-2 (Collins et al., 1988) and Gln 141 in the D helix of mouse IL-2 (Zurawski et al., 1990) appear to be critical for the interaction of IL-2 with high affinity IL-2 receptors but not with the combination of IL-2Ra and IL-2Rb. Based on their spatial location (Plate 8.1), Leu 17 and Trp 121 are presumably not involved in direct interaction with receptor chains, but rather their mutation likely results in an altered conformation that no longer allows binding to high-affinity receptors. These data are consistent with an early observation that monoclonal antibodies raised against either of two peptides spanning amino acids 8–27 or 33–54 can block the binding of human IL-2 to its receptor (Kuo and Robb, 1986). However, more investigation of the structural and functional relationship as to how IL-2 interacts with each of the low-, intermediate-, and high-affinity IL-2 receptors is still needed.

IL-2 is primarily expressed by CD4 T cells Activation of mature, resting T cells by an antigen via specific T cell antigen receptor (TCR)/CD3 complexes initiates a complex cascade of signaling pathways that eventually leads to cellular responses, including proliferation and differentiation (Cantrell, 1996). Following antigen stimulation, a number of cellular genes essential for T-cell activation are coordinately induced. Among these genes are those encoding IL-2, IL-2Ra, and IL-2Rb (Waldmann, 1989; Cantrell, 1996). Rapid induction of IL-2 gene expression, mainly by CD4 T cells, has long been shown in vitro by stimulating T lymphocytes with antibodies to CD3 to mimic antigen stimulation, which activates a number of signaling pathways, including protein kinase C (PKC) and calcium pathways (Cantrell, 1996). A combination of the phorbol ester phorbol 2-myristate 3acetate (PMA), which activates PKC, and ionomycin, which increases intracellular calcium concentration, are sufficient to mimic TCR activation and induce IL-2 gene expression. Both PKC and calcium signals together are required for regulation of IL-2 gene

169 expression; neither signal alone is sufficient (Cantrell, 1996). Induction of IL-2 by anti-CD3 or PMA plus ionomycin is sensitive to the inhibitory effects of the immunosuppressive agents, cyclosporin A (CsA) and FK506 (Flanagan et al., 1991). CsA and FK506 are such potent inhibitors of transcription of the IL-2 gene that they can both prevent transcription initiation if added before stimulation with anti-CD3 and arrest the ongoing transcription if added after anti-CD3 stimulation. It should be emphasized that CsA and FK506 do not directly interfere with transcription of the IL-2 gene. Instead, CsA and FK506 first need to form a complex with their corresponding cellular binding proteins, immunophilins. These complexes then bind and sequester cytosolic calcineurin, a calcium-dependent phosphatase, thereby inhibiting the calciummediated signaling pathway (Clipstone and Crabtree, 1992; Schreiber and Crabtree, 1992; Bram et al., 1993; Rao et al., 1997). Activation of calcineurin is an essential step in controlling the translocation of NF-AT (nuclear factor of activated T cells) family transcription factors from the cytoplasm to the nucleus in response to an increase in intracellular calcium. NFAT family proteins can in turn bind to the NF-AT sites in the IL-2 promoter region and, together with other transcription factors, regulate the expression of the IL-2 gene (Schreiber and Crabtree, 1992; Cantrell, 1996; Rao et al., 1997; Kiani et al., 2000). Induction of IL-2 expression in T cells by antiCD3 stimulation can be markedly enhanced by costimulation of the cells with antibodies to CD28, whereas anti-CD28 antibody cross-linking alone is inefficient in inducing IL-2 expression (June et al., 1989; Thompson et al., 1989). The cooperative effects between TCR and CD28 signals are at least in part attributed to their ability to synergistically activate the MAP kinases JNK1 and JNK2 (Jun N-terminal kinases) (Su et al., 1994), which potently activates the transcription factor, AP-1 (Davis, 1994; Su and Karin, 1996). Induction of IL-2 gene expression by anti-CD28 is resistant to the inhibitory effects of CsA or FK506 (June et al., 1989; Thompson et al., 1989), reflecting the different signaling pathways activated by CD28 versus TCR stimulation. Although CD4 T cells are the major source of IL-2 production in response to TCR stimulation, it has recently been shown that a transient induction of IL-2

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mRNAs and production of the protein can be detected in murine dendritic cells, but not in macrophages, that have been activated by Gram-negative bacteria (Granucci et al., 2001). This suggests an important role of IL-2 in conferring T-cell stimulatory capacity to dendritic cells. The biological significance of this transient IL-2 production by dendritic cells is demonstrated by the impaired ability of dendritic cells from IL-2/ mice to induce T-cell proliferation despite the lack of other apparent abnormalities in dendritic cells from IL-2/ mice (Granucci et al., 2001). IL-2 has also been shown to be produced in certain murine B lymphoma cell lines induced by Staphylococcus aureus (Walker et al., 1988) and to be constitutively produced by a mature murine B cell line that expresses high affinity IL-2 receptors when IL-2Rb cDNA was stably transfected into these cells (Gaffen et al., 1996). However, production of IL-2 by primary B cells has not been demonstrated.

Regulation of IL-2 gene expression The vital roles played by IL-2 in the immune system have stimulated extensive investigation of the mechanisms as to how this gene is regulated during T-cell activation and how its induction can be prevented by immunosuppressive agents (Jain et al., 1995; Abraham and Wiederrecht, 1996; Cantrell, 1996; Rao et al., 1997). The induction of IL-2 mRNA in response to anti-CD3 or PMA/ionomycin is almost entirely regulated at the level of transcription (Shaw et al., 1988; Kronke et al., 1985; Tocci et al., 1989; Brorson et al., 1991; Garrity et al., 1994), whereas anti-CD28 affects the expression of IL-2 gene at both transcriptional and post-transcriptional levels (Lindstein et al., 1989; Fraser et al., 1991, 1992; Umlauf et al., 1995; Ragheb et al., 1999). Detailed analyses of the IL-2 promoter region have revealed that both stimulationdependent transcription factors, including NF-AT, AP-1, NF-jB and Egr-1, and constitutively expressed transcription factors, including octamer (Oct) and Sp1 proteins, are required for anti-CD3-mediated induction of the IL-2 gene (Schreiber and Crabtree, 1992; Jain et al., 1995; Skerka et al., 1995; Cantrell, 1996) (Figure 8.3). Rel/NF-jB family proteins and AP-1 proteins are involved in modulation of the anti-CD28 response (Jain et al., 1995; Cantrell, 1996) (Figure 8.3). The DNA binding sites for all of these

factors are located within a 300-bp proximal promoter/enhancer region, and the integrity of these binding sites is crucial for transcriptional activation of the IL-2 gene (Jain et al., 1995; Rao et al., 1997). Interestingly, almost all of these factors appear to be the targets for the immunosuppressive agents CsA and FK506, with NF-AT being the major target (Schreiber and Crabtree, 1992; Jain et al., 1995; Cantrell, 1996; Rao et al., 1997). The activation-dependent chromatin changes in the IL-2 promoter region were first identified by DNase I hypersensitivity assays (Siebenlist et al., 1986). Data from in vivo footprinting analyses further show that binding of these transcription factors to DNA occurs in a highly coordinated fashion that is strictly stimulation-dependent (Garrity et al., 1994). Recently, CHART-PCR (chromatin accessibility by real-time PCR) analyses have revealed that the IL-2 gene undergoes substantial chromatin remodeling upon T-cell activation, and this is limited to the same 300-bp proximal enhancer/promoter region identified by in vitro assays (Rao et al., 2001). Interestingly, restimulation of the cells triggers a much more rapid chromatin remodeling process than is seen upon primary stimulation (Rao et al., 2001). The longer lag period in chromatin remodeling following primary stimulation is likely due to the requirement for both pre-existing and newly synthesized transcription factors for the coordinated occupancy of the IL-2 proximal promoter/enhancer region. Interestingly, either removal of the stimulus or treatment of the cells with known inhibitors of IL-2 transcription, such as CsA or cAMP, can reverse this activation-dependent chromatin remodeling process. These data reveal why these immunosuppressive agents so potently inhibit IL-2 transcription.

Actions of IL-2 As mentioned above, there are three classes of IL-2 receptors, the low affinity (the IL-2Ra chain), the intermediate affinity (the IL-2Rb and cc chains), and the high affinity (IL-2Ra, IL-2Rb, and cc chains) receptors. The functional IL-2 receptors are the intermediate- and high-affinity forms. The bestknown action of IL-2 is to augment the proliferation of T lymphocytes in response to antigenic stimulation, including the generation of both cytotoxic and sup-

THE CYTOKINES AND CHEMOKINES

IL - 2

NFAT

ZIP

A

OCT

NF-AT

Sp1

171

NF-jB

CD28RE

NF-jB

Rel/NF-jB

NFAT

AP-1

AP-1/OCT

NF-AT

Oct

IL-2 300 Egr-1

AP-1

Oct

TATA

AP-1 UEI/NFIL-2RA

PRRIII

CD28rE

B

8483

8689 AP-1

3780 Stat5

PRRI 3703 Elf-1

PRRII

299

228 137 SRF

PRRIV

64 Elf-1

3515

3587 Stat5

IL-2Ra 8942

CREB/ATF

736

C

625

GATA-1-Like

363

NF-jB

Sp1

251

170

HMG-I(Y)

139

Sp1 Egr-1

TATA

56

HMG-I(Y)

3719

34

GABP

IL-2Rb 857

80

D

58

97 Ets-1

Ets

cc 670

FIGURE 8.3 The known regulatory elements and corresponding factors identified in the genes encoding human IL-2 (panel A), IL-2Ra (panel B), IL-2Rb (panel C), and cc (panel D). pressor T cells (Smith, 1989; Waldmann, 1989). Thus, IL-2 controls the ‘amplification’ phase of the T-cell immune response. In the case of adult T-cell leukemia (caused by infection with human T-cell lymphotrophic virus type I, HTLV-I), IL-2 is also important in the early phase growth of leukemic T cells (Maeda et al., 1987). In addition to its function as a T-cell growth and survival factor, IL-2 plays a key role in Fas-mediated activation-induced cell death (AICD) of CD4 T cells in response to antigen restimulation. This is critical in peripheral tolerance for the elimination of auto-reactive T cells (Lenardo, 1991; Critchfield et al., 1994; Refaeli et al., 1998; Van Parijs et al., 1999; Lenardo et al., 1999). This role of IL-2 likely explains the development of autoimmunity in the absence of IL-2, IL-2Ra or IL-2Rb in both humans and mice (see below). IL-2 can also promote the growth and differentiation of mitogen- or antigen-activated B cells in vitro

(Mingari et al., 1984; Nakanishi et al., 1984; Tsudo et al., 1984; Waldmann et al., 1984; Zubler et al., 1984). Expression of IL-2Ra and IL-2Rb in B cells can be upregulated by a combination of antigen stimulation with cytokines, including anti-IgM and IL-4/IL-2 (Loughnan and Nossal, 1989; Nakanishi et al., 1992). The magnitude of IL-2-induced B-cell growth correlates with the level of IL-2Rb expression on these cells. During B-cell differentiation, induction of immunoglobulin J-chain expression by IL-2 (Blackman et al., 1986; Lansford et al., 1992) appears to result, at least in part, from down-regulation by IL-2 of a repressor protein, BSAP (B cell lineage-specific activator protein), which binds to a repressor element located between 126 and 113 in the J-chain promoter (McFadden and Koshland, 1991; Rinkenberger et al., 1996). In addition to its actions on T and B cells, IL-2 augments the cytolytic activity of natural killer (NK) cells

THE CYTOKINES AND CHEMOKINES

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(Domzig et al., 1983; Ortaldo et al., 1984; Trinchieri et al., 1984; Lanier et al., 1985), induces lymphokineactivated killer cell (LAK) activity (Grimm et al., 1985), and the proliferation of large granular lymphocytes (LGL) (Harel-Bellan et al., 1986; London et al., 1986; Talmadge et al., 1986). In the early phases of these processes, which cannot be inhibited by anti-Tac antibody to IL-2Ra and where there is no detectable IL-2Ra expression, intermediate affinity IL-2 receptors (IL-2Rb together with cc) appear to play a very important role (Dukovich et al., 1987; Siegel et al., 1987; Tsudo et al., 1987). Induction of IL-2Ra expression by IL-2 (Harel-Bellan et al., 1986; London et al., 1986) then leads to the generation of the high affinity receptors, which can further facilitate induction of LAK activity and proliferation of LGL (Siegel et al., 1987). The actions of IL-2 on monocytes include the stimulation of cytotoxic activity against tumor targets, induction of GM-CSF, IL-1b and IL-6 mRNA and protein, and enhancement of c-fms mRNA (Malkovsky et al., 1987; Espinoza-Delgado et al., 1990; EplingBurnette et al., 1993; Saraya and Balkwill, 1993; Musso et al., 1995). In neutrophils, IL-2 can enhance the growth-inhibitory activity of an opportunistic fungal pathogen, Candida albicans, in vitro, and this effect can be inhibited by a blocking antibody to IL-2Rb but not by anti-Tac monoclonal antibody to IL-2Ra, indicating a role for intermediate, but not high affinity IL-2 receptors in this process (Djeu et al., 1993). However, the physiological importance of IL-2 for neutrophil function still requires further analysis.

IL-2 RECEPTORS Different combinations of three IL-2 receptor subunits form three classes of IL-2 receptors To modulate immune responses, IL-2 first needs to interact with its cognate receptor on the effector cells. Receptor binding assays using radio-labeled IL-2 showed that high affinity IL-2 receptors were only detected on activated T cells (Kd  1011 M) (Robb et al., 1981). It was subsequently revealed that low affinity receptors (Kd  108 M) were also expressed on activated T cells, while intermediate affinity receptors (Kd  109 M) were identified on resting

lymphocytes. The development of a monoclonal antibody, anti-Tac (Uchiyama et al., 1981; Leonard et al., 1982) allowed the identification and subsequent cloning of the first recognized IL-2 binding protein (now known as the IL-2Ra chain) (Leonard et al., 1984; Nikaido et al., 1984). Interestingly, when expressed in non-lymphoid cells, the IL-2Ra chain only reconstituted the low-affinity receptor, but failed to reconstitute either high- or intermediate-affinity receptors (Greene et al., 1985). These observations led to the further efforts to search for additional IL-2R subunit(s). Chemical cross-linking experiments revealed a novel 70 to 75 kDa protein in the receptor–ligand complex (Sharon et al., 1986; Tsudo et al., 1986; Dukovicg et al., 1987; Teshigawara et al., 1987), now known as IL-2Rb. Although co-expression of IL-2Ra and IL-2Rb in lymphocytes yielded high affinity (Kd  1011 M) receptors, surprisingly, their co-expression only reconstituted IL-2 binding with a ‘pseudo-high’ affinity (Kd  1010 M) in non-hematopoietic cells (Arima et al., 1992). Moreover, although certain cells that expressed IL-2Rb but not IL-2Ra bound IL-2 with intermediate affinity (Kd  109 M), expression of IL-2Rb alone in non-lymphoid cells failed to reconstitute these receptors (Minamoto et al., 1990; Tsudo et al., 1990). In addition, the receptor complexes in these non-hematopoietic cells could not mediate IL-2induced receptor internalization. All of these observations led to the subsequent use of chemical cross-linking methods in the presence of both of IL-2 and an anti-IL-2Rb monoclonal antibody that led to identification of the 64-kDa (c chain) in the ligand– receptor complex (Takeshita et al., 1990; Saito et al., 1991). Interestingly, even prior to the cloning of IL-2Rb, a protein with an apparent molecular weight of 65 kDa (presumably the c chain) was detected as an IL-2interacting protein in a chemical cross-linking experiment in addition to the 70- to 75-kDa IL-2Rb chain, but at that time its identity was unknown (Sharon et al., 1986). Co-expression of the b and c chains reconstituted the intermediate affinity IL-2 binding and coexpression of all three subunits reconstituted high affinity binding (Takeshita et al., 1992a). Thus, the low affinity IL-2Rs contain only the a chain, the intermediate affinity receptors are composed of the b and c chains, and the high affinity receptors contain all three subunits (Figure 8.2). Interestingly, IL-2 has a

THE CYTOKINES AND CHEMOKINES

IL - 2 RECEPTORS

rapid on- and off-rate for IL-2Ra but a slow on- and off-rate for the binding to intermediate affinity receptors (Lowenthal and Greene, 1987; Wang and Smith, 1987; Smith, 1989). Thus, high affinity binding is achieved by a combination of the rapid on-rate to IL-2Ra and the slow off-rate from IL-2Rb/c.

Cloning and chromosomal localization of IL-2 receptors As mentioned above, monoclonal antibodies to IL-2Ra allowed its purification and cDNA cloning. The predicted amino acid sequence reveals that IL-2Ra contains 251 amino acids, comprising a 219-amino acid extracellular domain, a 19-amino acid transmembrane region, and only a 13-amino acid cytoplasmic domain (Cosman et al., 1984; Leonard et al., 1984; Nikaido et al., 1984) (Figure 8.4). The IL-2Ra gene is localized on human chromosome 10p14-p15 and murine chromosome 2 and has two potential TATA boxes, two transcription initiation sites, eight exons, seven introns and multiple putative polyadenylation signals (Ishida et al., 1985; Leonard et al., 1985a). The development of monoclonal antibodies to IL-2Rb, Mikb1 and Mikb2 (Tsudo et al., 1989), allowed

Conserved Cysteines

Sushi Domain 219 Amino Acids

a

214 Amino Acids

232 Amino Acids

b

c

WSXWS 13 Amino Acids

286 Amino Acids

86 Amino Acids S Region

A Region

Y338 Y355, Y358, Y361 Y392

Y281 Y303 Y335 Y341

H Region Y510

FIGURE 8.4 Schematic depicting regions of the IL-2Ra, IL-2Rb, and cc chains. Both IL-2Ra (shown) and IL-15Ra (not shown) contain sushi domains. For IL-2Rb, the ‘S’ indicates the ‘serine-rich’ region, the ‘A’ indicates the ‘acidic’ region, and the ‘H’ indicates the C-terminal region. The tyrosine residues in the cytoplasmic domains of IL-2Rb and cc are shown.

173

the expression-cloning of this subunit (Hatakeyama et al., 1989a). The predicted amino acid sequence of the human IL-2Rb chain revealed that the IL-2Rb chain contains 525 amino acids, comprising a 214-amino acid extracellular domain, a 25-amino acid transmembrane region, and a 286-amino acid cytoplasmic region (Figure 8.4). The IL-2Rb gene is localized on human chromosome 22q11.2-q12 and murine chromosome 15 and is organized in 10 exons and nine introns (Gnarra et al., 1990; Shibuya et al., 1990). Like many of the constitutively expressed genes, the IL-2Rb gene does not contain a TATA box; instead, it has GC rich sequences upstream of the transcription initiation sites, and it appears to be transcribed from multiple transcription initiation sites (Gnarra et al., 1990; Shibuya et al., 1990). Purification and molecular cloning of the third IL-2 receptor subunit (initially known as IL-2Rc, but now as cc for ‘common cytokine receptor c chain’, because it is also shared by receptors for IL-4, IL-7, IL-9, IL-15 and IL-21; see below) was made possible by a monoclonal antibody TU11 to IL-2Rb that could coprecipitate cc in the presence, but not the absence of IL-2 (Suzuki et al., 1989; Takeshita et al., 1992a). cc contains 347 amino acids, comprising a 232-amino acid extracellular region, a 29-amino acid transmembrane region, and an 86-amino acid cytoplasmic domain (Takeshita et al., 1992a) (Figure 8.4). The cc gene is on human chromosome Xq13.1 (Noguchi et al., 1993a) and murine chromosome X (Cao et al., 1993) and is organized in eight exons and seven introns (Noguchi et al., 1993b; Cao et al., 1995). Like the IL-2Rb gene, the cc gene does not contain a TATA box, but instead contains GC rich sequences upstream of the transcription initiation sites (Noguchi et al., 1993b). In their extracellular domains, IL-2Rb and cc both contain two pairs of conserved cysteine residues near their N-terminus and a conserved membrane proximal Trp-Ser-X-Trp-Ser (WSXWS) motif that are the common structural features shared by all type I cytokine receptors (Figure 8.4) (Leonard, 1999) (formerly known as the cytokine receptor superfamily) (Bazan, 1990). However, the IL-2Ra chain lacks these features and the only closely related known cytokine receptor is IL-15Ra. Both IL-2Ra and IL-15Ra instead share a conserved motif known as a GP-1 motif, or a SUSHI domain (Figure 8.4) (Giri et al., 1995).

THE CYTOKINES AND CHEMOKINES

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INTERLEUKIN - 2

Expression and regulation of IL-2 receptor chains cc is constitutively expressed on resting lymphocytes (Cao et al., 1995), IL-2Rb is constitutively expressed on NK cells, CD8 T cells, and some CD4 T cells (Sharon et al., 1988; Nakarai et al., 1994; Sugamura et al., 1996), explaining the intermediate-affinity binding of IL-2 to these cells, especially on NK cells (Dukovich et al., 1987; Siegel et al., 1987; Tsudo et al., 1987). IL-2Ra is not expressed on resting cells, but is potently induced after T-cell activation (Leonard et al., 1984, 1985b). Thus, co-expression of IL-2Ra with IL-2Rb and cc allows high-affinity binding (Takeshita et al., 1992a; Asao et al., 1993). IL-2Ra is so potently induced that low-affinity IL-2Ra receptors are approximately tenfold more abundant than high-affinity receptors on activated T cells (Robb et al., 1981, 1984; Nakarai et al., 1994). Interestingly, however, high-affinity receptors on activated T cells are five- to ten-fold more abundant than are intermediate-affinity receptors on resting lymphocytes (Nakarai et al., 1994; Waldmann, 1989). This is explained by the observation that IL-2Rb expression, while constitutive, is also induced after T-cell activation (Siegel et al., 1987).

IL-2Ra Similar to IL-2, expression of IL-2Ra is potently induced by activation. IL-2Ra is induced by anti-CD3, anti-CD3 plus anti-CD28, mitogens (e.g. PHA or ConA), the Tax transactivator protein of HTLV-I, PMA, ionomycin, the protein kinase A (PKA) activator forskolin and certain cytokines (including IL-1, TNFa and IL-2 itself ) (Waldmann, 1989). It should be emphasized that IL-2-induced expression of the IL-2Ra chain is an important mechanism for controlling the magnitude and duration of the T-cell immune response through high affinity receptors (see below). Like IL-2, regulation of IL-2Ra expression mainly occurs at the level of transcription (Leonard et al., 1985b). In contrast to IL-2, two signals are not obligately required for IL-2Ra induction. IL-2Ra transcripts can be detected in T cells as early as 1 h after PHA stimulation and IL-2Ra mRNA levels are sustained at high levels for at least 8–24 hr (Leonard et al., 1985b). The molecular mechanisms underlying how the IL-2Ra gene is regulated under various conditions

have been extensively investigated (Lin and Leonard, 1997a). Detailed analyses of the human and murine IL-2Ra promoter regions identified four positive regulatory regions in the 5 flanking region, denoted as PRRI, PRRII, PRRIII, and CD28rE (CD28 responsive element), and one in the first intron, denoted as PRRIV (Figure 8.3). PRRI (299 and 228) contains an NF-jB binding site (Bohnlein et al., 1988; Leung and Nabel, 1988; Ruben et al., 1988; Cross et al., 1989) and a CArG motif (Ballard et al., 1989; Toledano et al., 1990) that bind to Rel/NF-jB family proteins and serum responsive factor (SRF), respectively. However, the CArG motif is not conserved in mice (Sperisen et al., 1995). PRRI is required for IL-2Ra gene induction in response to PMA, PHA, IL-1, TNF-a, and the Tax protein of HTLVI. Within PRRI, a still uncharacterized factor binds upstream of the NF-jB site, denoted as UE1 (Ballard et al., 1989) or NF-IL-2RA (Toledano et al., 1990), and an Sp1 site overlaps the GG residues of CArG motif (Roman et al., 1990). PRRII (137 and 64) binds an Ets family protein, Elf-1, and the high mobility group proteins, HMG-I/Y (John et al., 1995). Like PRRI, PRRII is required for PMA-induced IL-2Ra expression. PRRIII is located between 3780 and 3703 in the human (John et al., 1996; Lecine et al., 1996) and between 1366 and 1319 in the murine (Sperisen et al., 1995) IL-2Ra genes. PRRIII binds Stat5 proteins (Stat5a and Stat5b), Elf-1, as well as a GATA-1-like protein. Interestingly, there are two tandemly linked Stat5 DNA binding sites within PRRIII, a consensus motif followed by a non-consensus motif. Both sites are required for efficient binding, indicating cooperative binding of Stat5 to these sites (John et al., 1999). This cooperative binding of Stat5 is at least in part mediated by tetramer formation of Stat5 dimers through protein–protein interactions. Moreover, binding of each factor to PRRIII appears to be important for IL-2-mediated IL-2Ra transcription (John et al., 1999). A novel IL-2 responsive element, denoted PRRIV, has been identified in the first intron of the human and murine IL-2Ra gene by DNase I footprinting analyses (Kim et al., 2001). PRRIV contains multiple binding sites for Stat5 and HMG-I(Y). Cooperation between these factors appears to be important for the full IL-2 responsiveness of PRRIV. Furthermore, the integrity of both of PRRIII and PRRIV is required for full promoter activity in response to IL-2, indicating a

THE CYTOKINES AND CHEMOKINES

IL - 2 RECEPTORS

functional cooperativity between these two widely separated regions, located upstream and downstream of the transcription initiation sites (Kim et al., 2001). Chromatin immunoprecipitation (ChIP) assays have provided direct evidence that occupancy of the binding sites for Stat5 proteins in both PRRIII and PRRIV in vivo is highly dependent upon IL-2 stimulation (Kim et al., 2001). The importance of Stat5 proteins in IL-2-mediated IL-2Ra transcription is further demonstrated by defective IL-2-induced IL-2Ra induction in Stat5 knockout mice (see below) (Nakajima et al., 1997a; Imada et al., 1998). However, activation of Stat5 alone is not sufficient for IL-2Ra induction. For example, in IL-3-dependent 32D cells transfected with the human IL-2Rb chain so that these cells can respond to both IL-2 and IL-3, only IL-2 induces IL-2Ra mRNA, even though both IL-2 and IL-3 can similarly activate Stat5 proteins (Ascherman et al., 1997). Recently, PRRIV has also been shown to function as a TCRresponsive element, at least in part due to its ability to mediate the TCR stimulation through NF-AT and AP-1 (Kim and Leonard, 2002). In DNase I hypersensitivity assays, a TCR/CD28-rE has recently been identified upstream of PRRIII, approximately 8.5 kb upstream of the major IL-2Ra transcription initiation site (Yeh et al., 2001). The function of this element appears to require cooperation between CREB/ATF and AP-1 family transcription factors. AP-1 is also important for PRRIV activity (Kim H.-P., unpublished observation).

IL-2Rb IL-2Rb is not only expressed in resting T cells, but is also potently induced by PHA, PMA, anti-CD3, antiCD28, or anti-CD28 plus anti-CD2 (Cerdan et al., 1995; Waldmann, 1989), and by cytokines, including IL-4 (Casey et al., 1992) and IL-2 (Siegel et al., 1987; Nakanishi et al., 1992). However, neither the Tax protein of HTLV-I nor activators of PKA induce IL-2Rb (Waldmann, 1989). Expression of IL-2Rb is regulated at both the transcriptional and post-transcriptional levels (Cerdan et al., 1995). As compared with our more detailed knowledge of the basis for regulation of the IL-2 and IL-2Ra genes, our understanding of IL-2Rb regulation is relatively limited. Analyses of a limited 5 flanking region of the IL-2Rb gene revealed

175

the region that mediates the basal promoter activity (Gnarra et al., 1990; Shibuya et al., 1990), as well as three PMA responsive enhancer-like regions: the 56 to 34 region that bind the Ets family proteins Ets-1 and GABP, the 170 to 139 region that bind Sp1 and the immediate–early gene product Egr-1 and the 363 to 251 region (Lin and Leonard, 1997b; Lin et al., 1993) (Figure 8.3). In addition, the 5 promoter region contains TG repeats (Gnarra et al., 1990; Shibuya et al., 1990) that are presumably able to form a Z-DNA structure, although the functional significance of this structural feature is not clear. Interestingly, promoters for IL-2Rb and IL-2 (Skerka et al., 1995) are both regulated by a combination of Egr-1 and Sp1, suggesting that both genes can be coordinately regulated. In addition, a region located between 763 and 625 appears to contain negative regulatory element(s) (Lin et al., 1993), which needs to be further characterized. As discussed below, markedly suppressed expression of the IL-2Rb chain has recently been reported in a patient with an NKcell deficient severe immunodeficiency disorder, suggesting that either the mutation(s) occur in the IL-2Rb regulatory region or those transcription factor(s) that are critical for expression of IL-2Rb are dysregulated, since the coding region of IL-2Rb appears to be normal in this patient. Therefore, the IL-2Rb promoter region clearly warrants further investigation. As discussed above, the approaches that were used to study the promoter regions for IL-2 and IL-2Ra, such as DNase I hypersensitivity analysis, as well as an analysis of the most highly conserved regulatory sequences in both human and murine IL-2Rb genes will undoubtedly provide valuable information to help further clarify the basis for IL-2Rb gene regulation.

cc cc is constitutively expressed in lymphocytes (Takeshita et al., 1992a; Cao et al., 1993), including expression in fetal thymus as early as day 13.5 (Cao et al., 1995). A low constitutive level of cc mRNA was also detected in fresh human monocytes, and its expression can be up-regulated in these cells at a posttranscriptional level by IL-2 or IFNc (Bosco et al., 1994). A limited 5 flanking region of the cc gene showed constitutive promoter activity (Noguchi et al., 1993b;

THE CYTOKINES AND CHEMOKINES

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INTERLEUKIN - 2

Ohbo et al., 1995). A region between nucleotides 80 and 58 relative to the transcription initiation site contains an Ets binding site (Figure 8.3), suggesting the involvement of Ets family proteins in regulation of basal cc promoter activity (Ohbo et al., 1995).

The critical roles played by IL-2Rb and cc in transducing IL-2 signals Although reconstitution of IL-2 receptor complexes with intermediate and high affinities in nonhematopoietic cells can lead to the induction of some IL-2-responsive genes, this was apparently not sufficient to transduce IL-2-induced proliferative signals in these cells (Asao et al., 1993; Minami et al., 1994), indicating that additional cell-type specific signaling molecule(s) were required. IL-2 signaling requires juxtaposition of the cytoplasmic regions of IL-2Rb and cc (Kawahara et al., 1994; Nakamura et al., 1994; Nelson et al., 1994), suggesting that both of these receptor components interacted with the downstream signaling molecules. Apparently, the principal role of IL-2 is to induce the hetero-dimerization of receptor chains, given that IL-2Rb and cc do not stably associate in the absence of IL-2 (Takeshita et al., 1990), analogous to the growth hormone (GH)/GH receptor model (de Vos et al., 1992), a model that may apply to all type I cytokines. Interestingly, binding assays using mutant recombinant IL-2 and BIAcore experiments demonstrate that IL-2Ra and IL-2Rb can pre-exist as a complex prior to the binding of IL-2 (Grant et al., 1992; Roessler et al., 1994; Myszka et al., 1996); if it is true under physiological concentrations of receptors, this would allow even more efficient binding of IL-2 to the receptor, at which point cc could be efficiently recruited. The short 13 amino acid long cytoplasmic domain of IL-2Ra is unlikely to interact with critical signaling molecules. Instead, with six positively charged residues (five arginines and one lysine), the cytoplasmic domain likely serves a cytoplasmic anchoring function. Although both IL-2Rb and cc contain much longer cytoplasmic domains (286 and 86 amino acids, respectively), unlike receptors for certain growth factors (such as insulin, EGF and PDGF), these proteins lack known catalytic domains typical of intrinsic protein kinases (Hatakeyama et al., 1989a; Takeshita et al., 1992a). Instead, both IL-2Rb and cc belong to the

type I cytokine receptor superfamily that include the receptors for GH, prolactin, erythropoietin (Epo), thrombopoietin, granulocyte–macrophage colonystimulating factor (GM-CSF), and many interleukins (Bazan, 1990; Sprang and Bazan, 1993; Leonard, 1999). All of these receptors contain tyrosine residues in their cytoplasmic domains and some of these tyrosine residues are targets for phosphorylation by nonreceptor protein tyrosine kinases. Stimulation of T cells by IL-2 triggers rapid tyrosine phosphorylation of a number of protein substrates, including IL-2Rb and cc themselves (Saltzman et al., 1988; Sharon et al., 1989; Farrar et al., 1989; Merida et al., 1990; Augustine et al., 1990; Asao et al., 1990, 1992; Kumaki et al., 1992). Consistent with these findings, experiments with the tyrosine kinase inhibitor herbimycin A demonstrated the importance of tyrosine phosphorylation for proliferative responses to IL-2 (Otani et al., 1993).

IL-2Rb chain Early experiments intended to delineate the functional domains of human IL-2Rb divided the cytoplasmic domain of IL-2Rb into three broad regions based on the locations of convenient restriction sites, the ‘serine-rich’ region (S region, amino acid 267 to 322), the ‘acidic’ region (A region, amino acid 313 to 382), and the ‘proline-rich’ region (H region, amino acid 378 to 525) (Hatakeyama et al., 1989b) (Figure 8.4). These regions do not necessarily correspond to discrete functional motifs or domains. For example, the principal known function of the S region is to recruit the Janus family tyrosine kinase (Jak) Jak1 via a proline-rich ‘Box 1’ motif contained within this region (Fukunaga et al., 1991; Murakami et al., 1991), while the well-known function of the H region is based not at all on proline residues, but instead relies on the presence of two tyrosine residues, Y392 and Y510, that are critical for Stat5 protein docking and activation (Goldsmith et al., 1995; Lin et al., 1995; Friedmann et al., 1996; Lin et al., 1996). In addition to these two tyrosines, human IL-2Rb contains four other tyrosines, Y338, Y355, Y358 and Y361 (Figure 8.4), all of which, except Y361, are conserved in mouse and rat (Page and Dallman, 1991). As these four tyrosines are all located in the A region, it is important to recognize that deletion or mutation of the A region may result in the loss of potential phosphotyrosine docking sites for signaling molecules.

THE CYTOKINES AND CHEMOKINES

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cc Chain The cytoplasmic domain of cc contains a region that was noted to have a limited sequence homology to SH2 domains (Takeshita et al., 1992a). However, this region is much shorter than the consensus SH2 domains and lacks the conserved arginine residue that is required for interaction of the SH2 domains with phosphorylated tyrosine residues. Nevertheless, this region is conserved in human and mouse and may play a role in transducing IL-2 signals, albeit presumably not as a phosphotyrosine binding region. cc has four tyrosine residues in its cytoplasmic region (Figure 8.4). As will be discussed below, cc associates with Jak3.

Signaling molecules associated with IL-2Rb and cc Jak family kinases Jak kinases are non-receptor protein tyrosine kinases. Four mammalian Jaks, Jak1, Jak2, Jak3 and Tyk2, have been identified. Jak1, Jak2 and Tyk2 are ubiquitously expressed, while Jak3 is mainly expressed in hematopoietic cells (Leonard and O’Shea, 1998). Interferons (IFNs) are the prototype cytokines (also known as type II cytokines) that cause rapid activation of Jak kinases. IFNa activates Jak1 and Tyk2, whereas IFNc activates Jak1 and Jak2 through a ligand-mediated receptor dimerization (for IFNa) or tetramerization (for IFNc) (Bach et al., 1997). IL-2 activates both Jak1 and Jak3 (Boussiotis et al., 1994; Johnston et al., 1994; Miyazaki et al., 1994; Russell et al., 1994; Witthuhn et al., 1994). Jak1 is primarily associated with the S region of IL-2Rb (Miyazaki et al., 1994), presumably not because of serine residues, but because this region contains the proline rich ‘Box 1’ motif typical of type I cytokine receptors. Jak3 appears primarily to form contacts with both proximal and distal regions of the cytoplasmic domain of cc (Russell et al., 1994) and seems to interact with IL-2Rb as well (Zhu et al., 1998). Consistent with the importance of Jak3, expression of Jak3 in an NIH3T3 cell line that is already transfected with all three IL-2R subunits and expresses Jak1 (Minami et al., 1994) allows an IL-2-induced proliferative responsiveness in these cells (Miyazaki et al.,

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1994). The importance of Jak3 in IL-2 signaling has been further substantiated by recent findings that homozygous point or deletion mutations in Jak3 are found in autosomal recessive TBNK severe combined immunodeficiency patients (Macchi et al., 1995; Russell et al., 1995) and mice lacking Jak3 are immunodeficient. Thus, data derived from both in vitro and in vivo studies firmly establish the physiological role of Jak kinases in IL-2 signaling pathways.

STAT proteins STAT (signal transducers and activators of transcription) proteins are transcription factors that are present in a latent form in the cytosol (Horvath and Darnell, 1997). STATs are rapidly activated through tyrosine phosphorylation (generally by Jak kinases in the cases of IFNs and cytokines) following stimulation with IFNs, cytokines and certain growth factors. The STATs are then translocated into the nucleus and can modulate target gene expression (Horvath and Darnell, 1997). STATs were the first identified known classes of transcription factors that are activated by tyrosine phosphorylation. At present, seven genes encoding mammalian STAT proteins (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b and Stat6) have been identified (Horvath and Darnell, 1997; Leonard and O’Shea, 1998). Analogous to IFNs (which activate Stat1 in the case of IFNc or both Stat1 and Stat2 in the case of IFNa/b), IL-2 rapidly activates Stat1, Stat3, Stat5a and Stat5b in lymphocytes (Nielsen et al., 1994; Fujii et al., 1995; Hou et al., 1995; Lin et al., 1995; Yu et al., 1996). The activation of STAT proteins is typically initiated by the phosphorylation of cytoplasmic tyrosine(s) on a receptor chain, resulting in the generation of docking sites for the SH2 domains of STAT proteins (Horvath and Darnell, 1997). Following IL-2 stimulation, either Y392 or Y510 of the IL-2Rb chain can serve as a docking site for binding and subsequent activation of Stat5a and Stat5b (Fujii et al., 1995; Goldsmith et al., 1995; Lin et al., 1995, 1996; Friedmann et al., 1996). Simultaneous mutation of both Y392 and Y510 results in a substantial decrease in IL-2-induced proliferation, suggesting the importance of STAT activation by IL-2 (Goldsmith et al., 1995; Friedmann et al., 1996). Both Jak1 and Jak3 are required for IL-2-mediated Stat5 activation (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995; Rodig et al., 1998), although which

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of the Jak kinases phosphorylates IL-2Rb versus Stat5 proteins in vivo remains to be determined. It has nevertheless been hypothesized that Jak3 is the kinase that phosphorylates Stat5 proteins after their docking on the IL-2Rb chain (Lin et al., 1996), a notion consistent with the observation that Jak3 and Stat5 can both be coprecipitated with IL-2Rb (Migone et al., 1995). The docking sites on the receptors determine the specificity of STAT activation by cytokines. cc is a shared receptor component for IL-2, IL-4, IL-7, IL-9 and IL-15, and all of these cytokines activate Jak3. However, IL-2, IL-7, IL-9 and IL-15 mainly activate Stat5 because of the existence of similar tyrosinephosphorylated motifs in the cytoplasmic domains of the IL-2Rb, IL-7Ra, and IL-9Ra that can serve as docking sites for Stat5 activation (Lin et al., 1995; Demoulin et al., 1996), while IL-4 mainly activates Stat6 due to the properties of the phosphotyrosine docking sites on IL-4Ra (Lin et al., 1995; Schindler et al., 1995; Wang et al., 1996). These data suggest that neither Jak kinases nor cc determines the specificity of STAT activation by IL-2. The biological significance of Stat5 proteins in IL-2 signaling is further established by analyses of genetargeted mice that lack expression of Stat5 proteins (Liu et al., 1997; Udy et al., 1997). Both Stat5a/ and Stat5b/ mice exhibit marked defects in IL-2 signaling. In Stat5a/ mice, IL-2-induced expression of IL-2Ra is impaired (Nakajima et al., 1997a). Consistent with this observation, splenocytes from Stat5a/ mice exhibit markedly decreased proliferation to low concentrations of IL-2, although maximal proliferation is still achieved at concentrations of IL-2 high enough to titrate the intermediate affinity receptors. This defect also correlates with defective Vb8 CD8 T-cell expansion in vivo in response to the superantigen, staphylococcus enterotoxin B (Nakajima et al., 1997a). Moreover, although the number of thymocytes is normal, the number of splenocytes is modestly reduced in Stat5a/ mice (Nakajima et al., 1997a). In Stat5b/ mice, there is a greater defect in proliferation in response to IL-2 than is seen in the Stat5a/ mice. Both Stat5a and Stat5b contribute to normal NK cell development, with a greater defect being seen for NK function in Stat5b/ mice (Imada et al., 1998), which appears to be explained at least in part by a marked defect in the induction of perforin expression by IL-2 and IL-15 in these mice. In

addition, expression of both IL-2Ra and IL-2Rb is diminished in Stat5b/ mice, at least in part explaining their defects in IL-2 and IL-15 responsiveness. Perhaps in part for this reason, and in contrast to the situation for Stat5a/ mice, high-dose IL-2 cannot compensate for the defective proliferation in Stat5b/ splenocytes. In Stat5a//Stat5b/ double knockout mice, the phenotype is more severe than that found in either of the individual knockout mice (Teglund et al., 1998; Socolovsky et al., 1999; Moriggl et al., 1999a, 1999b). In these mice, peripheral T cells are profoundly deficient in their proliferative potential and fail to undergo cell cycle progression. These phenotypes indicate that Stat5 proteins play a vital role in IL-2 signaling in T cells. One of the striking phenotypes in the double knockout mice is the absence of NK cells. Thus, Stat5a and Stat5b together are vital for NK-cell development, whereas Stat5b appears to be more important for perforin gene induction and cytolytic activity. Presumably, Stat5 is a key mediator for transducing IL-15 signals in support of the development of NK cells.

SHP-2 and p97/Gab2 In addition to direct activation of Stat5 proteins by JAK kinases in response to cytokines, Stat5-mediated transactivation can be augmented by other substrates of JAK kinases. In this regard, stimulation of T cells or NK cells by IL-2 induces tyrosine phosphorylation of SH2 containing phosphatase (SHP) 2 and the scaffolding protein p97/Gab2 (Gadina et al., 1998, 1999; Gesbert et al., 1998; Gu et al., 1998). Tyrosine phosphorylation of p97/Gab2 is most likely mediated by Jak3 and seems to be specific for certain cytokines, since tyrosine phosphorylation of p97/Gab2 is not induced by IL-4 (Gadina et al., 1999). SHP-2 is constitutively associated with Jak1 and Jak3; however, it is not yet clear whether it is phosphorylated by these JAK kinases. Over-expression of SHP-2 augments STAT-mediated reporter activity (Gadina et al., 1998), whereas over-expression of the dominant negative forms of either SHP-2 or p97/Gab2 diminishes IL-2induced STAT-mediated reporter activity (Gadina et al., 1998; Gu et al., 1998). These data suggest that SHP-2 and p97/Gab2 are both involved in augmenting Stat5-mediated transactivation in response to IL-2

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stimulation. In contrast to these findings, however, it has also been reported that SHP-2, but not SHP-1, can directly dephosphorylate Stat5 proteins in vitro, implying that it may be instead involved in the negative regulation of Stat5 activity (Yu et al., 2000). Therefore, the role of SHP-2 and p97/Gab2 in regulating Stat5-driven transcription in response to IL-2 requires additional investigation.

Shc-Ras-Raf-MAP kinase pathway The adaptor protein Shc is tyrosine phosphorylated within 1 min following IL-2 stimulation of T cells (Burns et al., 1993; Cutler et al., 1993; Liu et al., 1994; Ravichandran and Burakoff, 1994; Zhu et al., 1994). In addition to Shc, the adaptor protein Grb2 and guanine nucleotide exchange factor mSOS can also be detected in IL-2R-immune complexes prepared from cells treated with IL-2 (Zhu et al., 1994; Ravichandran et al., 1996). These findings provide a connection between IL-2 receptor complexes and IL-2-mediated activation of Ras (Satoh et al., 1991; Duronio et al., 1992; Welham et al., 1994), the serine/threonine kinase Raf1 (Turner et al., 1991), MEK (Karnitz et al., 1995), and MAP kinase (Perkins et al., 1993; Karnitz et al., 1995). Substitution of tyrosine 338 with phenylalanine abolishes the interaction of Shc with IL-2Rb and Shc tyrosine phosphorylation, and also diminishes IL-2-induced proliferation (Friedmann et al., 1996). The association of Shc with IL-2Rb is via the PTB domain rather than the SH2 domain of Shc (Ravichandran et al., 1996). Thus, although the diminished proliferation after deletion of the A region of IL-2Rb was originally attributed to the loss of p56Lck activation (Taniguchi, 1995), it is possible that it instead results at least in part from a loss of Shccoupled signaling (Friedmann et al., 1996).

PI 3-kinase Activation of phosphatidyl inositol 3-kinase (PI3K) has been demonstrated in a number of cytokine and growth factor systems. In most cases, PI3K activation is well correlated with transducing proliferative signals (Carpenter and Cantley, 1996; Zvelebil et al., 1996; Ward and Cantrell, 2001). Within 1 min of IL-2 stimulation of T cells, PI3K activity can be detected in complexes immunoprecipitated by an anti-IL-2Rb

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antibody (Augustine et al., 1991; Merida et al., 1991; Remillard et al., 1991). IL-2 can trigger tyrosine phosphorylation of the p85 component of PI3K in BAF-3 cells transfected with IL-2Rb (Merida et al., 1993). Moreover, two serine/threonine kinases, p70 S6 kinase (p70S6K) (Calvo et al., 1992; Kuo et al., 1992; Sawami et al., 1992; Monfar et al., 1995) and Akt (protein kinase B) (Ahmed et al., 1997; Reif et al., 1997), are downstream substrates of PI3K (Franke et al., 1997) and activated by IL-2 in T cells. These findings are further substantiated by an observation that IL-2induced anti-apoptotic and proliferative signals are transduced via the PI3K/Akt pathway in BAF/3 cells (Ahmed et al., 1997). Furthermore, an in vitro study has identified E2F as a target for PI3K/Akt that transduces the proliferative signals in response to IL-2 in T cells (Brennan et al., 1997), which may at least in part explain the ability of IL-2 to regulate the expression of a number of cell cycle-related genes (see below). It is well known that E2F family transcription factors are important regulators of proliferation, differentiation and apoptosis (Dyson, 1998; Helin, 1998; Nevins, 1998). However, it is not yet clear how PI3K interacts with IL-2Rb, as it had been proposed that PI3K could interact either directly with the phosphorylated Y392 of the IL-2Rb chain (Truitt et al., 1994), or indirectly through the Src-family kinases Fyn (Karnitz et al., 1994) or Lck (Taichman et al., 1993). Moreover, Jak1 has been shown to be important for recruiting PI3K (Migone et al., 1998).

Src-family kinases The Src-family of non-receptor protein tyrosine kinases (PTK) are plasma membrane-associated proteins, some of which are ubiquitously expressed, whereas others are preferentially expressed in certain tissues (Bolen et al., 1991; Pawson, 1995). Each Src family kinase contains an N-terminal unique domain that confers kinase-specific function to each enzyme, a 60-amino acid Src homology 3 (SH3) domain, a 100amino acid SH2 domain and a conserved C-terminal SH1 kinase domain (Cohen et al., 1995; Pawson, 1995). The SH2 and SH3 domains mediate interaction with other signaling molecules: SH2 domains interact with phosphorylated tyrosine residues and SH3 domains with proline-rich regions (Ren et al., 1993). Stimulation of IL-2-dependent human T-cell clones

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by IL-2 causes a rapid phosphorylation of p56Lck (Horak et al., 1991), which is mainly expressed in T cells and NK cells (Bolen et al., 1991). p56Lck can also be co-precipitated with IL-2Rb in the YT NK-like leukemia cell line in the absence of IL-2 (Hatakeyama et al., 1991), indicating that although activation of p56Lck depends on IL-2, the interaction of p56Lck with IL-2Rb chain does not. Furthermore, it appears that the interaction is mediated by the A region of IL-2Rb and the N-terminal region in the kinase domain of p56Lck (Hatakeyama et al., 1991), which is different from the region involved in p56Lck association with CD4 or CD8 co-receptor molecules (Barber et al., 1989; Rudd et al., 1989; Veillette et al., 1989). The specific interaction sites on IL-2Rb and p56Lck have not been defined. The physical interaction between p56Lck and the A region of IL-2Rb is apparently not sufficient for IL-2-induced p56Lck activation, however, as activation does not occur when the S region of IL-2Rb is deleted (Minami et al., 1993). Despite the ability of IL-2 to activate p56Lck in vitro, the physiological significance of p56Lck in IL-2 signaling remains unclear. First, although thymic maturation is blocked in p56Lck/ mice, the remaining peripheral T cells can still proliferate in response to IL-2 (Molina et al., 1992) and a p56Lck-deficient cell line derived from CTLL-2 cells proliferate in an IL-2dependent fashion (Karnitz et al., 1992). Second, a deletion mutant of the IL-2Rb chain lacking the A region is unable to activate p56Lck (Minami et al., 1993), but can still transduce IL-2-induced proliferative signals when expressed in BAF-B03 cells (Hatakeyama et al., 1989a), albeit at diminished levels (Shibuya et al., 1992). An in vivo study shows that in fact, T cells from IL-2RbDA transgenic mice on the IL-2Rb/ background proliferate even better than those from the wild-type mice in response to IL-2 (Fujii et al., 1998), indicating a discrepancy between data derived from cell lines versus mice regarding the role of the A region. Third, when the human IL-2Rb chain is expressed in 32D murine myeloid progenitor cells that lack detectable p56Lck, these cells can proliferate in response to IL-2 (Otani et al., 1992). Therefore, it is clear that p56Lck is not required for IL-2-induced proliferation, although perhaps other member(s) of Src-family kinase can substitute for the function of p56Lck in cells lacking p56Lck, or maybe these discrepancies simply reflect a usage of different signaling

molecules in different cell types. Indeed, other Srcfamily kinases can also be activated by IL-2 in IL-2Rbtransfected BAF-B03 cells. These Src-family kinases include p59Fyn that is expressed by a number of cell types, including both T and B cells, and p53/56Lyn that is mainly expressed in B cells (Bolen et al., 1991). Furthermore, p53/56Lyn can also associate with IL-2Rb (Torigoe et al., 1992; Kobayashi et al., 1993). Therefore, the physiological significance of each member of these Src-family kinase in IL-2-signaling pathways remains to be further evaluated and the question as to which Src-family kinases, if any, contribute to proliferation, differentiation and/or cytolytic activity remains to be addressed.

Protein tyrosine kinase Syk The Syk protein tyrosine kinase (PTK) has also been reported to interact with IL-2Rb and to be activated by IL-2 in PBL (Minami et al., 1995). Syk is a member of ZAP-70/Syk family of PTKs and is known to play an important role in signaling from Fc receptors and B cell antigen receptors (Chan et al., 1992; Wange et al., 1992; Straus and Weiss, 1993; Law et al., 1994). The interaction of IL-2Rb with Syk was mapped to the S region of the IL-2Rb chain (Minami et al., 1995), which is the same region of IL-2Rb that is essential for the association of Jak1. Deletion of this region results in a loss of c-myc induction by IL-2 in BAF-B03 cells. Activation of Syk seems to be dispensable for c-myc induction, as IL-2-induced c-myc expression can still be detected in a Syk-negative cell line (Takeshita et al., 1997); thus it seems more likely that Jak1 is the critical molecule. Furthermore, B-cell differentiation is impaired in Syk-deficient mice (Cambier et al., 1994; Cheng et al., 1995; Turner et al., 1995), while IL-2 response appears to be normal in these mice (Turner et al., 1995). The fact that IL-2Rb does not contain an immunoreceptor tyrosine-based activation motif (ITAM), as is typically required for the recruitment of Zap70 or Syk (Cambier et al., 1994), also raises doubts about the physiological role of Syk in IL-2 signaling.

STAM STAM (signal transducing adaptor molecule) was initially identified as an IL-2-induced tyrosine phosphorylated protein containing SH3 and ITAM

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domains. STAM has an apparent molecular weight of 70 kDa (Takeshita et al., 1996), is phosphorylated by Jak3 upon IL-2 stimulation, and can associate with either Jak3 or Jak2 via its ITAM domain, but not with Jak1, Lck or Syk (Takeshita et al., 1997). When expressed in BAF-B03 cells, a deletion mutant of STAM lacking its SH3 domain inhibits IL-2-induced proliferation. In addition, wild-type STAM, but not STAM mutants lacking either the SH3 or ITAM domain, can increase IL-2-induced c-myc promoter activity, suggesting that STAM may be involved in transducing signal(s) that regulate expression of the c-myc gene and proliferative response. Despite these in vitro findings, neither lymphocyte development nor responses to cytokines including IL-2 is compromised in STAM1/ mice (Yamada et al., 2001). It remains to be determined whether or not the discrepancy between the in vitro and in vivo data is due to the redundancy of STAM proteins, as additional STAM proteins has lately been identified (Endo et al., 2000; Pandey et al., 2000).

Negative regulation of cc by proteolysis Using a yeast two-hybrid trap with the cytoplasmic domain of cc as the bait, it has been shown that the small subunit of calpain, a calcium-activated protease, can associate with cc (Noguchi et al., 1997). Moreover, T cell receptor stimulation (which increases intracellular calcium) induces cleavage of cc (Noguchi et al., 1997). Calpain has been implicated in T-cell apoptosis (Martin and Green, 1995), but the relevant substrates for calpain in this process have been unclear. Additional studies are still required to clarify the physiological significance of this finding, but it is possible that TCR-induced, calpain-mediated cleavage of cc is a mechanism by which cc-dependent signals can be controlled. In summary, the IL-2Rb chain interacts with Jak1, Shc, STATs, Lck and Syk, whereas cc interacts with Jak3 and the small subunit of calpain. Jak1 and Jak3 are clearly important for signaling. Moreover, Y338 mediates Shc interaction and tyrosine phosphorylation of Shc, while Y392 and Y510 mediate STAT protein activation. When these three tyrosines are simultaneously mutated, there is a complete loss of IL-2-induced proliferation, indicating that Shc-coupled and STATcoupled pathways are both vital for proliferative

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response (Friedmann et al., 1996). In contrast, mutation of Y355, Y358 and Y361 does not affect proliferation (Friedmann et al., 1996). Thus, much has been learned about IL-2-mediated proliferation from mutations that abolish docking sites for phosphotyrosine. Figure 8.5 summarizes our current understanding about IL-2 signaling. The role of STATs in IL-2-induced signaling warrants further discussion. Clearly, Stat5 activation regulates expression of target genes such as IL-2Ra, and more importantly, STAT activation appears to more directly contribute to proliferation. It is therefore interesting that although normal cells contain Stat5 DNA binding activity only after IL-2 stimulation, in vitro transformation with HTLV-I results not only in IL-2-independent growth, but also in constitutive activation of Jaks and STATs (Migone et al., 1995). Moreover, constitutively activated Jaks and STATs have been found in malignant cells of patients with adult T-cell leukemia, which is caused by HTLV-I (Takemoto et al., 1997). This suggests that constitutively activated Jaks and STATs may contribute to the transformation process. Activation of STATs has also been found in cells transformed by v-abl (Danial et al., 1995), spleen focus forming virus (Ohashi et al., 1996), and v-src (Yu et al., 1995).

IL-2-activated genes involved in proliferation and IL-2 signaling pathways To achieve its biological effects, IL-2 increases expression of IL-2Ra and IL-2Rb, and also modulates expression of many other cellular genes, including those critical for cell cycle progression and anti-apoptosis (Figure 8.5). Among these genes are the protooncogenes c-myc (Reed et al., 1985; Pauza, 1987; Shibuya et al., 1992; Miyazaki et al., 1995), c-fos (Pauza, 1987; Shibuya et al., 1992), c-jun (Shibuya et al., 1992; Miyazaki et al., 1995), c-myb (Pauza, 1987), bcl-2 (Otani et al., 1993; Broome et al., 1995; Miyazaki et al., 1995), and bcl-XL (Broome et al., 1995). IL-2 also induces expression of the genes encoding cyclin family proteins A, B, C, D2, D3 and E, and the cdc2 family kinases cdc2 and cdk2 (Shibuya et al., 1992). In BAF-B03 cells, the ability to induce expression by IL-2 of the proto-oncogenes c-myc, c-fos, c-jun (Hatakeyama et al., 1992; Shibuya et al., 1992; Shibuya

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IL-2

IL-2 Ras

IL-2Ra Jak1

cc

Akt

SOS Grb2

Jak3

Jak3

Shc

P13K

Jak1

Rap

CIS

SOCS

Raf

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PTKs

S6K

p70 IL-2Rb

MEK

Stat5 Stat5

MAPK

Gene Expression Nucleus Basal Complex Co TF TF TATA

Genes encoding: Pol II

Biological Response

IL-2Ra c-Fos CIS/SOCS family proteins IL-2Rb c-Myc protein kinases c-Jun protein phosphatases, etc. c-Myb Bcl-2 Bcl-XL cyclins cdc2 family kinases Oncotatin M

FIGURE 8.5 The known signaling molecules associated with IL-2Rb and/or cc. PTKs represent the Src family protein kinases Lck, Fyn and Lyn. In the nucleus, transcriptional activation mediated by STAT proteins and other transcription factors are indicated by ‘TF’ and a co-activator of transcription is shown as ‘Co’. Inhibition of the PI3K pathway by rapamycin (Rap) is indicated. et al., 1994), and bcl-2 (Fujii et al., 1995) is correlated with IL-2-induced proliferative signals (Taniguchi, 1995). Almost all IL-2-induced signals examined so far seem to require the integrity of the membrane proximal S region of IL-2Rb (Minami et al., 1993; Fujii et al., 1995), suggesting a central role for Jak1. Induction of c-fos and c-jun also requires the A region (Hatakeyama et al., 1992; Shibuya et al., 1992; Shibuya et al., 1994). This suggests that Lck or one or more of the three conserved tyrosines in this region of IL-2Rb contribute to induction of these genes. In addition to the importance of IL-2Rb, cc also plays pivotal roles in IL-2-induced gene expression. Deletion of 68 amino acids in the cytoplasmic domain of cc results in the loss of induction of c-myc, c-fos and c-jun as well, whereas deletion of the 30 C-terminal amino acids causes the loss of induction of c-fos and c-jun but the

ability of IL-2 to induce c-myc is retained (Asao et al., 1993). These findings are consistent with recruitment of Jak3 by cc. Indeed, over-expression of a dominant negative mutant Jak3 in BAF-B03 cells also results in inhibition of IL-2-induced c-fos and c-myc expression (Kawahara et al., 1995). Data derived from transfection of chimeric constructs into CTLL-2 cells demonstrate that the membrane proximal domain of cc is required for induction of c-fos and c-myc and the transduction of proliferative signals (Nelson et al., 1997). Interestingly, the fact that truncations of cc affect bcl-2 induction, whereas a dominant negative mutant of Jak3 does not, indicates the possibility that some functions of cc are independent of Jak3. Nevertheless, mice lacking either cc or Jak3 or both proteins have indistinguishable phenotypes. Oncostatin M (OSM) is also induced as an imme-

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diate–early gene by a number of cytokines, including IL-2 (Yoshimura et al., 1996). Although the physiologicalrolesofOSMinIL-2signalingarenotyetunderstood, it has been proposed to function as a mediator for cell–cell communication in other systems (Brown et al., 1991) and play a role in osteogenesis (Malik et al., 1995).

CIS/SOCS/SSI/JAB proteins CIS (cytokine inducible SH2-containing protein) is the prototype member of a novel family of cytokineinduced proteins. CIS is induced by a number of cytokines and growth factors, including IL-2, IL-3 and Epo (Yoshimura et al., 1995; Chida et al., 1998; Kovanen and Leonard, 1999). An in vitro study shows that CIS (also known as CIS1) protein associates with IL-2Rb through the A region of the IL-2Rb chain (residues 313-382) (Aman et al., 1999). When over-expressed, the wild-type form of CIS, but not a mutant form of CIS in which the critical arginine in the SH2 domain is mutated to alanine, inhibited IL-2-induced Stat5 activation. This indicates that CIS might be involved in negative regulation of Stat5-mediated IL-2 signals and that the SH2 domain of CIS is required for this activity. Although the basis for the inhibitory effect of Stat5 activation by CIS is unclear since it does not seem to either associate with JAK kinases or inhibit the catalytic activity of Jak1 (Aman et al., 1999), expression of IL-2Ra in T cells and proliferation of T cells in response to IL-2 are partially suppressed in CIS transgenic mice (Matsumoto et al., 1999). This is most likely explained by a marked inhibition of Stat5 activation by IL-2 in T cells from these mice, consistent with the in vitrofindings.UnlikeCIS,whichassociateswithIL-2Rb, but not Jak kinases, SOCS3 (also known as CIS3 or SSI-3) primarily interacts with Jak1 and can inhibit Jak1 activation. When over-expressed in BaF3 cells, SOCS3 suppresses phosphorylation of Stat5 and proliferation in response to IL-2 (Cohney et al., 1999). Analyses of either SOCS3 transgenic or SOCS3/ genetargeted mice indicate that SOCS3 plays a critical role in negatively regulating fetal liver erythropoiesis (Marine et al., 1999a), but the IL-2 signaling pathway in these mice remains to be evaluated. SOCS1 (also known as JAB or SSI-1) is also highly induced by IL-2 and associates in vitro with IL-2Rb, Jak1 and Jak3. It potently inhibits Jak1 activity, suggesting an important role for SOCS1 in regulating T

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cell responses (Sporri et al., 2001). Mice lacking SOCS1 exhibit abnormal thymocyte differentiation and spontaneous activation of peripheral T cells so that they proliferate in response to IL-2 in the absence of priming by anti-CD3. These data indicate that SOCS1 plays a vital role in directing thymocyte differentiation, as well as in controlling signaling pathways of certain cytokines, including IL-2 (Marine et al., 1999b). Thus, more than one CIS/SOCS/SSI family protein may be involved in negative regulation of IL-2 signaling in vivo. As discussed above, at least in vitro studies show that CIS and SOCS3 can influence Stat5 signals through inhibition of the different steps of the activation cascade. The exact mechanism of how CIS/SOCS/SSI family proteins mediate their inhibitory effects, as well as the proteins that are recruited to the SH2 domain of this family of proteins remain areas for further investigation.

IL-2 regulates similar sets of genes to IL-7 and IL-15 but not to IL-4 in activated T lymphocytes Recently, analysis of the gene expression profiles regulated by IL-2 and other cc-dependent cytokines in human T cells using DNA microarray methodology has elucidated the identities of many of the genes that are positively or negatively regulated by these cytokines (Kovanen et al., in press). Of 7269 genes analysed, 137 genes were induced and 34 genes were repressed by these cytokines. Interestingly, IL-2 induces a very similar set of genes to IL-7 and IL-15, which also activate Stat5 proteins, whereas IL-4, which activates Stat6, regulates a more distinct set of genes. Among those genes repressed by IL-2, IL-7Ra is the most potently suppressed. Down-regulation of IL-7Ra expression by IL-2 depends upon activation of PI 3K, but not activation of either MAPK or Stat5 (Xue et al., 2002). This finding possibly reveals an important and previously unknown type of cross-talk between IL-2 and IL-7 signaling, although whose biological significance remains to be further established.

SOLUBLE IL-2Ra A soluble form of IL-2Ra (sIL-2Ra) with a molecular weight of 45 kDa has been discribed (Fernandez-

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Botran et al., 1996; Rubin and Nelson, 1990). This is generated by proteolytic cleavage of membrane bound IL-2Ra. Although it can bind IL-2, as expected, it does so with a low-affinity, making it unlikely that it can compete with IL-2 binding to high affinity receptors on the cell surface. Although sIL-2Ra may not have a physiological role, its presence at high levels can be used as a marker for T-cell activation in vivo and has been correlated with a number of disease states, including allograft rejection, some forms of leukemia, certain viral infections, autoimmune disorders and in vivo administration of IL-2 (Rubin and Nelson, 1990; Fernandez-Botran et al., 1996; Leonard, 1999; Morris and Waldmann, 2000).

PHENOTYPES ASSOCIATED WITH DEFECTS IN IL-2 AND IL-2 RECEPTOR CHAINS Above, we have reviewed much of what is known about the IL-2/IL-2R system from in vitro investiga-

IL-2Ra

tions. A critical complementary line of investigation was to clarify the function(s) of these molecules in vivo during development of the immune system and/or in the host defense against pathological challenges. As summarized in Figure 8.6, identification of naturally occurring mutations in the genes involved in the IL-2R system in immunodeficiency patients in humans and evaluation of the phenotypes of the knock-out mice have made it possible to directly address these issues. In mice lacking IL-2 expression, thymocyte and peripheral T-cell subset compositions are normal, but in vitro polyclonal T-cell responses are reduced and the isotype levels of serum immunoglobulins are dramatically changed (Schorle et al., 1991). Although in vivo immune responses to pathogenic challenges in IL-2-deficient mice are not severely impaired (Kundig et al., 1993), these mice develop autoimmunity, including ulcerative colitis-like inflammatory bowel disease, underscoring the importance of IL-2 in vivo in modulating immune responses (Sadlack et al., 1993). These observations also suggest that other

IL-2Rb

cc

IL-2 Jak3

Mouse: Normal T-, B-, and NK-cell development, reduced T-cell response in vitro, and developing autoimmunity

Normal T-, B-, and NK-cell development, polyclonal T- and B-cell expansion and impaired AICD in vivo

Human: Immunodeficiency with reduced T-cell response to antigen stimulation

Immunodeficiency with reduced T-cell response in vitro

No NK cells, spontaneously activated T cells, and B cells terminally differentiated into plasma cells, with a markedly altered Ig profile

Hypoplastic thymuses, age-dependent increase  of CD4 T cells, decreased B cells and absence of NK cells

T âB NK SCID

T B NK SCID











FIGURE 8.6 Phenotypes in mice and humans associated with known defects in IL-2, IL-2Ra, IL-2Rb, cc, and Jak3. THE CYTOKINES AND CHEMOKINES

PHENOTYPES ASSOCIATED WITH DEFECTS IN IL - 2 AND IL - 2 RECEPTOR CHAINS

IL-2Rb not only plays a vital role both in formation of functional intermediate and high affinity receptors and in transduction of the IL-2-induced signals, but it is also a shared receptor component for IL-15, which like IL-2, was also originally discovered as a T-cell growth factor (Bamford et al., 1994; Burton et al., 1994; Grabstein et al., 1994). In addition to its unique actions on mast cells and other non-lymphoid cells, IL-15 has both shared actions with IL-2 and its distinct ones on both T cells and NK cells (Tagaya et al., 1996; Waldmann and Tagaya, 1999; Ma et al., 2000). In a patient with T BNK SCID, expression of the IL-2Rb chain was markedly decreased, but expression of cc, Jak3, IL-15 and IL-15Ra all appeared normal (Gilmour et al., 2001). There were no apparent abnormalities found in the coding region of IL-2Rb gene, so it is not clear what caused the defective expression of IL-2Rb chain in this patient. Mice lacking expression of IL-2Rb manifest severe effects, including deregulated T-cell activation and autoimmunity. These mice die at about 12 weeks of age (Suzuki et al., 1995), indicating that IL-2Rb plays important roles in controlling the activation programs of T cells and in maintaining homeostasis and preventing autoimmunity. Interestingly, expression of IL-2Rb transgenes that lack either the A region or the C-terminal H region can prevent IL-2Rb/ mice from the developing autoimmunity (Fujii et al., 1998). The T cells from IL-2RbDA transgenic mice actually respond better than those from wild-type mice in terms of IL-2-mediated proliferation. This can at least in part be correlated with a constitutively active receptor-associated PI 3K/Akt activity in the cells expressing IL-2RbDA (Cipres et al., 2001). The IL-2Rc gene was localized to human chromosome Xq13 (Noguchi et al., 1993a), in the region previously determined to be the locus for X-linked severe combined immunodeficiency (XSCID, also known as SCID-X1) (de Saint Basile et al., 1987). Patients with SCID-X1 have profoundly decreased numbers of T cells and NK cells, while B-cell numbers are normal that are nonfunctional (TBNK SCID). Noguchi et al (1993) demonstrated that SCID-X1 patients had mutations in their IL-2Rc genes. This was unexpected given the much greater severity of the clinical and immunological phenotype in SCID-X1 patients than was seen in IL-2-deficient patients and mice, and suggested that IL-2Rc might be shared by other cytokine

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→

cytokine(s) may be able to compensate partially for the absence of IL-2 in vivo. A severe combined immunodeficiency patient with defective expression of IL-2, IL-3, IL-4 and IL-5 was also reported. Interestingly, this patient had normal development of lymphocytes, but impaired in vitro proliferation of T cells to mitogen and hypogammaglobulinemia (Chatila et al., 1990). Apparently, the defective expression of these cytokines was due to a selective defect in NF-AT DNA binding activity (Castigli et al., 1993). Importantly, the abnormal T-cell immune response in this patient was restored by administration of IL-2, even though defective expression of the other cytokines was not corrected (Chatila et al., 1990), suggesting that defective expression of IL-2 played a critical role in causing the immunodeficiency. The importance of the IL-2Ra chain is underscored by the analysis of IL-2Ra-deficient mice (Willerford et al., 1995). Although development of T and B cells is normal in young mice lacking expression of IL-2Ra, as adults, these mice typically develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion and exhibit impaired activation-induced T cell death in vivo. Older mice also develop autoimmune disorders, including hemolytic anemia and inflammatory bowel disease. Thus, IL-2Ra is essential for the regulation of both the size and content of the peripheral lymphoid compartment, probably by influencing the balance between clonal expansion and elimination of the effector cells following reactivation of T lymphocytes by antigen stimulation. Presumably, these defects result from the lack of high affinity IL-2 receptor formation. A truncation mutation of IL-2Ra has also been reported in a human immunodeficiency patient with increased susceptibility to viral, bacterial and fungal infections (Sharfe et al., 1997). This patient manifested decreased numbers of peripheral T cells and reduced in vitro proliferation in response to mitogen stimulations, including anti-CD3 and PHA, but normal B cell development. Although T-cell development was normal, cortical thymocytes did not express CD1. CD1 is normally induced in cortical thymocytes prior to a dramatic decrease in expression of the antiapoptotic protein bcl-2 in these cells (Gratiot-Deans et al., 1994; Vanhecke et al., 1995). As the result, downregulation of bcl-2 expression in this IL-2Ra-deficient patient is not observed.

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receptors. Indeed, IL-2Rc chain was later shown to be shared by the receptors for IL-4, IL-7, IL-9, IL-15 and IL-21 as well as IL-2 (Kondo et al., 1993; Russell et al., 1993, 1994; Noguchi et al., 1993c; Giri et al., 1994; Kondo et al., 1994; Kimura et al., 1995; Asao et al., 2001); as a result, it is now known as cc (for common cytokine receptor c chain) (Leonard et al., 1994). Mice lacking expression of cc have hypoplastic thymuses; splenic T cells are diminished at 3 weeks of age, but CD4 T cells are markedly increased by 4 weeks. B cells are greatly diminished in number, in contrast to the situation in human SCID-X1 patients. NK cells, cd intestinal intraepithelial lymphocytes, dendritic epidermal T cells, peripheral lymph nodes, and gutassociated lymphoid tissue are all absent (Cao et al., 1995; DiSanto et al., 1995). These findings underscore the importance of cc in the lymphoid development. It is striking that cc-deficient mice unexpectedly exhibit an age-dependent accumulation of CD4 T cells (Cao et al., 1995; Nakajima et al., 1997b). These cells exhibit both augmented proliferation (presumably mediated by a cc-independent cytokine) and augmented apoptosis, suggesting a vital role for cc in regulating lymphoid homeostasis (Nakajima et al., 1997b). The major defect in T-cell development in SCID-X1 is believed to result from defective IL-7 signaling. Indeed, humans with mutations in the IL-7Ra chain gene exhibit TBNK SCID (Puel et al., 1998). In contrast, the defect in NK-cell development is likely due to defective IL-15 signaling (Carson et al., 1994; Cavazzana-Calvo et al., 1996; Leclercq et al., 1996; Puzanov et al., 1996; Lodolce et al., 1998; Kennedy et al., 2000; Ma et al., 2000). Moreover, differences between humans and mice lacking cc expression indicate species-specific differences in the roles of ccdependent cytokines or the existence of redundant pathways. Because cc associates with Jak3, it was hypothesized that autosomal recessive TBNK SCID, which is clinically and immunologically indistinguishable from SCID-X1, would result from mutation in Jak3. Indeed this was found to be the case (Macchi et al., 1995; Russell et al., 1995). Moreover, Jak3-deficient mice (Thomis et al., 1995; Nosaka et al., 1995; Park et al., 1995) and mice lacking both Jak3 and cc (Suzuki et al., 2000) have a phenotype indistinguishable from cc-deficient mice (Cao et al., 1995; DiSanto et al., 1995; Ohbo et al., 1996). These data further establish

that Jak3 is the major transducer of cc-dependent signals.

CORRECTION OF X-LINKED SEVERE COMBINED IMMUNODEFICIENCY IN HUMANS The demonstration that mutations in cc cause SCIDX1 in humans provided the molecular basis for designing a clinical protocol potentially to correct this inherited immunodeficiency disorder. In theory, the shared nature of cc by the receptors for a number of immunologically important cytokines and the absence of T cells and NK cells in SCID-X1 suggested that this disease might be an ideal candidate for human gene therapy. The reason was that expression of cc was predicted to provide a growth advantage to cc transduced lymphoid progenitor cells. In this regard, correction of the immunodeficiency of cc-deficient mice was achieved by ex vivo cc gene transfer into hematopoietic precursor cells and this could work with human or murine cc (Lo et al., 1999; Soudais et al., 2000). Furthermore, long-term expression of human cc has also been achieved following retroviral infection of canine bone marrow (Whitwam et al., 1998). All these studies have contributed to a recent success in the correction of T and NK cell deficiencies in SCID-X1 patients (Cavazzana-Calvo et al., 2000; Fischer et al., 2001).

IMMUNOTHERAPY TARGETED TO THE IL-2/IL-2R SYSTEM As discussed above, the immunosuppressive agents CsA and FK506 can very efficiently inhibit the T-cell immune response by blocking the production of cytokines, including IL-2, through inhibition of the calcium signals, which in turn prevents certain transcription factors from activation by antigen stimulation. Although structurally similar to FK506, rapamycin inhibits the T-cell immune response by blocking the progression of IL-2-stimulated T cells from G1 to S phase of the cell cycle (Abraham and Wiederrecht, 1996). Interestingly, rapamycin needs to

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first form a complex with the same immunophillins FKBP12 as FK506, but instead then interacts with mTOR (the mammalian target of rapamycin) rather than calcineurin. The inhibitory effect of rapamycin is believed to block the PI3K-p70S6K pathway (Figure 8.4) (Abraham and Wiederrecht, 1996). These immunosuppressive agents are apparently not specific for inhibition of IL-2 production or IL-2 action, but instead have broader inhibitory effects on the immune response. In an effort to more specifically target the IL-2 response, humanized monoclonal anti-Tac (antiIL-2Ra antibodies were developed (Queen et al., 1989). Administration of the humanized anti-Tac to block IL-2-mediated T cell response appears to be effective to reduce the incidence of allograft rejection, and both radio-labeled anti-Tac and toxin-conjugated human recombinant IL-2 have been tested to treat certain T cell leukemia/lymphomas (Waldmann and O’Shea, 1998; Morris and Waldmann, 2000; Waldmann et al., 2001). The reason to choose IL-2 and IL-2Ra as the targets for the immunotherapy is based on their being the only known unique components in the IL-2/IL-2R system. More importantly, the IL-2Ra chain is not expressed by normal resting lymphocytes, but is constitutively expressed by malignant T and B cells involved in leukemias and lymphomas. Furthermore, its expression is tightly correlated with T cell activation that is always observed in those pathological conditions, including allograft rejection and autoimmunity. Mikb1 anti-IL-2Rb antibodies were also humanized (Hakimi et al., 1993). A combination of the humanized Mikb1 and anti-Tac antibodies not only synergistically inhibits IL-2-induced proliferation of activated T cells in vitro, but also markedly reduces the inflammatory reaction due to experimental autoimmunity in vivo. However, the humanized Mikb1 alone seems to be inefficient (Guex-Crosier et al., 1997). Another target for immunotherapy is likely to be Jak3. Although Jak3 can be activated by a number of cc-dependent cytokines, its expression appears to be largely restricted in lymphocytes and hematopoietic cells (Johnston et al., 1994; Witthuhn et al., 1994). More importantly, unlike the fetal or prenatal mortality found in Jak1- and Jak2-deficient mice models (Parganas et al., 1998; Rodig et al., 1998), Jak3 deficiency in humans and mice mainly manifests the

defects in lymphoid and hematopoietic systems (Macchi et al., 1995; Nosaka et al., 1995; Park et al., 1995; Russell et al., 1995; Thomis et al., 1995). Thus, development of specific inhibitors for Jak3 is likely to become another useful approach to modulate the immune responses. Above, we have reviewed the approaches to negatively modulate IL-2-mediated immune responses. Enhancement of the IL-2-mediated T cell immune response has also been applied in combination with other therapeutic regimens to the treatment of individuals with HIV infection and those with certain forms of tumors. In the case of HIV infection, although the antiviral therapy can effectively inhibit viral replication, its effectiveness is limited due to the impaired T cell immune response, especially the CD4 T cell response. The combination of low-dose IL-2 with anti-viral therapy is therefore beneficial in boosting the CD4 T cell response (David et al., 1998; Paiardini et al., 2001; Smith, 2001). Administration of high-dose recombinant IL-2 has also been used to treat selected patients with metastatic melanoma, kidney cancer and non-Hodgkin’s lymphoma (Rosenberg et al., 1994, 1998; Fyfe et al., 1995; Atkins et al., 1999). In this case, the ‘anti-tumor’ effect of IL-2 is likely due to its ability to expand lymphocytes in vivo and boost their functions, thereby inhibiting the tumor growth.

CONCLUSIONS It has been 25 years since IL-2 was first discovered as a T-cell growth factor that was able to specifically promote normal T lymphocyte growth in vitro. The cloning of the cDNAs encoding IL-2, all three IL-2 receptor chains, now known as IL-2Ra, IL-2Rb and cc, and the discovery of the critical signaling pathways, have allowed the major advances in this field. Among the signaling pathways analyzed so far, the JAK-STAT, Shc-Ras-Raf-MAP kinase and PI3K-Akt-p70S6K pathways have been demonstrated to be important for transducing IL-2 signals in vivo, whereas the in vivo function of Src family kinases and other signaling molecules remains unclear. The greater severity of defects associated with IL-2Rb- or cc-deficiency than those seen in IL-2- or IL-2Ra-deficiency is most likely due to the fact that IL-2 and IL-2Ra are the unique

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components in IL-2 system, whereas IL-2Rb and cc are shared with other cytokine systems. The finding that mutations in cc cause SCID-X1 led to the discovery that IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 share a common cytokine receptor component, and also led to the discovery of mutations in Jak3 and IL-7Ra as causes of TBNK and TBNK SCID, respectively (Leonard, 2001). Despite our current knowledge of IL-2 actions and its signaling pathways, a number of important questions still remain to be addressed regarding how IL-2 achieves its pleiotropic actions on a variety of cell types. First, how are IL-2-specific signals achieved? While IL-2 activates many signaling pathways, these signaling pathways are also activated by other cytokines or growth factors that have very different biological actions. For example, Jak1 and Jak3 are also activated by IL-4, IL-7, IL-9, IL-15 and IL-21; Stat5a and Stat5b are activated by growth hormone, prolactin, Epo, IL-3, IL-5, GM-CSF, IL-7, IL-9, IL-15 and IL-21; Stat3 is activated by some of these cytokines plus IL-6, IL-11, leukemia inhibitory factor, OSM, ciliary neurotrophic factor, and cardiotrophin-1; and both Shc and PI3K are activated by many cytokines and growth factors. Thus, ligand-specific signaling pathway(s) for IL-2 have not been identified so far and perhaps there will not be any that are truly specific. Instead, specificity is in part determined by the selective cellular distribution of IL-2 receptors and the specific spatial and temporal considerations that regulate production of IL-2. IL-2-specific signaling is more likely determined by the coordinated activation of different pathways, and by the magnitude and duration of their activation. A related issue is how cross-talk is achieved among these important signaling pathways in response to IL-2? Second, what is the full range of the substrates for kinases activated by IL-2? For example, are there other substrate(s) for JAK kinases besides IL-2Rb, cc, STATs, PI3K, SHP-2, p97/Gab2 and STAM? Third, are there other signaling molecules that are associated with IL-2Rb and/or cc? Fourth, a more complete study of the genes modulated by IL-2 in normal human T cells has recently been completed by DNA microarray methodology (Kovanen et al., in press). These IL-2-regulated genes not only include those previously identified by others (Beadling et al., 1993) but also novel ones, including genes whose functions have never been correlated

with IL-2 actions. It will require more investigation to determine which of these genes are functionally important for transducing IL-2 signals in vivo, not only those induced by IL-2, but also those suppressed by IL-2. Another important area is to achieve more detailed structural and functional analysis of how IL-2 interacts with its three classes of receptors. Such information will be critical in achieving a better understanding of the signaling pathways and actions induced by IL-2. Moreover, our knowledge from these investigations will collectively allow the design of new agents that can selectively either interfere with or enhance IL-2-mediated immune responses. Such agents may allow novel therapeutic approaches to more successfully correct dysfunctions of the immune system, including for example allograft rejection, autoimmunity, immunodeficiency, certain kinds of leukemia/lymphomas, certain forms and stages of the malignant tumors, and the acquired immunodeficiency that is found in AIDS.

ACKNOWLEDGEMENTS We thank David Margulies of Laboratory of Immunology, NIAID, NIH, for creating Plate 8. and Panu Kovanen, John Kelly and Hai-Hui Xue for their thoughtful comments.

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9 Interleukin-3 John W. Schrader University of British Columbia, Vancouver, BC, Canada

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INTRODUCTION

STRUCTURE

Interleukin-3 (IL-3) acts on numerous target cells within the hemopoietic system, so it is not surprising that it was discovered independently by a number of laboratories studying different biological activities on a variety of cell types. These activities went under a variety of names including persisting cell-stimulating factor, histamine-producing cell-stimulating factor, multi-CSF, multilineage hemopoietic growth factor, Thy-1 inducing factor, CFUs stimulating activity, CSF-2a, CSF-2b, hemopoietic-cell growth factor, mast cellgrowthfactor,eosinophil-CSF,megakaryocyte-CSF, erythroid-CSF, burst-promoting activity, neutrophilgranulocyte-CSF, hemopoietin-2 and synergistic activity. It was only with the biochemical purification (Ihle et al., 1983; Clark-Lewis et al., 1984), molecular cloning and expression (Yokota et al., 1984; Fung et al., 1984), and chemical synthesis (Clark-Lewis et al., 1986) that it was conclusively established that a single protein mediated all of these bioactivities.

IL-3 has broad structural similarities with other interleukins and hemopoietic growth factors. It is a relatively small protein – with a polypeptide chain of 140 amino acids in the mouse (Yokota et al., 1984; Fung et al., 1984) and 133 in the human (Yang et al., 1986), and is heavily glycosylated. There are no marked amino acid sequence homologies with other cytokines, although the fact that in the human the genes for GM-CSF and IL-3 are closely linked on chromosome 5 at 5q23-q31 (Yang et al., 1988) supports the notion of a common evolutionary ancestry. Moreover, the three-dimensional structures are highly related, reflecting the fact that the receptors for the two cytokines share a common subunit and closely related specific subunits (see below). The amino acid sequences of mouse and human IL-3 exhibit only 30% identity, reflecting the lack of cross-species biological activity (Yang et al., 1986). Interestingly, IL-3 and a number of other cytokines, including IL-1b, IL-2,

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GM-CSF and erythropoietin, share a short motif of amino acids at the amino terminus. This is characterized by an N-terminal alanine followed in most instances by a proline (Schrader et al., 1986a). The functional significance of this structural feature is obscure. At least in the case of IL-3 and GM-CSF it can be removed without affecting biological activity in vitro (Clark-Lewis et al., 1986, 1988). NMR studies (Feng et al., 1995) have confirmed that IL-3 has the basic four-helix bundle three-dimensional structure, that is characteristic of members of a family including hemopoietins and many cytokines and hormones. The prototype structure was that of growth hormone, and prolactin, erythropoietin, IL-2, IL-4, IL-5, IL-6, and GM-CSF have all been shown to share this basic pattern of three-dimensional folding. Interestingly other hemopoietic growth factors, such as CSF-1 and SLF that as discussed below, interact with a family of receptors quite distinct from that utilized by most hemopoietins and cytokines, nevertheless share the same overall three-dimensional structure, again pointing to a shared evolutionary ancestor. Analysis of structural analogues of IL-3 and of the effect on biological activity of antibodies specific for defined parts of the polypeptide chain have yielded information on the structural determinants of IL-3 bioactivity. IL-3 was the first protein of its size to be successfully synthesized by automated chemical methods (ClarkLewis et al., 1986). This technique, shown by Clark-Lewis to be useful for the synthesis of cytokines in general, allowed a relatively rapid examination of the effects of deleting parts of the IL-3 molecule on bioactivity. In the case of mouse IL-3, these studies showed that the first 16, and the final 22 amino acids, could be deleted with very little loss of biological activity, suggesting that residues 17–118 could form all the structures essential for interaction with the receptor (Clark-Lewis et al., 1986). The notion that the extreme amino-terminus of IL-3 is not involved in interactions with the receptor, is supported by the fact that polyclonal antibodies specific for a peptide corresponding to residues 1–29 of IL-3 had relatively weak ability to neutralize IL-3 bioactivity (Ziltener et al., 1987). Moreover antibodies to peptide 1–29 bind to IL-3 molecules that have been allowed to first interact with the IL-3 receptor (Duronio et al., 1991). In contrast, antibodies to peptides corresponding to

residues 44–75 (Ziltener, unpublished data) and 91–112 (Ziltener et al., 1987) strongly neutralize bioactivity, suggesting that these residues are part of or are close to the site or sites which interact with the IL-3 receptor. McKearn and colleagues have mutated multiple amino acid residues in human IL-3 and obtained analogues of IL-3 with increased potency (Thomas et al., 1995). Because the IL-3 receptor is made up of at least two distinct polypeptide chains, both of which associate closely with IL-3 (Itoh and Yonehara, 1990; Duronio et al., 1991; Miyajima and Mui, 1993), distinct regions of the IL-3 molecule will be involved in binding to the two chains of the receptor termed IL-3Ra and bc. Based on modeling and mutagenesis studies, Bagley and colleagues (1996) have proposed that eight discontinuous residues, Ser17, Asn18, Asp21, Thr25 in helix A and Arg108, Phe113, Lys116 and Glu119 in helix D, mediate binding of IL-3 to IL-3Ra. Binding to bc involves Glu22 but, in contrast with the results of studies on the analogous residue in GM-CSF, replacement of Glu22 with other residues failed completely to abrogate the biological activity of the mutant IL-3 molecules. A model for interaction of Glu22 has been proposed (Klein and Feng, 1997). There are other important properties of the IL-3 molecule, apart from its ability to interact with its receptor, that will be regulated by its structure. These include its clearance and half-life in the plasma, and its ability to interact with extra-cellular matrix. Natural IL-3 occurs in a diversity of glycoforms generated by the addition of carbohydrate groups. Whereas the synthesized polypeptide has a Mr of 14 K upon SDS-polyacrylamide gel electrophoresis, murine IL-3 released from its natural source, activated T lymphocytes, runs as multiple bands, with major groups of bands with Mr around 22 K, 28 K and 36 K (Ziltener et al., 1988). Different T-cell clones appear to produce different proportions of the differently glycosylated forms (Ziltener, unpublished data). Carbohydrate on murine IL-3 of T cell origin is exclusively N-linked (Ziltener et al., 1988). The glycosylation patterns of IL-3 produced by other physiological sources, such as the activated mast cells, have not been examined. IL-3 produced by recombinant DNA techniques in unnatural sources such as Chinese hamster ovary cells or COS cells, exhibits on SDS-gel electrophoresis a broad smear of differently glycosylated species, quite differ-

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ACTIONS ON HEMOPOIETIC STEM AND PROGENITOR CELLS

ent from the pattern of distinct bands seen with IL-3 from the natural source T lymphocytes (Ziltener et al., 1988). The function of these extensive carbohydrate modifications of the IL-3 polypeptide is unknown. Chemically synthesized IL-3 (Clark-Lewis et al., 1986) or IL-3 produced in E. coli (Kindler et al., 1985) exhibit all the biological activities of naturally glycosylated IL-3. Ziltener and colleagues purified heavily, lightly and moderately glycosylated forms of IL-3 from T lymphocytes and demonstrated that, at least in vitro, they had the same specific activities and target specificities as deglycosylated material (Ziltener et al., 1988). The rate of clearance of naturally glycosylated IL-3 from the blood was approximately half that of non-glycosylated IL-3, but was still rapid with a b-half-life of 20 min compared with 10 min for non-glycosylated IL-3 (Ziltener et al., 1994). It is conceivable that the degree or type of glycosylation could regulate interaction with the extracellular matrix and influence diffusion or localization in tissues. One study of in vitro interactions of IL-3 with extra-cellular matrix material found that glycosylated and non-glycosylated IL-3 was bound equally well (Roberts et al., 1988). Ziltener et al. (1994) saw no differences in the stimulatory effects on hemopoietic progenitor cells of long-term treatment with glycosylated or non-glycosylated murine IL-3.

ACTIONS ON HEMOPOIETIC STEM AND PROGENITOR CELLS IL-3 has the broadest target specificity of any of the hemopoietic growth factors. The range of target cells can be summarized as including progenitor cells of every lineage derived from the pluripotential hemopoietic stem cells. Thus IL-3 can stimulate the generation and differentiation of macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes and erythroid cells (summarized in Schrader et al., 1988). IL-3 stimulates the generation of dendritic cells from human CD34 cells in the presence of TNFa, (Caux et al., 1996) and in vivo in mice (Storozynsky and Woodward, 1999). Moreover IL-3 acts on more primitive pluripotential stem cells. IL-3

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stimulated the growth in vitro of colonies containing mixtures of myeloid and erythroid cells and stimulates both in vitro and in vivo the division of cells (CFUs) that form splenic colonies in irradiated mice (Iscove et al., 1989; Iscove and Yan, 1990). IL-3 also stimulates the growth of human hemopoietic stem cells with significant capacity for self-renewal (Haylock et al., 1992; Brugger et al., 1993). Stimulation with IL-3, however, may result in a decreased ability of stem cell populations to self-renew as assessed by long-term repopulating capacity (Yonemura et al., 1996; Peters et al., 1996). IL-3 also directly or indirectly promotes the survival in vitro of cells able to repopulate mice with T and B lymphocytes (Schrader et al., 1988). In vitro, hemopoietic stem and progenitors cells rapidly die if cultured in tissue culture medium alone. Like other hemopoietic growth factors, IL-3 prevents death by apoptosis and promotes survival in vitro (Williams et al., 1990). Populations of mast cells generated by culturing murine bone marrow cells in IL-3 remain dependent on IL-3, not only for their continual proliferation, but also for their survival (Schrader, 1981), and when deprived of IL-3, IL-3dependent cells undergo apoptosis (Williams et al., 1990). This reaction to the withdrawal of IL-3 may be a control mechanism to ensure that the massive proliferation of cells of the hemopoietic system that can be induced by the release of IL-3 (and other hemopoietic growth factors) during emergency situations like infections, is rapidly terminated when the emergency is over and levels of the growth factors drop. Experiments demonstrating the rapid disappearance of IL-3-dependent cells that have been injected into normal animals lacking detectable leads of serum IL-3 (Schrader and Crapper 1983; Crapper et al., 1984a), are consistent with this notion. As is common among cytokines, IL-3 shows strong synergistic activities with other cytokines. For example in man, IL-3 synergizes with CSF-1 in producing macrophages, and with G-CSF in producing neutrophils and with IL-11 in producing megakaryocytes. Both in humans and in mice, IL-3 synergizes with a number of cytokines such as CSF-1, steel locus factor (SLF) and IL-1 in maximally stimulating the growth of primitive hemopoietic stem cells. In the mouse, the effect of IL-3 in promoting the growth of mast cells can be enhanced by IL-4, IL-9 and IL-10.

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EFFECTS OF IL-3 ON MATURE CELLS OF HEMOPOIETIC ORIGIN In common with other hemopoietic growth factors, IL-3 affects not only immature hemopoietic cells, but also the mature members of some lineages. For example the subset of mast cells associated with mucosal surfaces depend upon IL-3 for survival (Crapper et al., 1984a; Schrader et al., 1988). IL-3 also regulates the levels of major histocompatibility antigens on these mast cells, blocking the increased levels of expression induced by interferon-c (Wong et al., 1984). This may reflect the action of IL-3 in inducing expression of members of the SOCS family which inhibit the activation of JAK kinases and activation of STATs. IL-3induced up-regulation of SOCS-3 accounted for the IL-3-induced inhibition of activation of STAT-3 by IL-11 (Magrangeas and Boisteau, 2001). IL-3 induces limited division of well-differentiated murine macrophages and enhances their phagocytosis of yeast (Crapper et al., 1985; Chen et al., 1988). IL-3 stimulation of macrophages results in increased levels of class II major histocompatibility complex antigens and LFA-1 (Frendl and Beller, 1990a), and increased levels of mRNA encoding IL-1 (Frendl and Beller, 1990a), IL-6 and TNF-a (Frendl et al., 1990b). In vivo, ectopic production of IL-3 by tumor cells results in increased numbers of antigen presenting macrophages in the tumors (Pulaski et al., 1996). Treatment of op/op osteoporotic mice lacking CSF-1 with IL-3 increased osteoclast numbers (Myint and Miyakawa, 1999). IL-3 also blocks the rapid apoptosis of so-called ‘plasmacytoid T cells’, CD4, CD3, CD11c cells present in secondary lymphoid tissue (Grouard et al., 1997). In the presence of IL-3 and CD40 ligand these cells differentiate into an important class of dendritic cells. Murine megakaryocytes differentiate in vitro in the presence of IL-3 (Ishibashi and Berstein, 1986). Human basophils are activated by IL-3 (Hirai et al., 1988; Kurimoto et al., 1989) and IL-3 stimulates the survival of human eosinophils (Rothenberg et al., 1988) as well as increasing antibody dependent cellmediated cytotoxity, phagocytosis, and superoxide anion production in response to stimulation with f-met-leu-phe (Lopez et al., 1987).

LYMPHOID CELLS The question of whether IL-3 has a key role in regulating the production of T or B lymphocytes has been controversial. Some of this confusion has stemmed from the fact that unfortunate misconceptions or errors in earlier literature have taken some time to be widely recognized. The early report that first used the term interleukin-3 (Hapel et al., 1981), erroneously ascribed to interleukin-3 the property of stimulating the growth of helper T lymphocytes. The cells misidentified as helper T lymphocytes were in fact contaminating cells of the myelomonocytic leukemia WEHI-3B, which had been used as a source for purification of the IL-3. The confusion in this study may in part have been related to the fact that WEHI-3B cells express the Thy-1 antigen. At that time the Thy-1 antigen was thought to be a specific marker for Tlymphocytes among lympho-hemopoietic cells in the mouse. However IL-3 induces the expression of high levels of the Thy-1 antigen on hemopoietic progenitor cells, including the precursors of macrophages and neutrophils (Schrader et al., 1982). Another erroneous link between IL-3 and lymphocytic cells was based on the notion that IL-3 played a critical role in T-lymphocyte development. This arose from the observation that IL-3 induced increased levels of the enzyme 20 a-hydroxy-steroid-dehydrogenase in spleen cells from athymic mice and the postulate that this enzyme was specific for the T lymphocyte lineage (Ihle et al., 1981). Ihle and colleagues (1983) used the induction of 20 a-hydroxy-steroid-dehydrogenase as the basis of the assay used for the first purification to homogeneity of IL-3. However the notion that this enzyme was restricted to T lymphocytes was disproved by the demonstration that IL-3 (and GM-CSF) induced this enzyme in cells of a number of myeloid lineages, including mast cells (Hapel and Young, 1988).

T LYMPHOCYTES As noted above, pluripotential stem cells capable of ultimately giving rise to T and B lymphocytes may be affected directly or indirectly by IL-3 (Schrader et al., 1988). Palacios reported the growth of IL-3 responsive lines with characteristics of prothymocytes (Palacios and Pelkonen, 1988), but these results have not been

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reproduced. There is evidence that relatively small subsets of mature T cells may respond to IL-3 in the human (Londei et al., 1989). However, at present there is no evidence that thymic development or the function of the common subsets of T lymphocytes are directly influenced by IL-3. It has also been demonstrated that treatment with IL-3 of mice lacking functional JAK-3 kinase genes and thus with severe defects in lymphopoiesis, results in increased production of thymocytes and T cells, presumably through substituting for the action of IL-7 on a lymphoid stem cell (Brown and Nosaka, 1999).

B LYMPHOCYTES As noted there is evidence that IL-3 directly or indirectly affects cells that can give rise to B lymphocytes in irradiated animals (Schrader et al., 1988). Palacios and colleagues reported that IL-3-responsive clones of pre-B lymphocytes could be obtained with high frequency from fetal liver (Palacios et al., 1984). However, there are no published accounts of the reproduction or extension of this work from other laboratories. Palacios has reported the generation of a small number of IL-3-dependent cell lines that had the capacity to give rise to B lymphocytes in irradiated animals (Palacios and Steinmetz, 1985). Furthermore, there are IL-3-dependent lines which can be induced to undergo B-cell differentiation and immunoglobulin gene rearrangement (Kinashi et al., 1988). However, it is unclear how frequently and how reproducibly such cell lines can be obtained. A proportion of a subset of human acute lymphoblastic leukemias classified as B cell precursors have also been shown to respond to IL-3 (Uckun et al., 1989). There are interesting data suggesting that IL-3 antagonizes development along the B-lymphocyte lineage. Ogawa and colleagues (Hirayama et al., 1992; Ball et al., 1995) have reported the in vitro growth of primitive lympho-hemopoietic stem cells that could give rise to both myeloid cells and B lymphocytes. The growth of these stem cells was optimally supported by combinations of Steel or stem-cell factor (SLF) with IL-6, IL-11 or GM-CSF. The generation of B lymphocytes in secondary cultures required the presence of IL-7 and SLF. Interestingly, IL-3 was ineffective, either alone or with SLF, in maintaining the potential to generate B

lymphocytes in the primary cultures and indeed appeared to inhibit the development of B lymphocytes. More detailed studies (Ball et al., 1996) suggested that, when given for a restricted period, IL-3 could enhance the generation of pre-B cell colonies, but that the overall effect of IL-3 was to inhibit B cell development. Winkler et al. (1995) have reported that purified, c-Kit B220 precursors of B lymphocytes could survive and proliferate in vitro in the presence of an IL-7 deficient stromal cell line and either IL-7 or IL-3. Rennick and colleagues (1989) have reported that IL-3 could synergize with a stromal cell factor in stimulating the proliferation of murine pre-B lymphocytes. It is possible that the difficulty in demonstrating the ability of IL-3 to support the growth of precursors of B lymphocytes in many systems relates to the actions of IL-3 on myeloid progenitors. Thus there is in vivo and in vitro evidence that myeloid cells inhibit B lymphopoiesis and that the absence of myeloid progenitors and macrophages enhances or permits the generation of B cells (Rico-Vargas et al., 1994; Nakano et al., 1994). However, there is evidence that the inhibitory effect of IL-3 on B lymphocyte development is a direct one. Thus IL-3 appears to suppress B cell development by acting on an early CD34-positive cell (Miyamoto and Tsuji, 2001). It is conceivable that this action occurs because of inference with other signals, for example by up-regulation of SOCS (Magrangeas and Boisteau, 2001). In summary, primitive hemopoietic stem cells that are ultimately capable of giving rise to cells contributing to the B or T lymphocyte lineages respond to IL-3. Such cells or their more committed progeny can give rise to immortal cell lines or leukemia cells. However overall, there is as yet no compelling evidence that IL-3 has a significant, direct influence on normal B- or T-lymphocyte development.

OTHER CELL TYPES The best characterized actions of IL-3 are restricted to derivatives of the pluripotential hemopoietic stem cell. However, IL-3 has been reported to up-regulate P-selectin expression on endothelial cells (Brizzi and Garbarino, 1993; Khew-Goodall et al., 1996) and vascular smooth muscle cells (Brizzi and Formato, 2001). There are reports of the expression of the IL-3

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receptor in cholinergic neurons in the brain and of protective effects of IL-3 on these neurons during cerebral ischemia neurons (Tabira and Chui, 1998; Wen and Tanaka, 1998). There are also reports that IL-3 may affect the growth of certain epithelial cells, e.g. in carcinomas of the colon (Berdel et al., 1989).

SOURCES OF IL-3 Cells of lympho-hemopoietic origin appear to be the only sources of IL-3. The major physiological source of IL-3 is the activated T lymphocyte (Schrader and Nossal, 1980; Schrader, 1981; Niemeyer et al., 1989). There is as yet no clear understanding of the mechanisms that regulate the spectrum of cytokines produced in response to a given antigen. It is evident that the type or form of antigen and the presence of adjuvants influence the range and quantity of cytokine produced. Mast cells produce IL-3 when IgE Fc receptors are cross-linked (Wodnar-Filipowicz et al., 1989; Burd et al., 1989; Plaut et al., 1989; Ishizuka and Okayama, 1999). The physiological significance of this phenomenon has yet to be established. It may serve to activate or prime other cells in the vicinity of an allergic response. These could include mast cells themselves, as well as macrophages and other hemopoietic cells. Production of IL-3 by antigen-mediated triggering of mast cells could be important in directing the differentiation of the IL-3 responsive subset of dendritic cells (Rissoan and Soumelis, 1999). Activation of mast cells and secretion of IL-3 may account for the rapid increase in histamine-producing cellstimulating activity observed in the serum of parasitized mice, stimulated 6 h before with parasite antigen (Abbud-Filho et al., 1983). Connective tissue mast cells that were cultured in contact with fibroblasts exhibited IL-3 mRNA (Razin et al., 1991), suggesting that IL-3 mRNA might be produced even in steady state conditions. The mRNAs for IL-3 resembles those encoding most cytokines in having a 3UTR that is characterized by AU-rich elements which are known to regulate the stability and efficiency of translation of the mRNA. Stabilization of IL-3 mRNA appears to be under the control of proteins regulated by JNK, p38 MAP kinase and PI-3K (Ming and Kaiser, 1998; Ming and Stoecklin, 2001). Eosinophils also produce IL-3 and other cytokines when activated. Stimuli

include cross-linking of FcR, and adherence to fibronectin (Moqbel et al., 1994). TNFa induces the rapid accumulation of IL-3 mRNA in eosinophils, a process which interestingly is blocked by inhibition of the enzyme p38 MAP-kinase (Tanaka and Schrader, unpublished data). As discussed, IL-3 may upregulate the production of cytokines that favor the development of TH2 cells (Rissoan and Soumelis, 1999). Interaction of mast cells with fibroblasts in vitro can also lead to the accumulation of IL-3 mRNA in mast cells (Razin et al., 1991). The physiological significance of this is unclear. It may depend upon the expression of the SLF on the surface of the fibroblasts and this may only occur in abnormal situations, for example during inflammation. As discussed below it is also possible that interaction of hemopoietic stem and progenitor cells, expressing the c-kit protein, with stromal cells expressing the kit-ligand, SLF could result in a similar phenomenon.

IL-3 IN NORMAL AND IMMUNOLOGICALLY STIMULATED ANIMALS IL-3 is undetectable in the blood of normal animals (Crapper et al., 1984b). In support of the notion that IL-3 is not present in significant quantities in the blood and extra-cellular fluids of normal mice, IL-3dependent cell-lines die when injected into normal mice although they survive if the mice are provided with an artificial source of IL-3 (Schrader and Crapper 1983; Crapper et al., 1984b). IL-3 can remain undetectable in the serum of animals undergoing immune responses (Crapper et al., 1984a). However in these instances, evidence for the local production of IL-3 at sites of immunological activation can be found (Crapper et al., 1984a). For example, cells from lymph nodes draining the site of injection of an antigen but not from normal lymph nodes, produce IL-3 when incubated overnight in tissue culture medium (Crapper et al., 1984a). The local release of IL-3 at sites where T cells are activated results in a characteristic histological ‘foot print’, namely the local accumulation of mast cells generated by the action of IL-3 on undifferentiated precursors (Crapper and Schrader, 1983). In cases where there is massive activation of T lymphocytes, for example graft-versus-host disease,

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small amounts of IL-3 can be detected in the serum (Crapper and Schrader, 1986). Another phenomenon which may have been accounted for by the release of IL-3 into the serum was the transient appearance of histamine-producing cell stimulating factor (HCSF) reported in the serum following the challenge of an immunized animal with a parasite antigen (AbbudFilho et al., 1983). As noted these observations may reflect the rapid release of IL-3, not from the T lymphocytes, but from mast cells activated by interaction of specific IgE with the injected antigen.

IL-3 IN THE SERUM The half-life of intravenously injected IL-3 is short, being in the order of only 40 min (Crapper et al., 1984a). A major part of this IL-3 is destroyed in the kidney. IL-3 does not appear to be bound to larger molecules in the serum (Crapper and Schrader, 1986) and enters the glomerular filtrate. Small amounts are detectable in the urine of animals with high serum levels of IL-3, but most of the filtered IL-3 appears to be resorbed and destroyed in the renal tubules (Crapper et al., 1984b). The release of IL-3 in vivo appears to be associated with stimulation of all of the various types of hemopoietic cells predicted from the in vitro activities of IL-3. For example, in certain phases of graft-versus-host disease in mice, increases occur in the number of mast cells and their precursors and immature myeloid and erythroid cells in the spleen (Crapper and Schrader, 1986). This coincides with the appearance of small amounts of IL-3 in the serum. Since T-cell activation results in the release of multiple cytokines affecting hemopoiesis, including IL-4, IL-5, IL-6 and GM-CSF, a clearer picture of the effects of the chronic release of IL-3 in vivo came from experiments in which mice were inoculated with WEHI-3B, a tumor which produces IL-3 constitutively as a result of insertion of a retroviral DNA into one copy of the IL-3 gene (Ymer et al., 1985). Mice with a localized, subcutaneous tumor of WEHI-3B showed dramatic stimulation of hemopoiesis in the spleen, with increased numbers of myeloid cells, mast cells and megakaryocytes (Crapper et al., 1984a). Interestingly, the levels of IL-3 in the serum of these mice were relatively low (less than 2 ED50 units per ml) suggesting that the chronic maintenance of low con-

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centrations of IL-3 in the serum could achieve marked effects on hemopoiesis. The effects of IL-3 vary in different tissues depending upon the local availability of the different types of target cells. For example, in the gut mucosa, committed mast cell precursors are relatively frequent, whereas progenitors of other hemopoietic lineages are relatively rare (Crapper and Schrader, 1983). In this tissue the local release of IL-3 induces a mastocytosis. On the other hand, in organs like the murine spleen where there is a higher frequency of hemopoietic stem and progenitor cells of various lineages, IL-3 stimulates increases of myeloid and erythroid cells, as well as more modest increases in mast cells and their progenitors (Crapper and Schrader, 1983).

ADMINISTRATION OF IL-3 Administration of IL-3 in vivo is complicated by the relatively rapid clearance of IL-3 from the circulation. The subcutaneous administration of 2000 ED50 units of chemically synthesized IL-3 three times a day for 3 days resulted in increases in splenic weight and in the number of mast cells and the progenitors of mast cells neutrophils and macrophages and in CFUs (Schrader et al., 1986b). Similar results were obtained usingE.coliderived material (Kindler et al., 1985; Metcalf, 1988). The administration of human IL-3 to primates and humans results in effects that are broadly similar to those seen in mice (Donahue et al., 1988; Mayer et al., 1989). In cynomolgus monkeys, IL-3 induced extramedullary hematopoiesis at sites of subcutaneous injection (Khan et al., 1996). IL-3 may have particular utility in stimulating platelet production (Ganser et al., 1990a). IL-3 potentiated the mobilization of stem cells into peripheral blood induced by G-CSF (Geissler et al., 1996; Huhn et al., 1996).

ROLE OF IL-3 IN STEADY-STATE LYMPHO-HEMOPOIESIS There is compelling evidence that IL-3 serves as a link between the immune system (that senses intrusion of foreign substances into the body) and the hemopoietic system that generates the phagocytic and granulocytic

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cells that mediate defense and repair. There is no evidence, however, that IL-3 is involved in steady-state production of blood cells, despite its potent ability to stimulate almost all phases of hemopoiesis. The absence of IL-3 in normal serum and the evidence that links its production, whether by T lymphocytes or mast cells to immunological activation, argue against a role for IL-3 in mediating steady-state hemopoiesis in unperturbed animals. Moreover, the production of a range of hemopoietic cells, including progenitor cells and stem cells capable of generating myeloid, erythroid and lymphoid cells, can occur in vitro in long-term bone marrow culture systems in which IL-3 bioactivity is undetectable (Eliason et al., 1988). These cultures can support the survival of IL-3dependent cells unresponsive to other growth factors like GM-CSF, CSF-1 or G-CSF despite the absence of IL-3 (Schrader et al., 1984), suggesting the presence of alternative mechanisms. One mechanism that permits the survival and limited growth of IL-3-dependent mast cells was clarified by characterization of the protein products of the W and Sl loci as, respectively, a growth-factor receptor and its ligand . Both W and Sl mutant mice exhibited a macrocytic anemia and a deficiency of mast cells that were caused in the case of W mice by a defect expressed in cells, including the hemopoietic stem cells and its derivatives and in the case of the Sl mice by a defect in the microenvironment expressed in tissues including the bone marrow, spleen and skin. Fujita and colleagues (1988a and b) showed that IL-3-dependent mast cells from normal mice but not W mutant mice could survive and proliferate in the absence of IL-3, provided they were allowed to contact fibroblasts from normal mice. Fibroblasts from Sl mice could not maintain mast cell survival. The demonstration that the W mutations involved the tyrosine-kinase receptor encoded by the c-kit gene (Chabot et al., 1988; Geissler et al., 1988) and that IL-3-dependent mast cells retained expression of this receptor, made mast cells an obvious substrate for assays designed to detect the ligand for this receptor. The kit ligand or steel locus factor (SLF), was shown to be a homodimer structurally unrelated to IL-3 and encoded, as expected, by the Sl locus (Anderson et al., 1990; Huang et al., 1990; Zsebo et al., 1990a, b; Williams et al., 1990; Copeland et al., 1990). Mice which lack functional IL-3 genes show no obvious defects in hemopoiesis (Nishinakamura et al.,

1996). In particular there were normal numbers of mast cells. When challenged by a nematode infection, mice lacking in IL-3 did not exhibit as high an increase in numbers of most cells or basophils, and were defective in their protective response (Lantz and Boesiger, 1998). There is also evidence for impairment in some delayed type hypersensitivity responses (Mach and Lantz, 1998). Curiously, mice lacking functional genes for both IL-3 and GM-CSF had increased numbers of circulating eosinophils (Mach and Lantz, 1998). Analysis of mice lacking both functional genes for Mpl (the thrombopoietin receptor) and for IL-3 or the IL-3 receptor has shown that IL-3 is not responsible for the platelet production observed in mice lacking Mpl alone (Gainsford and Roberts, 1998).

LINKS BETWEEN STRESS AND STEADY-STATE HEMOPOIESIS Whether or not small amounts of IL-3 play a subtle role in steady-state hemopoiesis, it is clear that IL-3 is one mediator of the response of the hemopoietic system to stress. The local release of IL-3 from activated T lymphocytes, and in severe immunological stress, its release into the serum, result in accelerated cycling of stem and progenitor cells and large increases in the production of differentiated cells of multiple lineages (Schrader et al., 1988). IL-3 and the other cytokines released during stress not only increase the blood cell production but also modulate the types of cells produced. The stress response thus involves not only acceleration of the normal mechanisms of hemopoiesis, but also overriding of some of the normal processes that regulate the proportions of the different cell types that are produced. Usually this is seen as the result of the positive effects of a lineage-specific factor, such as G-CSF or IL-5 that promotes the survival, growth and differentiation of committed progenitor cells expressing the respective receptors. However another component of this overriding process could be the disengagement of normal regulatory mechanisms. There is some evidence that IL-3 may be involved in such a disengagement of steadystate mechanisms. Thus exposure to high levels of IL3 leads to the down-regulation of c-kit mRNA and protein in both mast cells and cell lines correspon-

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ding to hemopoietic progenitor cells (Welham and Schrader, 1991). GM-CSF and erythropoietin have similar down-regulatory effects on expression of c-kit. The down-regulation of c-kit by high levels of IL-3 (as well as GM-CSF and erythropoietin) may be part of a mechanism that overrides steady-state regulatory processes and facilitates control of the rate and cellular composition of blood cell production by cytokines released during stress. One aspect of this overriding process may be simply the facilitation of the exit of stem and early progenitor cells from the bone marrow. The kit ligand exists in a cell-bound form as a trans-membrane protein (Anderson et al., 1990; Flanagan and Leder, 1990; Martin et al., 1990) and thus may function as one of the adhesion proteins that retains stem and progenitor cells in the bone marrow microenvironment. Down-regulation of c-kit by IL-3 may therefore facilitate the release of stem cells and early committed progenitors from the bone marrow microenvironment. The administration of IL3 has been shown to result in an increase of stem cells in the circulation (Huhn et al., 1996; Geissler et al., 1996). Seeding of stem and progenitor cells into the blood to sites where cytokine release is occurring, would allow the local generation of the appropriate effector cells at sites of inflammation.

RECEPTOR FOR IL-3 IL-3, like most other four-helix-bundle cytokines binds to a heterodimeric receptor, both chains of which belong to a large family of hemopoietin or cytokine receptors that have a common evolutionary origin and share distinctive structural features (Bazan 1990). The family includes receptors for IL-2, IL-4, IL-5, IL-6, IL-7,TSLP, IL-9, IL-11, IL-12, IL-13, IL-15, GM-CSF, G-CSF, ciliary neutrophilic factor, leukemia inhibitory factor, oncostatin M, thrombopoietin, erythropoietin, leptin, cardiotropin, prolactin and growth hormone. In some cases the receptors are homodimers (eg. those for growth hormone, prolactin, erythropoietin, G-CSF), but most are heterodimers made up of two subunits, each of which are members of the superfamily. As discussed below, in some cases there is evidence that the functional receptors are more complex oligomers of these basic subunits. In the human, the IL-3 receptor is made up of two subunits, an a- and a

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b-chain, each of which are members of the hemopoietin receptor super-family. The smaller, 70 K, a-chain (IL-3Ra) is a transmembrane protein that binds IL-3 with low affinity. It is homologous with two other a-chains that bind GM-CSF and IL-5, respectively. The larger, 125 K, b chain is also a component of the human receptors for IL-5 and GM-CSF. While showing no direct affinity for either human IL-3, IL-5 or GMCSF, this shared b chain, termed b common (bc), can interact with any of the three distinct complexes of a-chains and their respective ligands, to generate three specific, high-affinity ligand–receptor complexes (Gearing et al., 1989; Kitamura et al., 1991; Tavernier et al., 1991; Kitamura and Miyajima, 1992). An NMR structure of the cytokine-binding domain of the bc chain shared by the IL-3, IL-5 and GM-CSF receptors has been reported (Mulhern and Lopez, 2000). In the mouse, the situation is a little more complex. A duplication of the gene encoding bc has occurred. One gene (AIC-2B) encodes a b-chain which is functionally equivalent to that in the human, binding neither IL-3, IL-5 nor GM-CSF, but interacting with specific a-chains in the presence of the respective ligands to form three specific, high affinity receptors (Kitamura and Miyajima, 1992). The second gene (AIC-2A, now termed bIL-3), in contrast, has a low affinity for IL-3 (Itoh et al., 1990), and interacts only with the IL-3-specific a-chain. Thus in the mouse there are two types of high affinity IL-3 receptor; one corresponding to the human IL-3 receptor and made up of IL-3Ra and bc and the other, made up of IL-3Ra and bIL-3, peculiar to the mouse. The functional significance of this additional IL-3 receptor in mice is unknown. The intracellular portions of the bIL-3 and bc proteins are very similar and no differences in the signals they transmit have been detected. Both chains of the IL-3 receptor interact closely with IL-3 and can be detected by cross-linking studies with radio-labelled IL-3, as a 70 K and 120 K IL-3-binding species (Duronio et al., 1992). In the mouse there is a naturally occurring polymorphism of the IL3Ra gene which results in abnormally low expression of the IL-3Ra and defective responsiveness to IL-3 (Hara et al., 1995; Leslie et al., 1996). There is also a polymorphism in bIL-3 in the mouse, but this did not result in any defects in IL-3 function (Leslie et al., 1996). Mice which lack a functional bIL-3 gene exhibit no phenotype, whereas mice lacking a

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functional bc gene exhibit pulmonary alveolar proteinosis (Nishinakamura et al., 1995). This appears to reflect their defective response to GM-CSF as the same phenotype is seen in mice lacking functional GM-CSF genes. Interestingly, some patients with pulmonary alveolar proteinosis have been shown to have defective expression of bc (Nishinakamura et al., 1995).

IL-3-MEDIATED SIGNAL TRANSDUCTION Information on the amino-sequence of the a and b chains of the IL-3 receptor provided no clues to the mechanism by which ligand binding activates intracellular signalling. The cytoplasmic domain of the a-chain is relatively short and has no homology with known enzymes. However the cytoplasmic domain of the a-chain is essential for the generation of signals required for survival and proliferation (Orban and Levings, 1999). Like other members of this receptor super-family, the cytoplasmic domain of the IL-3Ra exhibits a region of homology, termed Box-1, which contains a Pro-X-Pro motif. Mutation of these prolines in the IL-5Ra abolishes IL-5-mediated signalling (Takaki et al., 1994). This region may be important for binding the tyrosine kinases that are activated by IL-3 signalling. As discussed below, the JAK-2 kinase is one candidate and members of the Src family of kinases, much as Lyn are others. The b-chain of the IL-3 receptor has a large cytoplasmic domain, but like other receptor subunits in the hemopoietin-receptor super-family, lacks any evidence of catalytic domains. Like the IL-3Ra, bc has a Box-1 region. In addition there are multiple tyrosine residues, some of which have been shown to be phosphorylated following binding of IL-3 and to be critical for recruitment of cytoplasmic signalling molecules to the receptor complex as discussed below. It is unclear how ligand binding activates intracellular events. Interaction of the complex of IL-3 and the a-chain with the b chain results in a stable complex. However by analogy with GM-CSF signaling, highaffinity binding of IL-3 is not likely to be essential for activation of the receptor. Thus, mutants of murine GM-CSF that bind with only low affinity can nevertheless stimulate the growth of murine factordependent cells (Shanafelt and Kastelein, 1992).

These experiments suggest that the key signalling event results from interaction of the a and b chains and does not depend directly on high affinity binding of the ligand and its interaction with the b chain. One model for receptor activation is that heterodimerization of the cytoplasmic domains of IL3Ra and bc generates or stabilizes sites for binding of cytoplasmic tyrosine kinases. Another, based on an analogy with the tetrameric structure of the active IL6 receptor, is that once formed, the IL-3Ra bc dimer would associate with another IL-3Rabc dimer (or alternatively a free bc chain), to generate a bc-bc dimer (Ward et al., 1994; Paonessa et al., 1995). Thus, in the case of IL-6 receptor, the key signalling event is dimerization of gp130 – corresponding to bc, and the cytoplasmic domain of IL-6Ra can be deleted without affecting signalling. In support of a role for dimerization of bc, there is some evidence suggesting that bc already exists as a homodimer in the absence of ligand (Muto et al., 1996). Also supporting the notion that IL-3 signalling involves preformed or induced dimers of bc, is evidence that the generation of dimers of the cytoplasmic domain of bIL-3 can induce mitogenic signals. One type of experiment used chimeric receptors in which the cytoplasmic domain of bIL-3 was fused with the extra-cellular domain of the erythropoietin or IL-4 receptors (Satamaki et al., 1993). The addition of erythropoietin or IL-4 to cells expressing these chimeras resulted in mitogenesis. However, these experiments did not exclude the possibility that mitogenesis depended upon recruitment of additional receptor subunits by the complexes of the ligands and the domains-domains of the erythropoietin or IL-4 receptors and their ligands. Indeed our experiments with a different design of chimeric receptor, in which the extra-cellular regions of the IL-3Ra or bIL-3 were replaced with domains that were not derived from hemopoietin receptors and so should not recruit subunits of the hemopoietin receptor family, have given different results. Thus expression in an IL-3-dependent cell line of a chimeric molecule made up of the extra-cellular domain of CD8 (CD8ED) and the cytoplasmic domain of bIL-3 (bIL-3CD) failed to result in factor-independent growth, despite the fact that CD8ED forms disulphidelinked dimers (Orban and Levings, 1999). Likewise expression of a similar chimera of CD8 and the cyto-

THE CYTOKINES AND CHEMOKINES

IL - 3 - MEDIATED SIGNAL TRANSDUCTION

plasmic domain of IL-3Ra did not confer factorindependent growth. However, co-expression of the CD8ED – bIL-3CD and the CD8ED – IL-3RaCD chimeras resulted in factor-independent growth. This result suggested that heterodimerization of IL3RaCD and bIL-3CD was both necessary and sufficient for the initiation of signal transduction. Similar results were obtained by expressing chimeras of the extra-cellular domain of CD16 and bIL-3CD or IL3RaCD. In this instance a monoclonal anti-CD16 antibody was used to induce cross-linking of the chimeric receptors. Once again, only if chimeras of the CD16ED–bIL-3CD and CD16ED–IL3RaCD were co-expressed and subsequently cross-linked with anti-CD16 antibodies, did growth occur. Patel et al. (1996) did observe factor-independent proliferation when leucine-zippers were used as extra-cellular domains to dimerize cytoplasmic domains of bc. In their studies even aCD–aCD dimers of the GM-CSF Ra had weak but detectable activity in promoting growth and viability. Other evidence that the generation of dimers of bcCD are sufficient to initiate proliferation come from experiments involving the expression of chimeras of the intracellular domains of GM-CSF-Ra or IL-5Ra and the cytoplasmic domains of bc in cells that expressed wild-type bc. In both types of experiment, the respective ligands GM-CSF (Muto et al., 1995) or IL-5 (Takaki et al., 1994) induced mitogenesis. Experiments indicating that expression of mutant bc that are predisposed to dimerize leads to factorindependent growth also suggest that dimerization of bc can result in mitogenesis (D’Andrea et al., 1996; Hannemann et al., 1995; Jenkins et al., 1995; Shikama et al., 1996). It is noteworthy however that bCD–bCD dimers appear to be active in some cell lines, but not others (Jenkins et al., 1995; Shikama et al., 1996). The simplest explanation for these seemingly contradictory results is to assume that aCDaCD, bCDbCD and aCDbCD dimers can all generate signals that suppress apoptosis and promote mitogenesis, but differ in their efficacy – the hierarchy being aCDaCD bCDbCD aCDbCD, with aCDaCD being barely active and aCDbCD being very effective. The events triggered by these dimers could differ only quantitatively, but are more likely to differ qualitatively in the number of signal transduction paths activated. One simple model is

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that both aCD and bCD have affinity for JAK-2 kinase. Thus the formation of dimers of aCD and bCD could lead to the juxtaposition of two JAK-2 kinase molecules and their transphosphorylation and activation. The greater efficiency of bCDbCD dimers as compared with aCDaCD dimers could reflect either a greater affinity of JAK-2 kinases for bCD, and thus greater efficiency in bringing together two JAK-2 kinase molecules, or alternatively a greater capacity of bCD to mediate downstream events, e.g. for example by providing multiple tyrosines which when phosphorylated generate docking sites for signal transduction molecules such as Shc, SHP-2, etc. In contrast to aCDaCD or bCDbCD dimers, aCDbCD dimers function in all cell types tested, consistent with the aCDbCD being the most efficient mechanism for initiation of a mitogenic signal. In terms of the above model we would postulate that aCDbCD provides the most stable site for docking of two JAK-kinase molecules, as well as the optimal set of sites for the docking of proteins involved in the activated receptor complex. However, the general arguments are the same if it is postulated that the key event is juxtaposition of other molecules, e.g. a Src family kinase plus JAK-2 kinase. On the whole, it seems likely that although b–b dimers can induce mitogenic signals, a–b dimerization is the physiological signal for assembly of active receptor complex and is probably sufficient. In support of the notion that ligand binding to receptors of this family induces formation of a only simple a–b dimer, and not more complex structures that include b–b dimers, are the observations of Behrmann et al. (1997). They co-expressed chimeras of the IL-5RaED and bcED with the cytoplasmic domain of gp130 and showed that IL-5 induced the activation of STAT-3 that was expected to follow dimerization of gp130CD. They then co-expressed the bc-gp130CD chimera and a chimera of IL-5RaED and a truncated gp130CD that lacked amino acid residues required for STAT-3 activation. IL-5 failed to induce activation of STAT-3, as would have been expected had higher-order complexes such as abb or abba been generated in the presence of IL-5. It is clear that one of the earliest detectable changes after binding of IL-3 to its receptor – occurring within seconds – is the phosphorylation of a set of proteins on tyrosine residues (Ferris et al., 1988;

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Morla et al., 1988). The clues to the likely nature of the tyrosine kinases involved, come from work on interferon-mediated signal transduction. The receptors of the hemopoietin-receptor super-family are close relatives of the receptors for the interferons. The demonstration that signal transduction via the receptors for IFNa/b or IFNc was critically dependent on a new family tyrosine kinases, the Janus or JAK-kinases (Schindler and Darnell, 1995), was rapidly followed by evidence that kinases of this family were also involved in signalling by the hemopoietin receptors. In particular, IL-3 was shown to induce tyrosine phosphorylation of one member of this family, JAK-2 kinase (Silvennoinen et al., 1993). In the case of the interferon receptors and some hemopoietin receptors, including the erythropoietin and growth hormone receptors (Witthuhn et al., 1993; Argetsinger et al., 1993), there is evidence for the preexisting association of JAK-family kinases with receptor subunits. In one in vitro study, JAK-2 was shown to bind bc (but not the GM-CSFRa) (Quelle et al., 1994) and in another, the cytoplasmic domain of bc was shown to interact with Src family kinases and more weakly, with JAK-2 kinase (Rao and Mufson, 1995). Ligand-induced dimerization of the receptors is postulated to bring the receptor-associated JAK-kinases into close proximity, leading to crossphosphorylation and association. The precise mechanism of activation of JAK-2 kinase by IL-3 is unknown. Other tyrosine kinases may also be involved. IL-3 results in modest activation of the Src-family kinase member Lyn (Torigoe et al., 1992; O’Connor et al., 1992). Comparison of the tyrosine phosphorylation events stimulated in mast cells by IL-3 with those stimulated by SLF, allowed identification of a set of IL-3 specific tyrosine-phosphorylated events (Welham and Schrader, 1992). One of these was tyrosine phosphorylation of the b chains of the IL-3 receptor (Isfort et al., 1988; Duronio et al., 1992). Over a period of 10 min, the b chain of the IL-3 receptor increases in apparent molecular weight, shifting from Mr 125 000, in unstimulated cells to Mr 135–150 000. Much of this increase in apparent molecular weight appears to be due to concomitant serine-threonine phosphorylation (Duronio et al., 1992). One candidate for the kinase involved is PKA (Chen and Yu, 2001), which may be responsible in particular for phosphorylation

of Ser 585, that forms a target for 14-3-3 zeta (Stomski and Dottore, 1999). There were other tyrosine phosphorylation events that were relatively specific for IL-3 stimulation, in that they were not observed with stimulation of cells with other hemopoietins, like SLF, CSF-1 or IL-2. However, in all cases these have involved proteins also involved in signalling by other stimuli. For example one protein that is prominently phosphorylated on tyrosine in response to IL-3, IL-5 and GM-CSF, but not IL-2 or SLF or CSF-1 was a protein with an Mr of 68 K (Duronio et al., 1992; Welham and Schrader, 1992). This protein was shown to be the tyrosine phosphatase SHPTP-2, or Syp, now known as SHP-2 (Welham et al., 1994a). Stimulation with IL-3 resulted in an increase in the enzymatic activity of SHP-2 and its association, via its SH-2 domains, with bc interacting with tyrosines 577, as well as other sites (Itoh et al., 1996; Bone et al., 1997). SHP-2 also became associated with the p85 subunit of PI-3 kinase and Grb-2 (Welham et al., 1994a). SHP-2 may thus have multiple functions in the receptor-complex – by recruiting Grb-2 contributing to the activation of Ras, and by recruiting p85 bringing PI-3 kinase to the membrane and closer to its substrates. Through its tyrosine phosphatase activity, SHP-2 may also down-regulate some signalling events. However SHP-2, while a prominent component of IL-3-stimulated signalling events, is by no means specific for IL-3 signalling, and is involved in signal transduction paths triggered by many growth factors and other stimuli. Indeed there is no evidence for any IL-3-specific component of the IL-3 signal transduction process, downstream of the IL-3 receptor itself. Rather IL-3 signalling conforms to the general model, where receptors for extra-cellular stimuli engage subsets of common intracellular signalling paths shared by many other receptors. In that IL-3 exerts effects common to all growth factors, such as stimulation of proliferation or enhanced survival, it would be expected that the intracellular signals triggered by IL-3 would include many that are shared by other growth factors. One such common event is IL-3-mediated activation of Ras (Satoh et al., 1992; Duronio et al., 1992). This is dependent on tyrosine kinase activity (Duronio et al., 1992). The prominent 55 K protein that is tyrosine phosphorylated in response to IL-3 (as well as GM-CSF, IL-5, IL-2, SLF and CSF-1) is Shc

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(Welham et al., 1994b). IL-3 stimulated tyrosinephosphorylation of Shc results in its association with Grb-2, a small adaptor protein that is constitutively associated with mSos-1. The latter is a guanine– nucleotide exchange factor and when it is brought to the membrane in association with Grb-2 with phosphorylated tyrosine residues in active receptors, it stimulates GDP–GTP exchange on Ras and the generation of active GTP-based Ras (Buday and Downard, 1993). The PTB domain of Shc binds to the bc when tyrosine-phosphorylated, the critical residue being tyrosine 577 (Durstin et al., 1996; Pratt et al., 1996), thus providing a mechanism to recruit Grb-2-m Sos-1 complexes to the receptor. The link between tyrosine phosphorylation of Shc and activation of Ras, is strengthened by the observation that IL-4 – which fails to activate Ras (Satoh et al., 1992; Duronio et al., 1992) – also fails to stimulate tyrosine phosphorylation of Shc (Welham et al., 1994b). Recently, the recognition that the monoclonal antibody used in the standard assay of p21Ras activation, also recognized M-Ras and other members of the Ras family (Ehrhardt and Leslie, 1999), prompted a re-evaluation of which Ras isoforms were indeed activated by IL-3. These studies showed that, whereas IL-3 strongly activated M-Ras, it was far less efficient at activating p21 H-Ras (Schallhorn and Schrader, unpublished). Another form of p21 Ras, K-Ras 4B was also efficiently activated by IL-3. CSF-1, another hemopoietic growth factor, that unlike IL-3 acts through a receptor tyrosine kinase exhibited the same pattern of activation of members of the Ras family. Susceptibility to activation by IL-3 appeared to correlate with carboxytermini that like those of M-Ras or K-Ras 4B, exhibited multiple basic amino acids, and lacked the sites for palmitoylation present in the carboxytermini of other p21 Ras species. There is evidence that these two types of carboxytermini target H-Ras and K-Ras 4B, respectively, to lipid rafts or disordered regions of the plasma membrane. (Roy and Luetterforst, 1999). IL-3 induces activation of endogenous Rac-1, Rac-2 and Cdc42, through a mechanism that is not dependent on IL-3-induced increases in PI-3K activity (Grill and Schrader, 2002). IL-4 induces increases in PI-3K activity (Wang et al., 1992; Gold et al., 1994), but does not activate Rac-1 (Grill and Schrader, 2002) suggesting that activation of the Ras path or of exchange factors activating both Ras and Rac proteins, such as

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mSos-1, may be needed for activation of Rac-1. IL-3 also induces activation of Rap-1, possibly through the adaptor CrkL and the exchange factor C3G (Arai and Nosaka, 2001). There was evidence that activation of Rap-1 was involved in IL-3-mediated activation of b1 integrin and induction of adhesion. It has also been reported that the IL-3-mediated activation of b1 integrin-dependent adhesion can be inhibited by dominant negative mutants of p21Ras (Shibayama and Anzai, 1999). Another event common to stimulation by IL-3 and growth factors in general, is activation of Erk/MAPkinases. Activation of Erk-1 and Erk-2 involves tyrosine phosphorylation and the two proteins of Mr 42 K and 44 K that are phosphorylated on tyrosine in response to the IL-3, were shown to correspond to Erk-2 and Erk-1 MAP kinases (Welham et al., 1992). IL-3 shared this action with GM-CSF, IL-5 and IL-2, as well as SLF and CSF-1 (Welham et al., 1992), although once again IL-4 and IL-13 were notable exceptions (Welham et al., 1994, 1995). The failure of IL-4 to activate Erk-1/Erk-2 MAP-kinases correlates with its inability to activate Ras. The Erk-1/Erk-2 kinases can be activated by a Ras-dependent path that involves Ras-mediated activation of the Raf-1 kinase. Raf-1 phosphorylates and activates MEK-1, a kinase that in turn phosphorylates and activates Erk-1/Erk-2. A minor part of the activation of Erk-1/Erk-2 kinases, however, was inhibited by a specific inhibitor of protein kinase C (Welham et al., 1992). This enzyme can also activate Raf-1 and thus lead to activation of the Erk-1/Erk-2 kinases. Activation of Erk-1/Erk-2 kinases seems to be an important component of the mitogenic signals stimulated by IL-3. Expression in a IL-3-dependent cell line of a dominant inhibitory mutant of MEK-1 that blocked activation of Erk-MAP kinases, resulted in decreased sensitivity to the stimulatory effects of IL-3 on proliferation and the suppression of apoptosis and in inhibition of IL-3-stimulated entry into cell cycle (Perkins et al., 1996). There is some evidence that the PI-3 kinase path may also contribute to the IL-3mediated activation of Erk (Craddock and Hobbs, 2001). IL-3 also activates the two other major families of MAP-kinases, the stress-activated protein kinases (SAPK) or Jun-N-terminal kinases (JNK), and the p38 MAP kinase or HOG-1 kinases. Both SAPK/JNK and p38

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MAP kinase families were initially identified as stressactivated kinases that were activated in response to stresses such as heat, UV-irradiation or hyperosmolarity. It is clear, however, that they are also involved in many physiological responses to growth factors and other stimuli. Thus p38 MAP-kinase is activated by IL-3 – both in primary bone marrow-derived mast cells and cell lines (Foltz et al., 1997; Nagata and Moriguchi, 1997). The enzyme is also activated in response to other growth factors like SLF, GM-CSF (Foltz et al., 1997), and erythropoietin (Nagata and Moriguchi, 1997) and to immunological stimuli like ligation of the antigen-receptors on T and B lymphocytes, of the Fc receptors on myeloid cells, or of CD40, or Fas on B cells and T cells, respectively (Salmon and Foltz, 1997). Likewise JNK-kinases are activated by stimulation with IL-3, as well as GM-CSF and SLF (Foltz and Schrader, 1997; Terada et al., 1997). Interestingly,aswas the case with the Erk-kinases, IL-4 was an exception and failed to induce activation of either p38 MAP kinases or JNK kinases (Foltz et al., 1997a; Foltz and Schrader, 1997b). This may reflect the involvement of the Ras pathway in activating p38 MAP-kinase or JNK kinases in response to hemopoietins. There is evidence that the activation of JNK kinases by IL-3 depends on activation of Ras (Terada et al., 1997). The function of p38 MAP-kinases and JNK kinases in IL-3 signalling is unclear. JNK kinases activate c-Jun and ATF-2, whereas p38 MAP kinases activate other transcriptional activators such as ATF-2, CREB and Elk-1 (Derijard et al., 1994; Kyriakis et al., 1994; Gupta et al., 1995; Raingeaud et al., 1995, 1996; Tan et al., 1996; Iordanov et al., 1997). IL-3 induced activation of MAPKAP-2 kinase and the subsequent phosphorylation of the small heat-shock protein Hsp 25/27 are completely dependent on activation of p38 MAP-kinase (Foltz et al., 1997). Both p38 MAP-kinase and JNK kinases have been postulated to play a role in apoptosis but IL-3, which activates these enzymes, inhibits apoptosis. Moreover, inhibition of p38 MAP-kinase activity failed to block apoptosis induced by withdrawal of IL-3 or by ligation of antigen receptors on T and B lymphocytes (Foltz and Schrader, unpublished). Thus, IL-3 activates, via a tyrosine kinase-dependent mechanism, one common path along which lie Shc, Grb-2, mSos-1, Ras, Raf-1 and Erk-1/Erk-2 MAPkinases. Erk-1/Erk-2 MAP kinases are known to activate the p90rsk S6 kinase which phosphorylates SRF, a

protein that regulates transcription of the c-fos gene. IL-3 mediated activation of the JNK and p38 MAPkinases which may at least in part depend on Ras activation, may also contribute to regulation of c-fos expression and the activity of transcriptional activation complexes like AP-1. Work on interferons not only demonstrated the role of JAK-kinases but also identified a new class of transcription factors, termed signal transducers and activators of transcription (STATs). This family of proteins is characterized by an SH-2 domain that recognizes specific phosphotyrosine residues in activated receptors. This SH-2-mediated recognition provides specificity so that different STATs are recruited from the cytoplasm to different receptors. Following binding to the active receptor, STATs are themselves phosphorylated on a tyrosine residue. This phosphotyrosine is within a recognition site for the SH-2 domain of that same STAT. This favors the formation of SH-2-stabilized STAT homodimers, although in certain situations heterodimers, e.g. of STAT-1 and STAT-3, are also formed. These STAT dimers are rapidly translocated from the cytoplasm to the nucleus where they bind to characteristic DNA motifs. IL-3 induces activation of STAT-5 (Azam et al., 1995; Mui et al., 1995), as do GMCSF, IL-5, prolactin, erythropoietin and IL-2. There are two closely related STAT-5 genes, STAT-5a and b (Mui et al., 1995). Expression of a dominant negative form of STAT-5 suppressed induction of osm, cis and pim-1 and partially inhibited IL-3-stimulated growth and induction of c-fos (Mui et al., 1996). One important class of genes induced by STATs are those encoding proteins of the SOCS family. These act as inhibitors of activation of the JAK-STAT pathway. They limit responses to interferons and cytokines and are likely to account for some instances of cross-inhibition of responses between cytokines and interferons. IL-3 like other growth factors, stimulates increased activity of the lipid kinase, PI-3 kinase (PI-3K) (Gold et al., 1994). This enzyme has multiple functions. One involves the suppression of apoptosis (KauffmannZeh et al., 1997). This occurs through a number of mechanisms, in many instances involving its activation of the protein kinase Akt (Songyang and Baltimore, 1997; Kuribara and Kinoshita, 1999; Gelfanov and Burgess, 2001; Sanz and Mellstrom, 2001). PI-3K also probably has an independent role in IL-3-mediated proliferation (Craddock and Orchiston,

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1999), at least in part through its contribution to activation of Erk (Craddock and Hobbs, 2001). It is also involved in the activation of many proteins such as Rac-1 (Hawkins et al., 1995). There is evidence that IL-3 results in translocation of protein kinase C from the cytoplasm to the cell membrane (Farrar et al., 1985; Whetton et al., 1988a; Pelech et al., 1990). No IL-3-induced increases in turnover of phosphatidyl inositol have been observed (Whetton et al., 1988b), although there is evidence for increased turnover of phosphatidyl choline (Duronio et al., 1989), which could account for generation of diacylglycerol and activation of protein kinase C. IL-3 results in upregulation of cyclin D2 and down-regulation of p27 (Kip1) (Parada and Banerji, 2001). IL-3 also activates protein kinase A, which may be involved in IL-3induced activation of CREB (Scheid and Foltz, 1999) and in survival signals (Chen and Yu, 2001). IL-3 stimulation may inactivate the double-stranded RNAdependent kinase PKR, relieving its inhibitory effect on protein synthesis (Ito and Jagus, 1994). IL-3 also resembles other growth factors in that it stimulates increases in levels of c-myc RNA (Chang et al., 1991). The mechanism is unclear, although there is evidence that PI-3K activity is necessary, although not sufficient (Weiler and Schrader, unpublished). Activation of Erk does not appear to be involved, in keeping with the observation that IL-4, which fails to activate Erk, nevertheless, induces increase in c myc mRNA (Weiler and Schrader, unpublished). IL-3induced increases in c-jun mRNA levels have been reported to be independent of tyrosine-kinase activity and to involve protein kinase C, although results of experiments using kinase inhibitors must be interpreted with caution (Mufson et al., 1992). Certainly Erk activity is likely to be involved, as once again IL-4 fails to induce increase in c-Jun mRNA (Weiler and Schrader, unpublished). IL-3 stimulates phosphorylation on tyrosine of the adaptor protein Cb1 and its association with Grb-2 and the enzymes Fyn and PI3kinase (Anderson et al., 1997). The enzyme phosphatidylinositol-3,4,5-triphosphate 5-phosphatase is constitutively associated with the IL-3 receptor, although its activity does not change following IL-3 stimulation (Liu et al., 1996). Stimulation with IL-3 (and GM-CSF and IL-5) induced expression of a gene DUB-1 that encodes a de-ubiquitinylating enzyme (Zhu et al., 1996).

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IL-3 suppresses apoptosis through multiple mechanisms. IL-3 increases expression of bcl-2 and bcl-xL through activation of the Ras pathway (Kinoshitu et al., 1995). IL-3 can also block apoptosis independently of bcl-xL through PI-3K and Raf-1-dependent up-regulation of NFIL-3 (Kuribara and Kinoshita, 1999). There is also evidence that IL-3 can up-regulate bcl-2 expression through a PKC-dependent mechanism (Rinaudo et al., 1995). One major mechanism through which IL-3 suppresses apoptosis involves its ability to induce increases in PI-3 kinase activity (Kaufmann-Zeh, et al., 1997; Songyang and Baltimore, 1997). This leads to activation of DREAM, a repressor of a proapoptotic protein (Sanz and Mellstrom, 2001), and in concert with Raf-1, to activation of NFkB (Besancon and Atfi, 1998) which induces the inhibitor of apoptosis cIAP (Gelfanov and Burgess, 2001). Increased PI-3K activity can also lead to phosphorylation and inactivation of the proapoptotic BAD (del Peso and Gonzalez-Garcia, 1997). IL-3 also maintains levels of Mdm-2 (Goetz and van der Kuip, 2001). In summary, the binding of IL-3 induces the generation of dimers or possibly higher-order oligomers of the cytoplasmic domains of the IL-3Ra and bc that result in activation of tyrosine kinases including JAK2 kinases and Src kinases. Some of the subsequent events, such as activation of members of the Ras family, increased activity of PI-3K and activation of STAT-5 clearly depend on tyrosine kinase activity. In other cases, such as activation of protein kinase C, it has not been formally shown that activation of an upstream tyrosine-kinase is involved.

CLINICAL SIGNIFICANCE OF IL-3 The ability of IL-3 to stimulate early members of hemopoietic differentiation pathways suggests that it may have specific clinical uses. Promising results in accelerating the recovery of bone marrow following bone marrow transplantation or damage to the bone marrow by cytotoxic drugs have been obtained with G-CSF and GM-CSF, which appear to be effective in reducing the period of neutropenia (Morstyn et al., 1990). However there are indications that, unlike G-CSF and GM-CSF, IL-3 may stimulate an increase in platelet levels (Ganser et al., 1990a). Animal experiments

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suggest that sequential administration of IL-3 and then G-CSF or GM-CSF may provide optimal stimulation of myelopoiesis (Donahue et al., 1988; Mayer et al., 1989). Similar results have been obtained in clinical trials in humans (Lemoli et al., 1996). Other potential uses for IL-3 are in the treatment of conditions, such as aplastic anemia (Ganser et al., 1990b) or other anemias (Halperin et al., 1989; Dunbar et al., 1992). In mice, it has been shown that the administration of IL-3 (but not erythropoietin) prevents death from acute anemia (Shibata et al., 1990). Because of the likelihood that optimal protocols will involve the use of multiple cytokines, it will be some time before the ultimate clinical potential of IL-3 and combinations of other cytokines are clear. However, at present there is no compelling evidence that the use of IL-3 is advantageous for stimulating marrow recovery (Hofstra and Kristensen, 1998; Brouwer and Vellenga, 1999; Mangi and Newland, 1999; Miller and Noyes, 1999; Palmeri and Leonardi, 1999). IL-3 may be useful in managing certain infections. There is some evidence that IL-3 may have a favorable influence on Herpes simplex infection in mice (Chan et al., 1991). Administration of IL-3 to mice infected with Trichinella spiralis accelerated the expulsion of worms (Korenaga et al., 1996a) and the development of IgE responses (Korenaga et al., 1996b). These data are consistent with in vitro experiments that suggest that IL-3 induces the release of IL-4 and IL-3 from non-T, non-B cells, and thus favors the development of a TH2 response (Aoki et al., 1996). IL-3 also induces expression of the TH2 cytokines IL-10 and IL-13 by mast cells (Marietta et al., 1996). Modification of a lung carcinoma by transfection of IL-3 DNA so that it secreted IL-3, resulted in decreased tumorigenicity and an increase in tumorinfiltrating cytotoxic T lymphocytes (Pulaski et al., 1996). This correlated with an increase in the tumor of macrophage-like cells that presented antigens to T lymphocytes. Similar results were obtained in other tumor models (Pulaski et al., 1996; de Wilt and Bout, 2001) suggesting that IL-3 may be effective in enhancing the generation of tumor-antigen-presenting cells. The role of IL-3 in TH2 responses and its stimulatory effects on mast cells and eosinophils suggest that IL-3 antagonists might prove useful in the management of diseases, such as bronchial asthma and allergies. Experimental models in which antibodies that neu-

tralize IL-3 have been used as models of IL-3 antagonists have given encouraging results. Anti-IL-3 antibodies in combination with anti-GM-CSF antibodies block the development of cerebral malaria in mice (Grau et al., 1988). Anti-IL-3 antibodies in combination with anti-IL-4 antibodies block the mastocytosis seen in the parasitized mice (Madden et al., 1991). The administrationofIL-3aggravatesleishmaniasisinmice (Feng et al., 1988). In vitro anti-IL-3 antibodies have been shown to synergize with anti-IL-4 antibodies in unmasking a macrophage-activating activity present in supernatants of cells from Leishmania-infected mice (Liew et al., 1989). Analysis of the relative resistance or susceptibility to various diseases of mice in which IL-3 genes have been artificially deleted should yield helpful information on possible therapeutic use of IL-3 agonists and antagonists. The fact that in man, the receptors for IL-3, IL-5 and GM-CSF share a common b chain, which plays a dominant role in signal transduction, raises the possibility of developing antagonists that will specifically block the activity of this trio of cytokines. Because IL-3, IL-5 and GM-CSF promote the production and survival of eosinophils, basophils and the mast cells that increase in number at sites of allergic reactions, drugs that block these actions could provide a new approach to the treatment of bronchial asthma or allergic diseases. Ideally such an antagonist would leave intact those signal transduction paths required for GM-CSF to block the development of pulmonary alveolar proteinosis. In the mouse there have been described a number of myeloid leukemias in which pathological activation of an IL-3 gene was a key oncogenic event. The constitutive production of IL-3 resulted in autostimulation of growth of the myeloid cell (Schrader and Crapper, 1983; Ymer et al., 1985; Leslie and Schrader, 1989). In some instances the growth of such autostimulatory leukemias may be blocked by anti-IL-3 antibodies (Schrader and Ziltener, unpublished). Autostimulatory production of IL-3, however, does not appear to be an important oncogenic mechanism in human myeloid leukemia, although the leukemic cells usually respond to IL-3 (Budel et al., 1989; Park et al., 1989b) and there has been a report of an acute lymphocytic leukemia in which a translocation joins the IL-3 and immunoglobulin heavy chain genes (Grimaldi and Meeker, 1989). However, there is evi-

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REFERENCES

dence that the primitive stem cells in chronic myeloid leukemia express IL-3 and G-CSF and that these can sustain their growth through an autocrine mechanism (Jiang and Lopez, 1999; Holyoake and Jiang, 2001). Expression of bcr-abl in murine bone marrow stem cells which were then transplanted into mice led to a myeloproliferative disease in which the tranfected cells secreted IL-3 and GM-CSF (Zhang and Ren, 1998). However, experiments using mice with ablation of IL-3, or GM-CSF genes or with ablation of both indicated that autocrine mechanisms were not essential for disease.

SUMMARY IL-3 functions as a link between the T-lymphocytes of the immune system, which sense invasion of the body by foreign materials, and the hemopoietic system, which generates the cellular elements that mediate defense and repair responses. IL-3 is also produced rapidly following stimulation of mast cells or eosinophils, for example by cross-linking Fc receptors and may promote the development of TH2 responses through its effects on a subset of dendritic cells. IL-3 stimulates the broadest range of targets within the hemopoietic system of any of the cytokines, and in addition, has the special ability to stimulate the growth of early stem cells and the progenitors of mast cells and megakaryocytes. IL-3 has no proven role in steady-state hemopoiesis. Evaluation of the clinical utility of IL-3 is still in progress. In the future, antagonists of IL-3 may provide new approaches to the management of allergic and inflammatory diseases.

ACKNOWLEDGEMENTS Experimental work in the author’s laboratory referred to was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada.

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10 Interleukin-4 Hideho Okada1, Jacques Banchereau2 and Michael T. Lotze1 1

University of Pittsburgh Medical Center, Pittsburgh, PA, USA; and 2

Baylor Institute for Immunology Research, Dallas, TX, USA

Gentleman, you can’t fight in here. This is the war room. 1963 Dr Strangelove or How I Stopped Worrying and Learned to Love the Bomb, Stanley Kubrick

INTRODUCTION Interleukin-4 (IL-4) is a glycoprotein of approximate molecular mass 15 kDa. Mouse IL-4 cDNA was first isolated from a T helper 2 (TH2) cell library as a cDNA encoding a unique mouse interleukin that expressed B cell-, T cell- and mast cell-stimulating activities. Subsequently, a human IL-4 cDNA was isolated by cross-hybridization from an activated human T cell clone cDNA library in 1986. In 1989, the genes encoding IL-4 high-affinity glycoprotein receptors (IL-4R) were identified. IL-4 production is restricted to activated T lymphocytes, mast cells, eosinophils and basophils. Precise molecular regulation of IL-4/IL-4R interactions occur at the level of both IL-4 gene transcription, as well as in the IL-4 signaling pathway. IL-4 induces specific biologic functions on a wide range of cells, including DCs and T cells, which are essential components of the adaptive immune response. Clinical trials are being performed which evaluate the therapeutic potential of IL-4 when delivered as a

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

recombinant cytokine or as a transgene in gene therapy trials.

GENE STRUCTURE The human IL-4 gene, composed of four exons and three introns, is located on the long arm of chromosome 5 on bands q23-31, together with genes of other related cytokines including IL-3, IL-5, IL-9, IL-13 and GM-CSF (Morgan et al., 1992) (Figure 10.1A). The IL-4 gene displays many potential binding sites for transcriptional factors, suggesting that its unique expression regulation is mediated by transcriptional factor complexes (Figure 10.1B).

Positive regulatory sequences The PRE-I (positive regulatory enhancer) element, located between nucleotide 241 and 223, is critical for IL-4 gene expression (Li-Weber et al., 1993), as

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Centromere

Telomere 200 kb

IL-13/4

IL-5

IRF-1 CDC25C

110-180 kb

100 kb

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-294 P3,

Neg-2

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-239

NFAT, AP-1, C/EBP β,γ

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IRF-2,NF-1-like

P3, CCAATd

-181

NF-Y, NFAT

CPRE

-176

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-59 -53

NFAT, PCC

MARE

-40

C-Maf, C/EBPβ

TATA

-23

Fra 1/2, JunB, JunD

FIGURE 10.1 (a) Genomic map demonstrating genes around chromosome 5q31-1. IRF-1, which encodes a transcription activator of IFNa/b and other IFN-inducible genes. CDC 25C, cell division cycle 25; YAC, yeast artificial chromosome. (b) Schematic design of the IL-4 promoter. The Figure is a comparison of studies of both the human and mouse IL-4 genes, and is not drawn to scale. Names of the different regulatory elements are indicated to the left. Numbers refer to the base pairs upstream from the transcription start site, and in general indicate the central nucleotide of each defined element. Factors that bind the promoter are shown to the right, with those shown to inhibit IL-4 transcription indicated in italics. Binding sites for some factors (e.g. octamer proteins) are not shown for clarity. The Figure is provided courtesy of Dr Steve N. Georas, Johns Hopkins University (Georas et al., 2000).

deletion or mutation in the PRE-I region abolishes basal and PHA/PMA (phytohemagglutinin/phorbol myristate acetate)-induced promoter activity. The PRE-I element interacts with at least two nuclear transcriptional complexes, POS-1 (positive element binding protein) and POS-2 (Li-Weber et al., 1993), assembled depending on cell type and including various transcription factors such as C/EBP-c (CCAAT/enhancer binding protein) (Davydov et al., 1995a), NF-IL-6 (nuclear factor) (Davydov et al., 1995b), NF-IL-6/3, jun or NF-AT (nuclear factors of activated T cells) factors (Li-Weber et al., 1997b). Of interest, the restricted expression of NF-IL-6 and POS-1 to T helper 2 (TH2) cells would indicate the importance of PRE-I and the composition of its nuclear transcriptional complexes in the cell type specific expression of IL-4 (Li-Weber et al., 1997a, 1998). The IL-4 gene also contains in its 5 flanking region, five sites of a purine-rich motif: ARE (activated responsive element) or P sequences that bind the NFAT transcription factor family, specific for T cells (Abe et al., 1992; Szabo et al., 1993). These P sequences and the IL-4 (741 to 60 base pairs) regions located outside the IL-4 promoter are IL-4 promoter elements conferring TH2-restricted expression (Wenner et al., 1997). Indeed, NF-AT DNA binding is equally induced in both TH1 and TH2 cells after antigen stimulation, but compared with that of TH1 cells only the NF-AT complexes present in TH2 cells promote a high level of IL-4 transcription coinciding with an increase in IL-4 production (Rincon and Flavell, 1997). This would suggest that regulation of NF-AT factors appears to be essential in TH1/TH2 differentiation. Moreover, the importance of NF-AT factors in IL-4 expression has been demonstrated in vivo in NF-ATp-deficient mice, which display a defect in IL-4 production as well as hyper-proliferation of both B and T lymphocytes (Hodge et al., 1996). A functional STAT6 binding site in the IL-4 promoter (located between nucleotides 167 to 135) promotes TH2 cell maturation (Lederer et al., 1996). Indeed, prolonged activation of STAT6 is characteristic of T cells undergoing TH2 differentiation. This observation may also explain the mechanism by which IL-4 regulates its own production. STAT6 interacts with three binding sites in the human IL-4 promoter. These sites overlap the P1, P2 and P4 NF-AT

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binding elements (Georas et al., 1998). A novel P element in the upstream region of the human IL-4 promoter P5 shares a core NFAT motif (353)GGAAA(357) and contributes to IL-4 promoter inducibility (Burke et al., 2000). The protooncogene c-maf, a basic region/leucine zipper transcription factor, interacts with a c-maf response element in the IL-4 promoter and also controls selective expression of IL-4 with restricted expression in TH2 cells (Ho et al., 1996, 1998). CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in TH cells in response to mitogenic stimulation. Overexpression of CP2 enhances activity of IL-4 promoter, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells (Casolaro et al., 2000). A zinc-finger protein YY-1 (Yin-Yang 1) can bind to multiple elements within the human IL-4 promoter, including (59)TCATTTT(53), which is essential for YY-1-driven IL-4 promoter activity in human T cells (Guo et al., 2001). More distant regulatory elements were also discovered using cross-species sequence comparisons of non-coding elements. Among the elements conserved in the mouse and human genome, characterization of the largest element in yeast artificial chromosome transgenic mice reveals it to be a coordinate regulator of IL-4, IL-13 and IL-5 (5q31 cytokines) spread over 120 kb (Loots et al., 2000). In a study using human yeast artificial chromosome transgenic mice that contained the cluster for 5q31 cytokines, human IL-4, IL-13 and IL-15, were expressed under TH2, but not TH1, conditions in vitro, suggesting that conserved sequences coordinate the cell-specific regulation within the cytokine cluster itself (Lacy et al., 2000). The transcription factor GATA-3 appears to be the key transcription factor inducing TH2 cell differentiation (Zheng and Flavell, 1997; Ouyang et al., 1998, 2000; Ranganath et al., 1998). GATA-3 is selectively expressed in TH2 cell. Antisense GATA-3 inhibits the expression of TH2 cytokines, and constitutive GATA-3 expression in CD4 T cells caused TH2 cytokine expression in TH1 cells in GATA-3 transgenic mice (Zheng and Flavell, 1997). Ectopic GATA-3 expression blocks IL-12 receptor expression and induces a TH2 phenotype in developing and committed TH1 cells (Ouyang et al., 1998; Lee et al., 2000). Overexpression of GATA-

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3 induces TH2-specific cytokine expression, not only in developing TH1 cells, but also in otherwise irreversibly committed TH1 cells. This phenomenon involves chromatin remodeling into an accessible form in committed TH1 cells (Lee et al., 2000) at the intronic and intergenic regulatory regions. Elements in the 3 region of the IL-4 gene contribute to TH2 specificity (Takemoto et al., 2000; Lee et al., 2001). Deletion of an evolutionarily conserved approximately 400-bp noncoding sequence in the intergenic region between the genes IL-4 and IL-13, designated conserved noncoding sequence 1 (CNS-1) results in compromised induction of TH2 response in vitro and in vivo (Mohrs et al., 2001). Despite the profound effect in T cells, mast cells from CNS-1 (/) mice are still able to produce IL-4. A T cell-specific element critical for the optimal expression of type 2 cytokines may represent the evolution of a common IL-4/IL-13 regulatory sequence exploited by adaptive immunity. In addition to the genomic DNA sequences, dsRNA, which could consist of a genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses stimulates TH1 responses. dsRNA also directly influences the IL-4 promoter and upregulates IL-4 expression through its effect on NF-jB and NF-AT activation (Kehoe et al., 2001).

Negative regulatory sequences The human IL-4 promoter also displays several negative regulatory elements: (1) a silencer region containing two protein binding sites NRE-I (negative regulatory element) and NRE-II, which interact with the T cell-specific Neg-1, as well as the ubiquitous Neg-2 nuclear factors (Li-Weber et al., 1992). They suppress the activity of the PRE-I enhancer element (Li-Weber et al., 1993); (2) an interferon (IFN)-stimulation response element (ISRE) in which selected mutations increase by two-fold the IL-4 reporter gene activity (Li-Weber et al., 1994), binding IRF-2 (interferon regulatory factor-2), a transcriptional repressor for interferon genes; and (3) a novel negative regulatory element (P2 NRE) adjacent to an NF-AT binding site, which binds to Rep-1 (Georas et al., 2000). Transfection of the nuclear tumor suppressor protein p53 in activated T cell lines downregulated IL-4 expression (Pesch et al., 1996). The use of a domain deleted p53 species demonstrates the

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importance of the transactivation and oligomerization domains of p53 for IL-4 transcriptional repression. These findings suggest the presence of regulatory p53 binding sites in the promoter of the IL-4 gene.

Other mechanisms of IL-4 gene regulation IL-4 production is also controlled post-transcriptionally through stabilization of IL-4 mRNA (Dokter et al., 1993b). IL-7 increases IL-4 mRNA expression in human anti-CD3/anti-CD28-activated T lymphocytes, via mRNA stabilization (Borger et al., 1996a). Enhanced accumulation of IL-4 mRNA is conversely down-regulated by prostaglandin (PG)E2 or 2-0dibutyryl cyclic AMP (Borger et al., 1996b), which activate the protein kinase A pathway, a negative regulator of IL-4 expression. Post-transcriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which both type I and II IFNs antagonize IL-4 production in human immune cells (So et al., 2000).

PROTEIN STRUCTURE Human IL-4 cDNA contains a single open reading frame (ORF) encoding 153 amino acids, yielding a secreted glycoprotein of 129 amino acids (Yokota et al., 1986). IL-4 displays 20% homology at the amino acid level with IL-13, another T cell-derived cytokine which shares numerous biologic properties with IL-4 (McKenzie et al., 1993; Minty et al., 1993). Expression of the recombinant IL-4 protein in mammalian cells generates three secreted variants with apparent Mr values of 15, 18 and 19 kDa. This microheterogeneity is related to N-linked oligosaccharides present in the 18- and 19-kDa species. Human IL-4 contains three disulfide bridges located between C3-C127, C4-C65 and C46-C99 (Le et al., 1991). X-ray diffraction of IL-4 crystals (Walter et al., 1992), as well as magnetic resonance spectroscopy of IL-4 in solution (Smith et al., 1992; Powers et al., 1992) confirm that IL-4 is another left-handed four a-helical bundle cytokine with short stretches of b sheets. The four a helical bundles are located between residues 9–21, 45–64, 74–96 and 113–129, and the mini antiparallel b sheets are located between residues 32–34 and 110–112 (Garrett et al.,

1992). This structure strongly resembles granulocytemacrophage colony-stimulating factor (GM-CSF), MCSF and growth hormone. The crystal structure of the intermediate complex formed between human IL-4 and IL4-receptor achain reveals a novel spatial orientation of the two proteins with a small, unexpected conformational change in the receptor-bound IL-4, and an interface with separate clusters of trans-interacting residues (Hage et al., 1999). Among the binding clusters, a recent study has demonstrated that the energetically most important group is the receptor carboxylate group of D72 forming an ion pair with IL-4 R88 in cluster II. The second main receptor determinant is the hydroxyl group of Y183 forming a hydrogen bond with IL-4 E9 in cluster I (Zhang et al., 2002). These findings may facilitate the design of effective IL-4 receptor antagonists with therapeutic potential. mRNA sequencing has revealed the existence of an additional IL-4 message lacking 48 base pairs coding for amino acid residues 22–37 by alternative splicing of exon 2 (Sorg et al., 1993). Such a transcript results in a mature protein lacking one of the cysteine bridges and part of the loop connecting helices 1 and 2. This natural splice variant of IL-4 mRNA, termed IL-4 d2, is expressed more strongly in thymocytes and bronchoalveolar lavage cells than IL-4 mRNA, suggesting tissue-specific expression. Unlike IL-4, IL4 d2 alone does not act as a costimulator for T-cell proliferation, but rather as an IL-4R antagonist, inhibiting T-cell proliferation induced by full-length IL-4 (Atamas et al., 1996). Recombinant IL-4 d2 blocks the inhibitory action of IL-4 on LPS-induced cyclooxygenase-2 expression and subsequent PGE2 secretion in monocytes. In B cells, IL-4 d2 is an antagonist of the IL-4-induced synthesis of IgE and expression of CD23 (Arinobu et al., 1999). Moreover, an IL-4 molecule in which the Tyr residue 124 was substituted with an Asp residue also acts as an antagonist of IL-4 (Kruse et al., 1992). Associations between polymorphism of IL-4 (see Chapter 2, Gallagher et al.) and increased prevalence of some autoimmune and infectious diseases have been reported. For example, Crohn’s disease has significant association with the 590 T allele of the interleukin-4 gene (P  0.03), that leads to reduced expression of IL-4 (Klein et al., 2001). The IL-4-524 T allele is at high frequency in the population and is

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associated with elevated antibody levels against malaria antigens (Luoni et al., 2001).

CELLULAR SOURCES OF IL-4 IL-4 is secreted by several hematopoietic cell types, including T cells, CD3 NK1.1 (NK-T) T cells (Yoshimoto and Paul, 1994), basophils, eosinophils and mast cells (Moqbel et al., 1995; Velazquez et al., 2000).

T lymphocyte production CD4 T cells There are two differentiation types observed in socalled helper CD4 T cells based on their pattern of cytokine production: TH1 cells secrete IL-2, lymphotoxin (LT)-a and IFN-c. TH2 cells secrete IL-4, IL-5, IL-6 and IL-10. Both cell types secrete some other cytokines including GM-CSF, IL-3 and IL-13 (Romagnani, 1992; Parronchi et al., 1992; Belardelli, 1995). CD4 T cells with an intermediate cytokine profile TH0 have also been described (Rocken et al., 1992a). TH1 and TH2 cells differentiate from a pool of multipotent precursors through differentiation pathways controlled by specific transcription factors, induced by cytokines and costimulatory molecules expressed by lymphocytes or antigen presenting cells. IFN-c, IL-12 and IL-18 promote differentiation of T helper precursors into TH1 cells (Trinchieri, 1993; Germann et al., 1993), whereas IL-4 induces differentiation into TH2 cells (Parronchi et al., 1992). However, the notion that IL-4 acts solely as a TH2 inducer has been reconsidered. In the presence of IL-4 and TGF-b, the nominal TH2 differentiation factors, naive CD4 T cells can differentiate into TH1 type cells in vitro (Lingnau et al., 1998). Naive CD4 T cells themselves can be a source of IL4 that leads to their own TH2 development. IL-6 is a potent inducer of this IL-4 (Rincon et al., 1997). This differentiation pathway can be mediated by activation of the NF-IL-6 family, whose binding sites are found in the IL-4 gene promoter (Davydov et al., 1995b). Moreover, anti-IL-4 antibodies block IL-6induced TH2 differentiation demonstrating that IL-6 promotes maturation of TH2 cells through induction of IL-4 production. Up-regulation of IL-4R expression is

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identified during T cell maturation, a mechanism mediated by IL-4 and not suppressed by IL-12 (Nakamura et al., 1997). Thus, the endogenous IL-4 autocrine loop favors TH2 cell maturation. The question of whether T cells have to be committed to TH2 response to produce high levels of IL-4 is still open to debate (Coffman and von der Weid, 1997). Conventional CD4 T cells (class II MHC-restricted and lacking NK markers) can act as sources of the initial IL-4 for TH2 induction. Transient, partial depletion of CD4 T cells from Balb/c mice with anti-CD4 antibodies at the time of L. major infection converted this strain from TH2 to TH1 responders (Titus et al., 1985). The early IL-4 source in this model is a CD4 T cell, an interpretation supported by direct demonstrations that the basal IL-4 level and the earliest IL-4 induced by L. major could be abrogated by anti-CD4 treatment (von der Weid et al., 1996). Likewise, TH2 development of naive CD4 T cells (CD62L) stimulated in vitro with immobilized anti-CD3 can be driven by IL-4 originating from CD4 T cells that had been previously activated, based on their low levels of CD62L expression (Gollob and Coffman, 1994). A few studies have suggested the possibility that the early source of IL-4 may originate from naive-responding CD4 T cells, possibly during CD4 T cell precursor differentiation (Rocken et al., 1992b; Kamogawa et al., 1993; Rincon et al., 1997).

Counter-regulation of TH1 and TH2 TH2 immune responses may be particularly dependent on the availability of coreceptor and costimulatory molecule interactions. B7 (CD80, CD86) ligand interactions are required for the development of the TH2 immune response. Interaction of CD28 with either CD80 or CD86 can provide sufficient signals for its initiation. In CD86-deficient mice, although the initial TH2 immune response is intact, the response is not sustained. This suggests that CD86 is important at later stages of the TH2 immune response (Gause et al., 1997, 1999). As expected from the pattern of cytokine production, TH1 and TH2 cells regulate distinct biologic functions. TH1 cells activate macrophages resulting in delayed-type hypersensitivity responses and lysis of intracellular parasites. In contrast, TH2 cells control more particularly humoral responses including the production of IgE and associated eosinophilia.

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An important feature of TH1, and TH2 cells is the ability of one subset to negatively regulate the activity of the other. This occurs directly at the level of these subsets, as the products of one subset can antagonize the activation of the other: IFN-c inhibits proliferation of TH2 cells (Gajewski et al., 1989), whereas IL-4 inhibits cytokine production by TH1 cells (Peleman et al., 1989). It also occurs at the level of the effector cells triggered by these subsets, as indicated by the inhibitory effects of IFN-c on IL-4-induced B-cell activation or those of IL-4 on IL-2-induced T- and B-lymphocyte proliferation.

CD8 T cells Naive CD8 T cells, similar to CD4 T cells, can differentiate into at least two subsets of cytolytic effector cells with distinct cytokine patterns: T cytotoxic-1 (Tc1) cells secrete a TH1-like cytokine pattern, including IL-2 and IFN-c. Tc2 cells produce TH2 cytokines, including IL-4, IL-5 and IL-10. Despite their distinctive cytokine profiles in vitro and in vivo, Tc1 and Tc2 cells induce similar DTH reactions (Li et al., 1997).Polarized mouse Tc1 and Tc2 phenotypes persisted in vivo for up to 13 weeks following adoptive transfer (Cerwenka et al., 1998). Extensive studies have been conducted using an ovalbumin (OVA) transfected tumor models to evaluate these cells. Titration of Tc1 and Tc2 effector cells showed that protection was dose-dependent with the former being five-fold more effective (Dobrzanski et al., 1999). Analyses of trafficking of the effector cells in vivo demonstrated that Tc1 cells injected intravenously entered the draining lymph nodes faster than Tc2 and resulted in a more rapid accumulation of host immune cells (Helmich and Dutton, 2001). In an autoimmune diabetes model, Tc2 cells were unexpectedly found to be just as cytotoxic as Tc1 cells, but with less rapid accumulation in the pancreas. This was thought to be a possible consequence of differential chemokine receptor expression (Vizler et al., 2000). Also in humans, significant functional differences are observed between the subsets. Tc2 and Tc0 clones expressed CD30 and CD40 ligand at a much higher level than Tc1 clones. All Tc1, Tc2 and Tc0 clones showed comparable cytotoxicity and produced similar levels of perforin and Fas L. However, Tc2 clones were much more resistant to activation-induced cell death and less susceptible to apoptosis following direct Fas

ligation. Moreover, Tc1 and Tc2 clones had restricted effects on the development of CD4 effectors, promoting type 1 and type 2 responses, respectively (Vukmanovic-Stejic et al., 2000).

NK-T cells CD3 NK 1.1  T cells (NK-T cells) are a unique subset of T cells that recognize lipid antigens presented by CD1d. Following TCR, NK T cells promptly produce large amounts of cytokines notably IL-4, which modulates the subsequent adaptive immune response (Yoshimoto and Paul, 1994). Stimulation of NK-T cells with CD1-presented a-galactosylceramide (a-GalCer), induced substantial IFN-c secretion in vivo. Recall stimulation with antigen in vitro leads to the synthesis of IL-4 and IL-10 in addition to IFN-c. Repeated exposure of mice to a-GalCer induces splenic T cells to secrete IL-4 and IL-10, but dramatically reduced levels of IFN-c, suggesting TH2 type polarization of NK-T cells with this ligand (Burdin et al., 1999). Furthermore, as early as 6 days after alpha-GalCer injection into mice, marked increases in serum IgE levels were observed, as well as IL-4 production from NK-T cells (Singh et al., 1999). IL-7 is also a potent IL-4 inducer in NK-T cells both in vivo and in vitro (Hameg et al., 1999). NK-T cells also play a role in various mouse disease models. Experimental allergic encephalitis (EAE) was induced in B10.PL mice with myelin-reactive T cells. Simultaneous activation of NK-T cells with a-GalCer upon vaccination aggravated disease induction. Prior activation of NK-T cells protects against development of disease. Exacerbation of EAE was mediated by an enhanced TH1 response to myelin basic protein and is lost in mice deficient in IFN-c. Protection was mediated by immune deviation of the anti-myelin basic protein (MBP) response and is dependent upon the secretion of IL-4 (Jahng et al., 2001).In humans, invariant NK-T cells rapidly produced large amounts of IL-4 and IFN-c and influenced TH1 and TH2 differentiation, respectively. In contrast, bone marrowderived CD1d-reactive T cells, the majority of which use a more diverse TCR repertoire, were TH2-biased and suppressed an MLR response (Exley et al., 2001). Increased IL-4 production by NK-T cells was also detected in systemic lupus erythematosus (Funauchi et al., 1999).

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INTERLEUKIN - 4 RECEPTOR COMPLEX

IL-18 is a potent inducer of IFN-c, GM-CSF, IL-13, IL1a and IL-1b, as well as enhancing FasL expression on NK and T cells. It promotes a TH1 response primarily with IL-12 (Okamura et al., 1995). Recent studies demonstrated that IL-18 acts in a more subtle manner in that both pro-TH1 and TH2 responses are promoted depending on the nature of the stimulation and the targeT cells. For example, IL-18 induces IL-4 expression from ligand-activated NK-T cells (Leite-DeMoraes et al., 2001). NK-T cells play a dynamic role in determining the initial immune response through expression of cytokines, including IL-4.

Basophils, eosinophils and mast cells Mast cells, basophils, and eosinophils produce significant amounts of IL-4 upon activation (Brown et al., 1987; Brunner et al., 1993). The ability of mast cells and eosinophils to produce IL-4 within the peritoneal cavity or at mucosal sites, makes them particularly good as candidates in initiating TH2 differentiation for antigenic challenge at these sites. The clearest evidence of the role of a non-T cells source of IL-4 in the initiation of a TH2 response was demonstrated in studies of the initial response to intraperitoneal injection of S. mansoni eggs (a strong TH2 stimulus) (Sabin and Pearce, 1995). The earliest IL-4 originates from eosinophils in this setting. Mast cells indirectly also play a role by secreting IL-5 and recruiting eosinophils to the site of egg injection. In patients with allergic asthma, mast cells within the airway mucosa express intracellular IL-4 (Bradding et al., 1993). IL-4 appears to be present within these cells in a preformed state. Recent studies, however, demonstrated that severe asthma, compared with mild asthma, is characterized by reduced mucosal eosinophilia and reduced number of IL-4 containing cells (Vrugt et al., 1999), perhaps down-regulated in the setting of chronic inflammation.

INTERLEUKIN-4 RECEPTOR COMPLEX High-affinity (Kd  40–120 pM) receptors of IL-4 are expressed in low numbers on a wide range of cell types, including T and B lymphocytes, monocytes granulocytes, fibroblasts, epithelial and endothelial

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cells (Cabrillat et al., 1987; Park et al., 1987). IL-4 upregulates expression of its own multimeric receptor, after inducing its transient down-regulation following receptor ligand internalization (Galizzi et al., 1989). Moreover, cross-linking studies show that IL-4 binds to at least three separate molecular species of molecular weight 140, 70–75 and 65 kDa (Foxwell et al., 1989; Galizzi et al., 1989). A specific cDNA encoding the human 140-kDa IL-4R (gp140/IL-4Ra/CDwl24) has been isolated (Galizzi et al., 1990; Idzerda et al., 1990) (Figure 10.2). The mature receptor is a glycoprotein composed of 800 amino acids, with an extracellular domain of 207 amino acids containing two motifs (four conserved Cys and a WSXWS box) characteristic of the cytokine receptor family (Miyajima et al., 1992), a single

Classical IL-4R

Alternative IL-4R IL-4

IL-13

γc IL-4Rα hematopoietic cells

IL-13Rα IL-4Rα non-hematopoietic cells including cancer cell

FIGURE 10.2 IL-4 receptor structure model. The classical IL-4R is predominantly expressed in hematopoietic cells and consists of the 140 kDa protein, IL-4Ra and the 65 kDa form is the c common chain (cc) which is shared with other cytokine receptors, including IL-2, IL-7, IL-9 and IL-15. Alternative form of IL-4R, on the other hand, is predominantly expressed in non-hematopoietic cells and consists of IL-4Ra and IL-13Ra chains. Both IL-4 and IL-13 signals are transmitted through the alternative form of IL-4R in non-hematopoietic cells. The structure and signal transduction through IL-4R and IL-13R appear to be different between hematopoietic and nonhematopoietic cells.

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24-amino-acid transmembrane domain, and a long 569-amino-acid intracellular domain. The 65-kDa protein is the IL-2 receptor cc chain, a component common to other cytokine receptors including IL-2, IL-7, IL-9, IL-15 and IL-21 (Noguchi et al., 1993). Binding of IL-4 to the IL-4Ra chain induces dimerization of the IL4Ra chain with the cc chain, which increases by two- to three-fold the affinity of IL-4 for gp140/IL-4Ra in lymphoid cells. This type, which consists of IL-4Ra and IL2Rcc chains, is called a classical, or type I IL-4R and is predominantly expressed in hematopoietic cells. Although the cc chain remains undetectable on human renal cell carcinoma cells, it still binds IL-4 efficiently (Obiri et al., 1993), suggesting participation of another subunit in the IL-4R complex. Binding experiments on various cell types have also demonstrated association of the gp140/IL-4Ra with one of the IL-13 receptor chains, IL-13Ra (Kruse et al., 1992; Zurawski et al., 1993; Aman et al., 1996). This represents another IL-4R termed the alternative, or type II IL-4R (Murata et al., 1998b). Shanafelt et al. (1998) created a mutant form of IL-4 that was substituted in the region of IL-4 interacting with the cc chain. The mutant, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete functional and binding selectivity for T cells, B cells and monocytes, but showed no activity on endothelial cells which express only the alternative form of IL-4R, suggesting its utility in the treatment of certain autoimmune diseases. Genetic variants within this signaling pathway might contribute to the risk of developing asthma. A number of polymorphisms have been described within the IL-4Ra gene. Several studies have suggested association and/or linkage between IL-4Ra gene variants and allergic disease, although not all studies have been positive. The Ile50 allele of the IL4Ra is associated with atopic asthma (Mitsuyasu et al., 1998). Functional evidence for the importance of these polymorphisms has been obtained with assays of signal transduction in vitro (Mitsuyasu et al., 1999). Low IgE levels appear to be associated with the Pro478 and Arg551 alleles (Kruse et al., 1999). The Arg551 allele is also associated with elevated IgE levels and atopic dermatitis in some populations (Hershey et al., 1997). Several other amino acid polymorphisms have been reported within this gene, although good functional evidence supporting their importance has not yet been obtained (Deichmann et al., 1997).

SIGNALING CASCADE OF IL-4 IL-4 binding induces dimerization of the IL-4Ra with the cc chain, as well as their cytoplasmic domains essential for IL-4-induced signaling (Harada et al., 1992; Russell et al., 1993). IL-4 induces tyrosine phosphorylation of multiple proteins, including IL-4Ra itself and species of 170, 130, 110–120, 100 and 92 kDa (Izuhara and Harada, 1993). The lack of consensus sequences established for catalytic activity in the cytoplasmic domain of gpl40 or that of the cc chain is consistent with signaling requiring recruitment of non-receptor tyrosine kinases. IL-4 activates members of the Janus tyrosine kinase (JAK) family that activates STAT6 and the p170/IRS-2 (insulin receptor substrate) (Welham et al., 1995). This molecule associates with the 85-kDa subunit of the phosphoinositol-3 kinase (PI-3 kinase) involved in cell proliferation. IL-4 stimulation also activates distinct PI3 kinase pathways inducing: (1) rapid association of c-fes proto-oncogene product (FES) with the src homology 2 (SH2) domain of PI3 kinase (Izuhara et al., 1996); (2) tyrosine phosphorylation of IRS-2; and (3) association of PI3 kinase with IRS-2, and FES or a FES-related protein. Further studies revealed the presence of both FES-dependent and -independent pathways. Overexpression of kinase-inactive FES blocks IL-4 activation of IRS-2 but not STAT6, and inhibits the recruitment of PI-3-kinase to the activated IL-4 receptor complex. This decreases the activation of p70 (S6k) kinase in response to IL-4, and correlates with a decrease in IL-4-induced proliferation (Jiang et al., 2001). Another important element of the IL-4 pathway consists in the activation of signal transducers and activators of transcription proteins, such as IL-4 STAT (later termed STAT6) (signal transducer and activator of transcription) (Hou et al., 1994) or STF-IL-4 (signal transducing factor) (Schindler et al., 1994). STAT6deficient mice demonstrate abrogation of IL-4induced increases in the cell surface expression of both MHC class II antigens and IL-4R, and lymphocytes from STAT6-deficient animals fail to proliferate in response to IL-4. STAT6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, STAT6-deficient T lymphocytes fail to differentiate into TH2 cells in response to either IL-4 or IL-13. These results demonstrate STAT6 is essential for mediating responses to IL-4 lymphocytes

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SIGNALING CASCADE OF IL - 4

(Kaplan et al., 1996). Co-transfection experiments with different chains of IL-4R and kinase-deficient JAK1 and JAK2 mutants in Chinese hamster ovary (CHO) cells show that JAK1 and JAK2 are required for optimal activation of STAT6 in the type II IL-4R transfectant, but only partially in the type I transfectant, suggesting that IL-13Ra chain is a novel functional component of the IL-4R system and that JAK1 and JAK2 mediate IL-4-induced optimal activation of STAT6 in non-hematopoietic cells (Murata et al., 1998c). Tissue specific signaling cascades exist with the type II IL-4R, consisting of type II IL-4 Ra/IL-13Ra, employing distinct signaling pathways from that of the type I, which consists of IL-4R, IL-4Ra and cc.

IL-4 signaling in B lymphocytes IL-4 augments phosphorylation of JAK1, JAK3 and TYK-2 in human normal B lymphocytes, in Epstein–Barr virus-immortalized B lymphocytes (Murata and Puri, 1997) and in malignant B lymphocytes (Tortolani et al., 1995). JAK3 is not expressed constitutively in plasmacytoid cells, which represent the final stage of B-cell differentiation (Tortolani et al., 1995). This suggests that down-regulation of JAK3 occurs during B-cell differentiation into plasma cells. IL-4 also induces phosphorylation of IRS-2 in the murine plasmacytoma cell line B9 and splenic B cells (Welham et al., 1995), requiring STAT6 for IL-4mediated maturation and cellular functions of B lymphocytes (Shimoda et al., 1996; Takeda et al., 1996). Indeed, IL-4-induced proliferation and CD23 or MHC class II expression is abolished in STAT6-deficient B lymphocytes. IL-4 induces serine phosphorylation of the STAT6 transactivation domain in B lymphocytes possibly by a novel-signaling pathway in addition to the IRS/PI 3-kinase pathway (Wick and Berton, 2000). IL-4 induces adhesion and locomotion in B cells accompanied by dramatic changes in B cell morphology including spreading, dendritic cell protrusions and producing microvilli-like structures. In B cells from STAT6-deficient mice, cell spreading and polarization are severely impaired and microvilli formation is reduced, suggesting that the STAT6 pathway is essential for this response (Davey et al., 2000). In splenic B cells from STAT6-deficient mice, IL-4 does not induce detectable levels of germline IgG1 or IgE transcripts. Germline transcript expression induced

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by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely abolished. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination (Linehan et al., 1998).

IL-4 signaling in T lymphocytes IL-4 induces tyrosine phosphorylation of JAK3 and to a lesser extent that of JAK1 in human and murine T lymphocytes (Witthuhn et al., 1994; Johnston et al., 1995), as well as that of IRS-2 in some murine splenic T cells (Welham et al., 1995; Izuhara et al., 1996). A domain between residues 353 and 431 of the IL-4Ra associates with p92 FES (Izuhara et al., 1994), which becomes phosphorylated and complexes with PI3 kinase (Izuhara et al., 1996). The noninvolvement of IRS-2 pathway in these T-cell lines indicates that p92 FES acts solely as an adapter molecule between PI3 kinase and the IL-4Ra. STAT6 proteins control the cytokine-induced proliferative response of activated T cells, by regulating the expression of cell cycle inhibitors, such as p27Kip1. Cyclin-cyclin-dependent kinase complexes may function to promote transition from the G1 to the S phase of the cell cycle (Kaplan et al., 1998). With regard to the direct roles of STAT6 in the TH1/TH2 differentiation, ectopic expression of STAT6 in TH1 cells induced TH2-specific cytokines and suppressed IFN-c production in the absence of IL-4. It also induced GATA-3 and c-Maf expression and down-regulated IL-12Rb2 chain expression, consistent with TH2 development (Kurata et al., 1999). STAT6-deficient mice are unable to mount efficient memory IL-4 responses, although STAT6-deficient CD4 cells produce high level IL-4 in response to primary stimulation (Finkelman et al., 2000). Although T cells from STAT6-deficient fail to differentiate into TH2 cells in response to IL-4 (Kaplan et al., 1996; Shimoda et al., 1996), the reduced level of IL-4 production in STAT6-deficient T cells is still significantly higher than that observed in TH1 controls. IL-4Ra-chain or STAT6deficient CD4 lymphocytes can develop a classical TH2 phenotype (IL-4, IL-5, IFN-c, IL-2) in vivo and in vitro, suggesting that IL-4R/STAT6 signaling, while influencing the final frequency of TH2 lymphocytes, is not essential for TH2 development (Jankovic

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et al., 2000). A cell surface affinity matrix technique revealed that IL-4-secreting STAT6-deficient T cells stably expressed GATA-3, as well as the TH2 phenotype. STAT6 is however necessary for complete in vitro TH2 differentiation. Introducing GATA-3 into STAT6deficient T cells completely restored TH2 development. The fact that GATA-3 fully reconstitutes TH2 development in STAT6-deficient T cells indicates it is the master switch in TH2 development. GATA-3 also exerts STAT6-independent autoactivation, creating a feedback pathway stabilizing TH2 commitment (Ouyang et al., 2000). GATA3 expression is dependent on the expression of the p50 subunit of nuclear factor B (NF-jB). CD4 T cells from p50/ mice failed to induce GATA-3 expression under TH2-differentiating conditions. Inhibition of NF-jB activity prevented GATA-3 expression and TH2 cytokine production in developing, but not committed, TH2 cells (Das et al., 2001). STAT6 is required for the in vitro differentiation of CD8 T cells into IL-4-secreting cytotoxic T cells type 2 cells. On the other hand, NK-T cells, that do not require IL-4 for their development, are still competent to secrete IL-4 in the absence of STAT6 (Kaplan et al., 1999).

IL-4 signaling in myeloid cells and dendritic cells (DCs) Tyrosine phosphorylation of JAK3 occurs in response to IL-4 in myeloid cells (Witthuhn et al., 1994). In the human erythroleukemia cell line TF-1, IL-4 stimulates increased tyrosine phosphorylation of JAK1 and TYK2, even if levels of phosphorylated JAK1 remain very low. Interestingly, IL-4 induces monocytes to release nitric oxide (NO) and this stimulation of NO synthase is likely to explain the accumulation of cyclic GMP (Kolb et al., 1994). In addition, the IL-4-induced inhibition of cytokine production by monocytes may be a consequence of the inhibition of c-fos and c-jun expression, whose complex constitutes the transcription factor AP-1 (Dokter et al., 1993a). With regard to the influence of other cytokines on STAT6 pathway in human monocytes, pretreatment with type 1 IFN or II interferon (IFN-b or IFN-c), but not IL-1, IL-2, GM-CSF, M-CSF, IL-6, or TGF-b suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6

(Dickensheets et al., 1999). Recent DNA array technology revealed up-regulation of NF-jB/Rel family genes during the differentiation of DCs from monocyte progenitors in comparison with macrophages derived from the same source using GM-CSF  IL-4 or M-CSF, respectively. NF-jB/Rel family gene expression, particularly c-rel was significantly up-regulated in maturing DCs compared to macrophages. NF-jB pathway genes are up-regulated in DCs when compared with macrophages and were constitutively expressed in monocytes, then selectively down-regulated during macrophage development, but not DC differentiation. The results illustrate the ability of the NF-jB pathway to respond to differentiation stimuli by activating, in a cell-specific manner, unique signaling pathways and subsets of NF-jB target genes (Baltathakis et al., 2001).

IL-4 signaling in other cell types In human fibrosarcoma cells, colonic carcinoma cell lines and vascular endothelial cells, IL-4 fails to phosphorylate JAK3 but induces that of JAK1, JAK2 and TYK-2 without expression of the cc chain (Murata et al., 1995). Tyrosine phosphorylation of IRS-2 induced by IL-4 in human colorectal carcinoma cell lines is associated with a growth inhibitory signal (Schnyder et al., 1996). Activation of IRS-2 may act both as a positive and a negative element in the control of cell proliferation. IL-4 activates the STAT6 pathway in human vascular endothelial cells (Palmer-Crocker et al., 1996) and colonic carcinoma cell lines (Murata et al., 1996). IL-4 itself is not necessary for the development of an IL-4-producing phenotype in mast cells. Bone marrow-derived mast cell precursors from STAT6/ mice can differentiate into mature cells that express IL-4 levels comparable to those of wildtype mast cells (Sherman et al., 1999). In fibroblast cell lines, mRNA for IL-13Ra, IL-13Ra and IL-4Rb chains were expressed. The IL-2R-c chain, which modulates IL-4 and IL-13 binding, is not expressed. JAK3 was present, but not phosphorylated, in any of the cell lines studied. Both IL-4 and IL-13 activate STAT6 and augment phosphorylation of IRS-1. IL-4 and IL-13 share similar transduction pathways in human fibroblasts and JAK2, rather than JAK3, plays the major role in IL-4/IL-13-induced signal transduction in human fibroblasts (Murata et al., 1998a).

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BIOLOGIC EFFECTS OF IL-4 ON B LYMPHOCYTES

Ontogeny of B lymphocytes IL-4 can inhibit the spontaneous proliferation of human B cells, as well as that induced by IL-7 (Pandrau et al., 1992; Ryan et al., 1994). This inhibition is also observed with the human progenitors of acute lymphoblastic leukemia, inducting apoptosis during the G0/G1 phase of the cell cycle (Manabe et al., 1994; Renard et al., 1994). IL-4 plays a role in the final maturation step during bone marrow B lymphopoiesis, by enhancing the output of surface IgM cells in murine

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cultures (Kinashi et al., 1988). Human bone marrow B-cell precursors differentiate into immunoglobulinsecreting cells when cultured with IL-4 in association with activated T cells (Punnonen et al., 1993).

B lymphocyte activation IL-4 increases the number of resting B lymphocytes in vitro (Valle et al., 1989), induces their homotypic aggregation (Elenstrom and Severinson, 1989), and induces hyperexpression of MHC class II antigens on murine B cells (Noelle et al., 1984). This effect is less pronounced on human resting B lymphocytes, which already express high levels of these surface molecules (Diu et al., 1990).

TABLE 10.1 Biologic properties of IL-4 ● ●

Human IL-4: Glycoprotein with molecular weight (MW) of 15–19 kDa encoded on chromosome 5 at q23.3–31.2 Mouse IL-4: MW; 19 kDa (EL-4 cells), 24 kDa (COS7 derived recombinant) encoded on chromosome 11

Cellular sources of IL-4 CD4 and CD8 T cells, CD3 NK1.1 (NK-T) cells, basophils and mast cells Effects on B cells Increases surface class II major histocompatibility complex antigens and co-stimulatory molecules ● Induces immunoglobulin isotype switching for IgG1 and IgE production ● Induces aggregation and microvilli formation ● Enhance Ig production and expression of CD23 on B cells ● CD40–CD40L dependent proliferation ●

Effects on T cells ● Stimulates the growth of both normal helper and cytotoxic T cells, including tumor infiltrating lymphocytes ● Promotes TH2 and TH1 cell differentiation and growth ● In concert with TGF-b, promotes the differentiation of naive CD4 T cells into TH1-type cells ● Roles in induction of TH1 type response and anti-tumor immune response shown in experiments using IL-4 gene knock-out mice Effects on NK cells ● Promotes the growth, however, suppresses IL-2-induced growth and cytotoxicity Effects on myeloid cells ● In combination with granulocyte–macrophage colony-stimulating factors (GM-CSF), promotes the differentiation bone marrow precursor cells to DC ● Up-regulates MHC class II expression and confers antigen-presenting capability ● Drive TH1 response by inducing apoptotic death of lymphoid DC and matures myeloid DC ● Enhance production of IL-12 p70 from CD40L-stimulated DCs Effects on other cells ● Induces histamine release from mast cells ● Blocks cytokine-induced proliferation of synoviocytes ● Up-regulates the expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelia, allowing enhanced recruitment of VLA-4 cells (memory T cells, eosinophils and DC) ● Inhibits haptoglobin, but enhances cytochrome P-450,2E1 expression from hepatocytes ● Suppresses the growth of IL-4R-expressing human cancer cells, including induction of apoptotic cell death ● Anti- and pro-angiogenesis THE CYTOKINES AND CHEMOKINES

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IL-4 strongly enhances the expression of CD23 (the low-affinity receptor for IgE-FceRII) on normal and leukemic B lymphocytes (Defrance et al., 1987a). Expression of CD23 mRNA on B cells isolated from atopic patients suggests a recent encounter with IL-4. Moreover, its association with MHC class II antigens (Bonnefoy et al., 1988b) appears to play an important role in the B-cell presentation of antigen to T lymphocytes (Flores-Romo et al., 1990). IFN-a and c block the IL-4-dependent increase of CD23 on B cells. IL-4 also induces the release of soluble CD23, which retains the capacity for IgE binding (Bonnefoy et al., 1988a). IL-4 up-regulates the expression of surface IgM (Shields et al., 1989), CD40 (Gordon et al., 1989), CD80 (Valle et al., 1991) and CD86. Mouse IL-4 induces Thy-1 on B cells (Snapper et al., 1988), but decreases that of CDw32-FccRII. IL-4 reverses the Fc receptormediated inhibition of B-lymphocyte activation (O’Garra et al., 1988). In addition, IL-4 induces activated B cells to produce IL-6 and TNF-a (Smeland et al., 1989). These effects of IL-4 support its role in enhancing antigen-presentation by B cells. Comparison of gene expression profiles of IL-4-stimulated B cells from STAT6/ versus STAT6/ mice led to identification of primary and secondary target genes of STAT6. Among 106 genes analyzed, 31 genes were expressed at higher levels in STAT6/ B cells, and 39 genes were expressed at higher abundance in STAT6/ B cells, implying both positive and negative regulatory functions of STAT6 in IL-4-mediated gene expression (Schroder et al., 2002).

B lymphocyte proliferation Antigen receptor triggering IL-4 enhances DNA replication of B cells costimulated or preactivated with insolubilized anti-IgM antibody (Defrance et al., 1987b; Clark et al., 1989). IL-4 does not costimulate with Staphylococcus aureus strain Cowan (SAC) (Jelinek and Lipsky, 1988), but enhances DNA replication of SAC-preactivated B lymphocytes (Defrance et al., 1987b). Addition of PGE2 and pharmacologic agents inducing intracellu-

lar cAMP (cholera toxin, dibutyryl cAMP, forskolin) enhances IL-4-induced DNA synthesis (Garrone and Banchereau, 1993). Paradoxically, IL-4 antagonizes the IL-2-induced DNA replication of B cells costimulated through their antigen receptor (Defrance et al., 1988). The inhibitory effect is particularly striking in nonHodgkin’s B-cell lymphomas (Defrance et al., 1988), but is less apparent on chronic lymphocytic leukemia B cells (Kaplan et al., 1996; Dickensheets et al., 1999; Daniel et al., 2000; Schaefer et al., 2001). This effect may be due to an IL-4-dependent sequestering of the cc chain, which is also a part of IL-2R (Lee et al., 1990). IL-4 also inhibits TNF-a-induced proliferation of phorbol ester activated B-CLL (B-chronic lymphocytic leukemia) cells (van Kooten et al., 1992). However, while blocking DNA synthesis, IL-4 protects B-CLL cells from death by apoptosis through increased expression of the Bcl-2 protein (Dancescu et al., 1992).

CD40-dependent activation Combinations of soluble anti-CD40 antibodies and either anti-IgM or phorbol esters induce DNA synthesis in B lymphocytes. Addition of IL-4 preferentially boosts proliferation (Gordon et al., 1988). Although neither of these conditions results in long-term B-cell proliferation, the addition of IL-4 to B lymphocytes cultured in the CD40 system (combining irradiated fibroblastic L cells transfected with human FccRII-CDw32 and anti-CD40 antibody) or CD40L-transfected L cells results in their sustained proliferation (Banchereau et al., 1991), generating factor-dependent long-term normal B cell lines. Moreover, agents increasing intracellular cAMP levels strongly enhance IL-4-induced cell proliferation (Garrone and Banchereau, 1993). IL-4 enhances B-CLL cells DNA synthesis in the CD40 system (Fluckiger et al., 1992b). This results in the expansion of viable leukemic cells, although the extent is lower than that obtained with normal B lymphocytes. Thus, the lack of growth promoting activity of IL-4 on B-CLL cells activated via surface immunoglobulin (sIg) may be due to altered signal transduction through sIg rather than to impaired IL-4R signaling.

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B-lymphocyte differentiation Antigen receptor triggering IL-4 induces IgG and IgM, but not IgE production, by SAC-preactivated B lymphocytes, most likely as a consequence of induced proliferation (Defrance et al., 1988; Jelinek and Lipsky, 1988). Moreover, IL-4 blocks IL-2-induced immunoglobulin secretion by SAC-costimulated B lymphocytes. Inhibitory effects of IL-4 on antigen-specific immunoglobulin production have also been observed. In particular, the secondary response of B lymphocytes to influenza virus, which requires both antigen and IL-2, can be inhibited by IL-4 (Fluckiger et al., 1992a). Likewise, the IL-2-dependent primary response to trinitrophenylated polyacrylamide beads is also inhibited by IL-4 (Llorente et al., 1989). IL-4 blocks IL-2dependent B-cell differentiation, whereas it stimulates the antigen-dependent B-cell proliferation (Llorente et al., 1990).

CD40-dependent activation and IL-4 and isotype switching Purified B lymphocytes cultured in the CD40 system produce low amounts of IgM, IgG and IgA. Addition of IL-4 increases the production of IgM and IgG and, more strikingly, the secretion of large amounts of IgE (Jabara et al., 1990; Rousset et al., 1991a). Thus, the IgE secretion dependent on T cells-induced IL-4 production requires CD40 triggering. Addition of IFN-c or IFN-a to anti-CD40-activated B cells surprisingly fails to inhibit IL-4-induced IgE production, thus contrasting with studies in which B cells were stimulated by T lymphocytes (Pene et al., 1988a, 1988b). A role for IL-4 in IgA switching has been reported for lymphoma B cells CH12F3 cultured in IL-4 alone (Nakamura et al., 1996). Many studies performed in both mouse and human have shown that IL-4-induced B lymphocytes produce IgE following isotype switching. Single sIgD B lymphocytes cultured for 10 days in the CD40 system with IL-4 yielded B-cell clones whose isolated cells expressed the same VDJ genes coupled to different constant region genes (Galibert et al., 1995). Isotype switching is associated with a DNA recombination

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event, in which CH (heavy chain) genes originally lying between Sl and the newly expressed CH gene are deleted by excision of a circular piece of DNA (Snapper et al., 1997) and is preceded by expression of germline Ce transcripts that initiate 5 of the switch region specific for the constant region to be expressed and which encompass the downstream Ce gene. IL-4 induces purified resting B lymphocytes to express 1.8-kilobase germline Ce transcripts (Gauchat et al., 1990). The IL-4-dependent expression of the germline Ce transcripts is enhanced by TNF-a, but decreased by TGF-b thus explaining its inhibitory effects on IgE synthesis (Gauchat et al., 1992). Interestingly, interferons and IL-6, which respectively block and stimulate IL-4 and T cell-dependent IgE synthesis, do not modify the levels of Ce germline transcripts. Molecular studies demonstrate that, in contrast to the mouse, the transcriptional activity of the human IgE germline induced by IL-4 is not mediated by the transcription factor B-cell activator protein (BSAP) (Albrecht et al., 1996), but requires an IFN-c-activated site in the IgE germline promoter (Ezernieks et al., 1996). Furthermore, protein tyrosine kinase, such as JAK3 (Fenghao et al., 1995) play an important role in both IL-4 and the CD40 signaling pathway that leads to IgE switching. Numerous studies in mice with parasitic infections or treated with anti-IgD have confirmed the fundamental role of IL-4 in the regulation of circulating IgE levels (Finkelman et al., 1990). Anti-IL-4 suppresses the eosinophilia, hyper-IgE and intestinal mastocytosis found in helminthic infections, but not the induction of IgG or protective immunity to the infection. Moreover, inactivation of the IL-4 gene in mice was associated with normal T and B cell development, but with substantial reductions in IgE levels after infection with the nematode Nippostrongylus brasiliensis (Kuhn et al., 1991) or with that of Brugia malayi (Lawrence et al., 1995). Infection of IL-4-deficient mice with a murine retrovirus, inducing an immunodeficiency syndrome, raised serum IgE levels comparable to those in control mice (Morawetz et al., 1996). This suggests that an IL-4-independent pathway for IgE switching occurs in mice following retroviral infection. Conversely, IL-4 transgenic mice had increased IgE levels and an allergic-like disease with ocular lesions infiltrated with mast cells and

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eosinophils (Tepper et al., 1990). Mice that have been induced to a hyper-IgE state display an increased production of IL-4 and a decreased production of IFN-c. Studies have been performed in humans attempting to correlate IL-4 status with IgE status. Some of these concluded that atopic patient blood mononuclear cells display an increased capacity to produce IL-4 and/or a decreased capacity to produce IFN-c in response to polyclonal activation (Rousset et al., 1991b). B cells derived from STAT6-deficient animals revealed that it is required for IL-4-induced germline Ig gene transcription and switch recombination (Linehan et al., 1998). B lymphocytes from a tyrosine kinase Lyn deficient (lyn/) mice require 10-fold less IL-4 to induce switching from IgM to IgG1 and IgE upon CD40 ligand stimulation. In vivo, lyn/ mice develop IgG1 Ab-forming cells prematurely and adult lyn/ mice have abnormally high IgG1 and IgE levels (Janas et al., 1999). A DNA binding protein Ku, is a heterodimer composed of p70 and p80 subunits, associates with CD40 in the cytoplasm of human B cells and translocates to the nucleus following incubation with IL-4 and anti-CD40 mAb (Morio et al., 1999). It likely plays a dynamic role in the signal transduction following IL-4 and CD-40 mediated stimulation.

BIOLOGIC EFFECTS OF IL-4 ON T-LYMPHOCYTES

CD1. Concomitantly, the CD4 CD8 subset disappears and CD4 CD8, CD4 CD8, CD4 CD8 cells are generated (Ueno et al., 1989). Paradoxically, IL-4 transgenic mice have involuted thymuses (Tepper et al., 1990) and IL-4 inhibits early T-cell development in fetal thymus organ culture, probably during the differentiation of CD4 CD8 cells into CD4 CD8 thymocytes. The generation of TCR-a/b thymocytes appears to be more impaired than that of TCR-cd thymocytes (Plum et al., 1990). Likewise human IL-4 induces a preferential differentiation of TCR-cd pre-T cells (Barcena et al., 1990). Murine CD8 T cells, activated by IL-4, develop into a CD8 CD4 population that is not cytolytic, lacks perforin and does not produce IFN-c. Thus CD8 T cells may also differentiate into Tc1 and Tc2 subsets (Seder et al., 1992). Furthermore, these cells produce large amounts of IL-4, IL-5 and IL-10, and can induce the growth and differentiation of activated B cells (Erard et al., 1994). In humans, IL-4-producing CD8 T cells have been observed in diseases such as leprosy (Salgame et al., 1991), acquired immune deficiency syndrome (AIDS) (Paganelli et al., 1995) and leishmaniasis (Uyemura et al., 1993). Cancer cells promote production of IL-4 and down-regulate the production of IFN-c in cervical cancer-infiltrating CD4 and CD8 T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-b plays only a synergistic role (Sheu et al., 2001). The emergence of such cells may contribute to the disease, possibly because of reduced overall cytolytic activity against microbe-infected cells and cancer cells.

Effects on thymocytes Both murine (Zlotnik et al., 1987) and human (Barcena et al., 1991) thymocytes proliferate in response to IL-4 and phorbol esters. In the mouse, IL-4 induces the proliferation of the most immature CD4 CD8 subset, which also has the ability to produce IL-4. The intermediate CD8 CD4 subset fails to proliferate under these conditions, whereas the most mature CD4 CD8 and CD4 CD8 subsets also proliferate in response to IL-4 and PMA. Fetal thymocytes also respond to IL-4, and in situ hybridization studies have demonstrated IL-4 transcripts in the fetal thymus (Sideras et al., 1988). In addition, IL-4 induces maturation of thymocytes through the induction of mature T-cell antigens (CD3, CD5, TCR) and loss of

Effects of IL-4 on mature T cells The T cell growth-promoting effects of IL-4 were initially discovered in continuous T cell lines (Mosmann et al., 1986), and subsequently confirmed in activated normal CD4 and CD8 T cells (Spits et al., 1987) in an IL-2-independent fashion. Studies with antisense oligonucleotides showed that IL-4 and IL-2 are autocrine growth factors of TH2 and TH1, T-cell clones respectively (Harel-Bellan et al., 1988). However, IL-4 blocks the specific IL-2-induced proliferation of peripheral blood naive CD4/CD45RA T cells (Gaya et al., 1990). IL-4 inhibits the production of IFN-c by activated T cells (Peleman et al., 1989), and thus favors the generation of T-helper (TH)2 cells,

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TABLE 10.2 Consequences of IL-4-knock-in and -out, IL-4R or STAT6 knockouts IL-4 TG ● IL-4 transgenic mice had increased IgE levels and an allergic-like disease with ocular lesions infiltrated with mast cells and eosinophils (Tepper et al., 1990) ● IL-4 transgenic mice display eye inflammation and conjunctivitis due to large numbers of eosinophils and mast cells (Tepper et al., 1990) IL-4 KO ● Failed to resist to the induction of EAE (Singh et al., 2001; Jahng et al., 2001) ● CD8 T cells from IL-4 tumor-immunized IL-4/ animals cannot mediate anti-tumor immunity (Rodolfo et al., 1999), nor TH1 anti-tumor immunity (Schüler et al., 1999) ● Resistant (Kopf et al., 1996) or susceptible (Noben-Trauth et al., 1996) to L. major ● Failed to mount protective TH1 immunity against Candida albicans (Mencacci et al., 1998) ● These mice have a normal T and B cell development, but IgG1 and IgE serum levels are reduced (Kuhn et al., 1991, 1996) ● A reduced cutaneous delayed-type hypersensitivity (Dieli et al., 1999) IL-4R KO ● Diminished but not absent TH2 responses in response to N. brasiliensis and IL-4 production by NK-T cells (Noben-Trauth et al., 1997) ● Failure to resist to N. brasiliensis like STAT6 / mice and in contrast to IL-4-deficient mice (Urban, J.F. et al., 1998) ● Lack of IFN-c response and variable resistance to L. major depending on the strains (Noben-Trauth et al., 1999) ● Lack of sensitivity to induction of asthma (Cohn et al., 1999) ● Progressive exacerbation of L. major LV39 infection (Mohrs et al., 1999) ● IL-4Ra on non-bone marrow-derived cells, but not on bone marrow cells are required for worm expulsion (Urban et al., 2001) STAT6 KO Lack of IL-4-mediated maturation and cellular functions of B lymphocytes (Shimoda et al., 1996) ● Abrogation of IL-4-induced increases in the cell surface expression of both MHC class II antigens and IL-4R, and lymphocytes from STAT6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6-deficient T lymphocytes fail to differentiate into TH2 cells in response to either response to either IL-4 or IL-13 (Kaplan et al., 1996) ● B cells do not proliferate in response to IL-4- or up-regulate CD23 or MHC class II expression (Shimoda et al., 1996) ● B cells have impaired microvilli formation in response to IL-4 (Davey et al., 2000) ● T lymphocytes that fail to completely differentiate into TH2 cells in response to IL-4 (Kaplan et al., 1996; Shimoda et al., 1996) ● Important role of STAT6 in vivo memory responses (Finkelman et al., 2000) ●

which produce predominantly IL-4, IL-5, IL-6 and IL-10, drive isotype-switching in the differentiation of B cells and also enhance CD8 T cell production of IL-4 (Tc2). IL-4 is not however solely a TH2 inducer. In the presence of IL-4 and TGF-b, both of which favor TH2 differentiation, a naive CD4 T cell can differentiate into TH1-type cells in vitro (Lingnau et al., 1998). The endogenous production of IL-4 in response to viral challenge is likely to play an important role in the generation of antigen-specific cytotoxic cells. T-cell clones cultured with IL-4 express a lipase, which may itself be involved in the cytolytic process. Such cells display higher cytolytic activity than those cultured in

IL-2 (Grusby et al., 1990). Administration of sIL4R to mice inhibits an allogenic response in vivo and enhances heart allograft survival, thus suggesting an in vivo role for IL-4 in the development of cytotoxic T cells (Fanslow et al., 1991).

Effects of IL-4 on NK cells CD3 NK cells proliferate in response to IL-4 (Spits et al., 1987). IL-4 inhibits the IL-2-dependent proliferation of these cells (Kawakami et al., 1989) at low doses of IL-4 but enhances their proliferation at higher doses. It accordingly blocks the IL-2-dependent

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generation of human lymphokine-activated killer (LAK) cells (Nagler et al., 1988). IL-4 also inhibits the production of TNF-a and serine esterase by NK cells (Blay et al., 1990). The blocking effect of IL-4 on IL-2induced cytotoxicity appears to be linked to its ability to raise cAMP levels in NK cells.

BIOLOGIC EFFECTS OF IL-4 ON MYELOMONOCYTIC CELLS Effects of IL-4 on hematopoiesis IL-4 can either inhibit or enhance myelopoiesis from bone marrow progenitor cells. It blocks the development of colonies dependent on M-CSF (Jansen et al., 1989),butstimulatestheformationofcoloniesdependent on G-CSF (Broxmeyer et al., 1988; Rennick et al., 1989). Furthermore, in combination with IL-3, IL-4 induces the generation of basophil/mast cells from human (Favre et al., 1990) and mouse (Rennick et al., 1987) progenitor cells and that of eosinophils (Favre et al., 1990) from human progenitor cells. IL-4 also acts on the generation of eosinophils in mice. Administration of plasmacytoma-expressing IL-4 results in eosinophil infiltration of tumors (Tepper et al., 1989, 1992). Furthermore, IL-4 transgenic mice display eye inflammation and conjunctivitis due to large numbers of eosinophils and mast cells (Tepper et al., 1990). IL-4 is an essential growth factor for the in vitro growth of connective tissue-type mast cells (Tsuji et al., 1990). Combinations of erythropoietin and IL-4 induce partially purified progenitor cells to generate erythroid colonies (Broxmeyer et al., 1988), but this may be due to an indirect effect of IL-4 on accessory cells. In contrast, IL-4 inhibits the formation of pure and mixed megakaryocyte colonies from enriched human hematopoietic progenitors (Sonoda et al., 1993).

Effects on monocytes–macrophages Activation and differentiation IL-4 up-regulates the expression of MHC class II antigens, LFA-1, CD13 and CD23 (Vercelli et al., 1988), but down-regulates CD14 (Lauener et al., 1990) and the

three Fcc receptors (te Velde et al., 1990). Accordingly, it blocks the antibody-dependent cytotoxicity of macrophages without affecting their phagocytic properties. Monocytes cultured in the presence of IL-4 acquire a macrophage-like dendritic cell (DC) morphology, as they increase in size and develop extensive processes (te Velde et al., 1988). Of interest, addition of both GM-CSF and IL-4 to blood monocyte cultures induces their differentiation into immature DCs that can efficiently present soluble antigen to specific T-cell clones (Romani et al., 1994; Sallusto and Lanzavecchia, 1994). Subsequent addition of TNF-a induces differentiation into mature CD83 dendritic cells that display increased antigen-presenting cell functions (Zhou and Tedder, 1995, 1996). Moreover, IL-4 appears to deliver a critical signal for maturation of monocytes into DCs, as DCs generated from monocytes in the presence of GM-CSF and TNF-a show only low accessory cell capacity (Pickl et al., 1996). Renal cell cancer or fibroblast-derived IL-6 and M-CSF interferes with generation of DCs from monocytes. IL-4 can reverse such suppression in a dose-dependent manner (5–500 IU/ml) (MenetrierCaux et al., 2001).

Production of mediators IL-4 blocks the spontaneous and lipopolysaccharide (LPS)-induced production of IL-1, IL-6, IL-8, IL-10, IL-12 and TNF-a by monocytes (Hart et al., 1989; Standiford et al., 1990). IL-4 also inhibits virusdependent production of IFN-a/b by monocytes (Gobl and Alm, 1992). Distinct subsets of DCs activate in turn distinct subsets of T cells (Kalinski et al., 1999). Rissoan et al. (1999) proposed that DC1 (myeloid DCs) and DC2 (lymphoid DCs) subpopulations promote TH1 and TH2 immunity, respectively. This model was supported by the observation that IL-12, an essential cytokine for TH1 responses, was released by DC1 but not by DC2 after stimulation with CD40 ligand (CD40L). The presence of IL-4 is critical in driving a TH1 response and IL-12 secretion from DCs upon CD40–CD40L stimulation, at least partially by inducing apoptotic death of DC2 and maturing DC1 (Rissoan et al., 1999). However, recent studies have revealed more flexibility and clarification of the model (Cella et al., 2000). Production of

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IL-12 appears to be exhausted in both subsets of DCs at certain time points after in vitro stimulation (Cella et al., 2000; Langenkamp et al., 2000). DC1 started producing IL-12 at 10 h after lipopolysaccharide (LPS) stimulation and stopped by 24 h. Delivery of a second stimulus failed to induce IL-12. TH2 cells interacting with monocyte-derived DC effectively induce bioactive IL-12p70 and revert to TH0/TH1 phenotype in an IL-4-dependent manner. While IL-4 suppresses lipopolysaccharide (LPS)-induced IL-12 production by human peripheral blood monocytes and in vitro monocyte-derived macrophages (Bonder et al., 1999), IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-c at low concentrations of both cytokines (Kalinski et al., 2000). Mouse IL-4 also induced high levels IL-12 p70 from bone marrow-derived DCs (Hockrein et al., 2000), and in vivo IL-4 administration at the priming phase of adaptive immune response induces TH1 response and induces resistance to Leishmania major (Biedermann et al., 2001). Recently, in the mouse a common progenitor for so-called myeloid and lyphoid DC has been identified (del Hoyo et al., 2002).

Fibroblasts are chemoattracted by IL-4 (Postlethwaite and Seyer, 1991). IL-4 also induces dermal fibroblasts to secrete extracellular matrix proteins, such as type I and type III collagen and fibronectin (Fertin et al., 1991). IL-4 also (Postlethwaite et al., 1992) stimulates fibroblast T cell lines to produce G-GSF and M-CSF (Broxmeyer et al., 1988). On TNF- or IL-1-stimulated fibroblasts, IL-4 increases synthesis of the complement protein C3, but decreases that of factor B (Katz and Strunk, 1990). IL-4 blocks cytokine-induced proliferation of synoviocytes (Dechanet et al., 1993). Rapid and persistent blocking of the G1 phase, accompanied by increased cell volume, is noted following IL-4 treatment. IL-4, in fact, inhibits IL-1 and platelet-derived growth factor-induced synoviocyte proliferation. IL-4 enhances synoviocyte IL-6 production and inhibits spontaneous or cytokine-induced leukocyte inhibitory factor and PGE2 production (Dechanet et al., 1994).

Effects on granulocytes

IL-4 and endothelial cells

Normal eosinophils are responsive to IL-4, as they down-regulate FccR expression upon IL-4 treatment, correlating with a decrease in secretion of glucuronidase and arylsulphatase in response to IgGcoated beads (Baskar et al., 1990). IL-4 also acts on neutrophils to enhance their respiratory burst and phagocytic properties (Boey et al., 1989). IL-4 inhibits the secretion of IL-8 (Wertheim et al., 1993) but enhances the expression and secretion of membrane and soluble type II ‘decoy’ IL-1R by neutrophils (Colotta et al., 1993), thereby blocking IL-1 effects. IL-4 also promotes proliferation of human mast cells and induces FceRI expression on their surface. Treatment of mast cells with IL-4 enhances the release of histamine after FceRI cross-linking (Toru et al., 1996). IL-4 also induces leukocyte functional antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) expression, resulting in homotypic aggregation of mast cells (Toru et al., 1997). IL-4 promotes maturation of mast cells, enhancing expression of both tryptase and chymase (Toru et al., 1998).

Treatment of endothelial cells with IL-4 increases their adhesiveness for T cells, eosinophils and basophils but not neutrophils, owing to increased vascular cell adhesion molecule-1 (VCAM-1) expression (Thornhill et al., 1991; Schleimer et al., 1992). IL-4 pretreatment of vascular constructs, composed of endothelial cells cultivated on extracellular matrix from human fibroblasts, induces adherence and layer penetration of eosinophils, but not of neutrophils. For layer penetration, blood eosinophils from nonallergic donors need priming with GM-CSF, IL-3 or IL-5, whereas those from allergic donors transmigrate spontaneously (Moser et al., 1992). Cytokine-induced up-regulation of ICAM-1 and endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells is inhibited by IL-4. This contrasts with the IL-4-induced increase of ICAM-1 expression observed on dermal fibroblasts, macrophages and mast cells (Thornhill et al., 1991; Valent et al., 1991; Piela-Smith et al., 1992). IL-4 synergizes with TNF or IFN-c to modify endothelial cell morphology

BIOLOGIC EFFECTS OF IL-4 ON OTHER CELL TYPES IL-4 and fibroblasts

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(Thornhill and Haskard, 1990), coinciding with reorganization of the intracellular vimentin matrix from a diffuse to a perinuclear pattern (Klein et al., 1993). Synergy between IL-4 and TNF-a, enhancing VCAM-1 expression on endothelial cells is observed (Iademarco et al., 1995). IL-4 increases IL-6 production by endothelial cells in synergy with IL-1 or IFN-c (Howells et al., 1991; Colotta et al., 1992), as well as IL-8 production by LPS-stimulated endothelial cells (De Beaux et al., 1995). In contrast, IL-4 decreases secretion of the chemokine RANTES (regulated upon activation, normal T expressed and secreted) induced by TNF-a and IFN-c (Marfaing-Koka et al., 1995). Finally, IL-4 counteracts the effect of LPS, IL-6 and TNF-a on the enhanced expression of procoagulant activity on endothelial cells and down-regulates the thrombomodulin anticoagulation pathway (Kapiotis et al., 1991).

IL-4 and epithelial cells On human thymic epithelial cells, IL-4 increases IL-1induced IL-6 production and inhibits that induced by GM-CSF (Galy and Spits, 1991). On tubular epithelial cells, IL-4 up-regulates protein expression, as well as enzymatic activity of both CD13 and CD26 peptidases (Riemann et al., 1995). Conversely, it inhibits inducible NO synthase expression in lung epithelial cells (Berkman, 1996), inhibits RANTES secretion on airway epithelial cells (Berkman et al., 1996), and decreases chloride secretion by intestinal epithelial cells (Zund et al., 1996).

IL-4 and hepatocytes IL-4 decreases the spontaneous production of haptoglobin in human hepatocytes in primary cultures and to a lesser extent that of albumin and C-reactive protein, while not affecting al-antitrypsin and fibrinogen production (Loyer et al., 1993). Furthermore, IL-4 antagonizes IL-6-enhanced secretion of haptoglobin, thereby demonstrating another antiinflammatory activity. In addition, IL-4 enhances the expression of cytochrome P-450, 2E1 in a specific manner, as levels of cytochromes P-450, 1A2, 2C and 3A are not affected or weakly inhibited (AbdelRazzak et al., 1993). Treatment of human hepatocytes in primary cultures by IL-4 up-regulates expression

of glutathione-S-transferases, enzymes involved in cellular defense against lipid peroxidation (Langouet et al., 1995).

IL-4 and cancer cells Human IL-4 has a direct growth-inhibitory effect on the in vitro growth of human colon (HT 29) carcinoma (Toi et al., 1992), human renal cell carcinoma, malignant melanoma (Obiri et al., 1993) and breast cancer cells (Topp et al., 1993, 1995a, 1995b; Gooch et al., 1998). These cells express the high affinity receptor for IL-4. In five of seven colorectal cancer cell lines, a dose-dependent reduction of proliferation was observed and those cells expressed functional IL-4 receptors (IL-4R) (Lahm et al., 1994). The growth inhibition mediated by IL-4 involves suppression of insulin-like growth factor II (Lahm et al., 1996). Similar observations were found in human astrocytes and astrocytic tumors (Barna et al., 1995). Growth inhibition of glioma cells by IL-4 involves up-regulation of p27Kip1, a cyclin dependent kinase inhibitor (Liu et al., 1997). Interestingly, growth suppression of breast cancer cells involves IL-4-induced apoptotic cell death (Gooch et al., 1998). Taken together, the anti-tumor effect of IL-4 on human cancer cells appears to be mediated, at least, in part by the direct inhibition of tumor growth via IL-4R-mediated signaling. However, IL-4 may also promote the growth of some types of human cancer cells, such as squamous cell carcinoma of the head and neck (SCCHN) cell lines (Myers et al., 1996). Anti-angiogenesis is an important strategy to control tumor growth in vivo. The inhibited growth of C6 rat glioma cells engineered to express mIL-4 in athymic mice is accompanied by markedly reduced levels of vascularization and down-regulated expression of vascular endothelial growth factor-receptor 2 (VEGF-R2), endothelial cells in vitro (Saleh et al., 1997). IL-4 also blocks the basic fibroblast growth factor-mediated neovascularization in rat cornea (Volpert et al., 1998), demonstrating unusual dose–response curves that were sharply stimulatory at a concentration of 0.01 ng ml1, but inhibitory over a wide range of higher concentrations. IL-13 inhibits the migration of human or bovine microvascular cells, as well as IL-4. However, using collagen gels with bovine aortic endothelial cells, as well as by human

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microvascular endothelial cells, human recombinant IL-4 at 50–500 U/ml stimulated by about two- to three-fold the formation of tubes by bovine aortic endothelial cells, and the effect was almost completely inhibited by the addition of IL-4 receptor neutralizing antibody (Fukushi et al., 1998). These contrasting observations suggest that further studies to determine the mechanisms and effects of IL-4 on neo-vascularization are warranted.

EXPERIMENTAL ANIMAL MODELS Systemic delivery of IL-4 protein, as well as local or systemic IL-4 gene delivery, have allowed investigations of the in vivo therapeutic effect of IL-4 in disease models. Conversely, soluble IL-4R has emerged as an attractive modality for treatment of allergic diseases. Disruption of IL-4 genes in vivo has also allowed investigators to analyze the impact of IL-4 in various disease processes.

Animal models for allergy, asthma and autoimmune diseases Treatment of experimental allergic encephalomyelitis by IL-4-expressing encephalitogenic T cells (Shaw et al., 1997) which allow local delivery of IL-4, limited the time course of the disease. Central nervous system (CNS) delivery of the Herpes simplex viral vectors encoding IL-4 vector, after EAE onset, induced in situ production of IL-4 by CNS cells residing in contact with the cerebrospinal fluid (CSF) and markedly reduced disease-related deaths (Furlan et al., 2001). Interestingly, protection of susceptible mice from EAE by activation of V(alpha)14 NK-T cells with a-GalCer was CD1d- and IL-4-dependent. CD1 or IL-4 knockout (KO) mice failed to resist the disease after a-GalCer administration (Singh et al., 2001; Jahng et al., 2001). Similarly, rheumatoid arthritis induced by injection of collagen in mice can be efficiently counteracted by intra-muscular injection of adenoassociated viral vectors encoding IL-4 (Cottard et al., 2000), or by i.v. injection of immature DC transfected with an adenoviral vectors expressing IL-4 (Kim et al., 2001). In addition, the protective effects of IL-4 have also been reported in nonobese diabetic mice

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(Rapoport et al., 1993). Splenocytes from nonobese diabetic mice overexpressing murine IL-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Immunoregulatory CD62L() cells following mIL-4 transduction inhibit disease transfer (Yamamoto et al., 2001). Constitutive expression of an IL-4 transgene by B cells completely prevents the spontaneous development of lethal lupus-like glomerulonephritis in the (NZW  C57BL/6) F1 murine model of systemic lupus erythematosus. IL-4 acts by down-regulating the appearance of TH1-mediated IgG3 and IgG2 autoantibodies, known to be especially nephritogenic (Santiago et al., 1997). Conversely, IL-4 promotes the development of lupus nephritis in NZB/W F1 mice by enhancing the production of IgG anti-doublestranded DNA antibodies. Administration of antibodies against IL-4 prevents the onset of lupus nephritis and decreases the production of autoantibodies (Nakajima et al., 1997). In mice from a different strain, BXSB, homozygous for IL-4 gene deletion have significantly lower serum levels of total IgG1 compared with wildtype BXSB, consistent with the lack of IL-4. However, no significant differences are observed in mortality, spleen weight, severity of glomerulonephritis, levels of anti-chromatin and anti-ssDNA Abs, or frequency of activated (CD44high) CD4 T cells when compared with controls. This suggests that the pathogenesis of systemic lupus erythematosus in this strain is independent of IL-4 production (Kono et al., 2000). In an asthma model induced by soluble egg antigens (SEA), antigen injection induces peak eosinophil extravasation into the airway at 48 h. Mice treated with anti-IL-4 antibodies demonstrated a 10-fold decrease in eosinophil influx and a reduction in total pulmonary leukocyte cellularity (Lukacs et al., 1994). Excess mucus production correlates with airway eosinophilia. Although IL-4/ TH2 cells can induce mucous at comparable levels in the absence of IL-4 production, IL-4/ TH2 cells do not induce mucous production in IL-4Ra / mice, and bronchial alveolar lavage (BAL) eosinophils are absent. In the absence of IL-4, IL-13 may indeed replace IL-4 for TH2induced mucous production and eosinophilia, signaling through IL-4Ra is critically important in TH2 cell stimulation of mucus production (Cohn et al., 1999). Recombinant soluble IL-4R (sIL-4R) was designed to

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neutralize secreted IL-4 molecules based on the molecular characterization of the soluble IL-4R (Renz, 1999). In a murine asthma model sensitized to OVA, challenge of animals with intranasal (i.n.) administration of allergen elicits pulmonary eosinophilic infiltration, with widespread mucus occlusion of the airways, resulting in bronchial hyperreactivity. Administration of exogenous sIL-4R is effective in blocking the late phase pulmonary inflammation (Henderson et al., 2000). Data from a study conducted in adult asthma patients suggest that IL-4R administration is safe and effective in the treatment of moderate persistent asthma (Borish et al., 2001). In a diabetic mouse model, in which mice carry an insulin promoter-driven transgene encoding a viral CD8 CTL epitope, introduction of another transgene encoding IL-4, also under the control of insulin promoter, inhibits the onset of diabetes. DCs treated with IL-4 have decreased CD80 expression and increased CD86. Treatment of DC with IL-4 inhibits the acquisition of cytolytic capacity by CD8 T cells, suggesting the role of these co-stimulatory molecules in the regulation of the CD8 T cell response. Injection of transgenic animals with DCs treated with IL-4 and pulsed with the epitope-peptide protects the animals from the onset of the disease associated with a decline in CD8 T cell numbers and antigen-specific CTL activity. This suggests that IL-4-stimulated DCs elicit qualitative differences in T-cell responses (King et al., 2001). In the liver, IL-4 secreted by CD4 T cells is a critical mediator of CD4/CD8 cross-talk enabling the full development of antigen-specific CD8 T cell responses to malaria (Carvalho et al., 2002).

Experimental infectious disease models Individual TH1 or TH2 subsets critically influence the outcome of several types of infection in mice. However, like autoimmune diseases, IL-4 can elicit both beneficial and detrimental effects depending on the nature of the infectious agent. During Borrelia burgdorferi infection, CD4 T cell-derived IL-4 plays a critical role in control of spirochete growth. Administration of IL-4 to mice during the early phase of B. burgdorferi infection significantly reduces joint swelling and spirochete numbers (Keane-Myers et al., 1996). This increased resistance is also associated

with a decrease in serum levels of specific IgG2a and IgG3 antibodies and an increase in specific IgG1 antibodies. IL-4 exerts protective effects against development of Toxoplasma gondii-induced encephalitis by preventing formation of cysts and foci of acute inflammation, and diminishing proliferation of tachyzoites in the brain (Suzuki et al., 1996). Control of parasitemia and survival during Trypanosoma brucei infection is also related to the ability of the mice to produce IL-4 (Bakhiet et al., 1996). The role of IL-4 in Leishmania major infection in mice were less clear, as both disease-promoting and protective effects had been reported (Muller et al., 1991). Disruption of the IL-4 gene in L. major-infected Balb/c mice has also given contradictory results reporting either enchanced susceptibility or resistance towards L. major infection (Kopf et al., 1996; Noben-Trauth et al., 1996). The mechanisms by which IL-4 influences TH1 responses has been poorly understood. IL-4 can induce IL-12 production by DCs (Kalinski et al., 2000; Hockrein et al., 2000) and in L. major infected Balb/c mice, IL-4 promotes IL-12 production by activated APC (Biedermann et al., 2001). This precedes T cell stimulation, and induces maturation of IFN-cproducing TH1 cells promoting resistance to L. major in susceptible Balb/c mice. During the later period of T cells priming, IL-4 induced TH2 differentiation and promoted progressive leishmaniasis in resistant mice. These contrasting effects of IL-4 on DC development and T cells differentiation led to immune responses with opposing functional consequences (Biedermann et al., 2001). IL-4 induces a protective TH1 response in IL-4 / animals infected with virulent Candida albicans. In the early stage of systemic infection with unopposed interferon IFN-c production, IL-4deficient mice were more resistant than wild-type mice to infection. Yet, IL-4-deficient mice failed to efficiently control infection in the late stages of disease and ultimately succumbed to it. Defective IFN-c and IL-12 production, but not IL-12 responsiveness, was observed in IL-4-deficient mice that failed to mount protective TH1-mediated acquired immunity in response to live pathogens (Mencacci et al., 1998). IL-4 can also exert in vivo inflammatory and pathogenic effects (Rankin et al., 1996). Epithelial cell hypertrophy, as well as accumulation of lymphocytes, eosinophils and neutrophils represent important fea-

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tures of the inflammatory responses induced by IL-4 overexpression in lung. Since 1997, studies with IL-4R-deficient mice have revealed the roles of IL-4R in host-immune response against infections. Two groups independently generated IL-4Ra-deficient Balb/c mice (Noben-Trauth et al., 1997; Mohrs et al., 1999). The IL-4Ra-deficient mice showed no overt phenotypic abnormalities and had normal lymphocyte cell numbers and a normal cell type composition in the thymus and spleen. Cells obtained from these mutant mice were impaired in IL-4- and IL-13-mediated functions, formally proving that the IL-4Ra chain is a crucial component of both the IL-4 and the IL-13 receptors. IL-4Ra-deficient mice have reduced but significant production of IL-4, indicating IL-4 producing cells can arise in the absence of IL-4R signaling (Noben-Trauth et al., 1997). While IL-4-deficient mice were able to expel Nippostrongylus brasiliensis like wild-type animals, expulsion was markedly suppressed in IL-4R- and STAT6-deficient animals (Urban et al., 1998). After infection with L. major, IL-4Ea / mice developed progressive disease with massive footpad swelling as late as 12 weeks post-infection. This was not observed in IL-4 / mice (Mohrs et al., 1999). The mechanisms controlling parasitic infections are most likely mediated by IL-13 signaling via the IL-4Ra chain and STAT6. In concordance with this hypothesis, administration of a soluble IL-13R inhibited N. brasiliensis expulsion in wild-type and IL-4-deficient Balb/c mice (Urban et al., 1998). IL-4Ra needs to be expressed on non-bone marrow-derived cells, but not on bone marrow to enable worm expulsion (Urban et al., 2001)

Experimental model of neoplasia Recombinant IL-4 in cancer models The availability of recombinant IL-4 led investigators to test its anti-tumor effect in vivo. The treatment of pulmonary metastases was assessed in a murine RENCA renal adenocarcinoma model (Hillman et al., 1995). Dose-dependent reduction of the number of metastasis and augmented survival was observed. Both CD8 T cells and asialoGM1 cells were necessary for rejection of tumors. When IL-4 was administered intralesionally to animals bearing RENCA tumors implanted below the renal capsule (6 lg

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animal1, single injection), marked inhibition of the primary tumor growth was noted, but little effect on the progression of distant metastatic sites was observed (Younes et al., 1995). Daily local subcutaneous injection of rIL-4 at the site of tumor-draining lymph nodes (0.00001 to 1000 pg day1) inhibited the growth of a chemically induced fibrosarcoma and a spontaneous adenocarcinoma (TS/A) and led to induction of immunologic memory specific for the tumor (Bosco et al., 1990). Delivery of 0.1 pg day1 of IL-4 led to the best survival among the dosages tested, suggesting a dichotomous response to IL-4. Interestingly, IL-4 inhibited tumor growth far better than the direct injection of the most effective doses of IL-1, IL-2 or IFN-c. Tumor-draining lymph nodes from animals treated in these studies displayed numerous macrophages, activated lymphocytes and eosinophils. Co-administration of IL-1 and IL-4 was noted to be much better at eliciting tumor rejection and a state of long-lived memory, superior to that observed with other cytokines used singly or in combination. Conversely, other reports demonstrated compromised tumor-rejection responses following systemic administration of IL-4. Systemic administration of high-dose rIL-4 (10 lg mice1) in mice harboring syngeneic p815 tumor lines transfected with B7.1 results in a compromised rejection response, despite the fact that the tumor line is nominally rejected in a T celldependent manner in non-treated animals (Terres and Coffman, 1998). IL-4 administration from days 7 to 22, every 3 days, induces remarkable splenomegaly characterized by a marked increase in neutrophils and NK activity. Suppressive effects of systemic IL-4 administration in anti-tumor immunity have been also demonstrated in a pulmonary metastasis model (Kobayashi et al., 1998). Higher numbers of pulmonary metastasis in the murine melanoma cell line B16F10 was associated with enhanced production of IL-4 and IL-10-secreting CD4 T cells in the spleen. When animals were treated with anti-IL4 antibody, the number of metastasis decreased. Animals with B16F1 cells had an increased number of metastasis following treatment with 10 lg kg1 of recombinant IL-4. This study suggested that IL-4 secreted from tumor-associated TH2 cells enhanced the metastatic ability of tumors in vivo, perhaps by activating endothelium. The impact of systemic IL-4 administration in tumor-bearing animals may differ depending on

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the cell line, animal strains, etc., as well as timing, dosage and the duration of administration. The complex immunobiology of IL-4 also mimics that of other cytokines, in which such issues as timing and location are important to the function of cytokines.

IL-4 gene therapy for neoplasia As is true for other cytokines, intrinsic potency and toxicity have complicated application of IL-4 as therapeutic agents when applied systemically. Many cytokines mediate a variety of immunologic effects (both antagonistic and synergistic) when encountered by immune cells at various stages subsequent to hematopoiesis and lineage commitment or which have been activated during the process of an ongoing immune response. Indeed, one of the major characteristics of most cytokines is that they regulate immunity at a local or regional level. Systemic levels provided by most conventional schema fail to mimic the induction of an effective immune response, particularly in the context of an established tumor. Tumor rejection, initiated with IL-4 transfection of murine tumors, is at least partially mediated by eosinophils and macrophage infiltration (Tepper et al., 1989). Transfecting the IL-4 dependent T cells line with IL-4 leads to autocrine growth in vitro (Li et al., 1990; Blankenstein et al., 1990), but these cells cannot grow in vivo even in nude mice presumably due to the local biologic activity of IL-4. Introducing the IL-4 gene into a murine renal cell tumor (Renca) or a colorectal carcinoma (CT-26), causes rejection and systemic immunity mediated predominantly by CD8 T cells. Therapeutic effects are observed even when administered as late as 9 days after establishment of the primary tumor. Macrophage and eosinophils infiltrate the tumors early, but T cells enter the tumor site especially during the second week (Golumbek et al., 1991). Locally produced IL-4 at the murine tumor site functions synergistically with systemic administration of IL-2 (Pippin et al., 1994). The individual roles of anti-tumor humoral and cellular responses have been addressed using IL-4-transfected TS/A tumor cells (Pericle et al., 1994). Although morphologic observation suggests that the rejection of IL-4-transfected TS/A tumor depends on eosinophil cytolysis, lymphocyte depletion experiments shows that tumor rejection also required CD8 lymphocytes. The memory

response induced on rejection appears to be mediated partially by a TH2-type humoral response, as well as antibody-dependenT cellsular cytotoxicity (ADCC) and other cellular effector mechanisms. Comparison of these mechanisms mediating memory with those elicited by IL-2 gene-transduced TS/A cells shows that TH2 memory is more efficient in protecting against a subsequent challenge with TS/A-pc than the TH1-type memory elicited by IL-2 gene-transduced TS/A cells. IL-4-transduced cancer cells also increases the influx of dendritic cells to the tumor site relative to other cytokines (Stoppacciaro et al., 1997). This suggests that local IL-4 expression at the vaccine site may enhance leukocyte recruitment including DCs and thereby enhance tumor-antigen uptake. The roles of eosinophils has been investigated using IL-5transfected TS/A (TS/A-IL5), in comparison with TS/A-IL-4. TSA-IL5 cells, despite the large eosinophil infiltrate, grow progressively in a manner similar to control tumors. This suggests that eosinophils per se do not play a crucial role in TSA tumor rejection. Rejection of TSA-IL4 appears to rely on expression of VCAM-1 and MCP-1 by endothelial cells, thereby recruiting basophils, mast cells, and macrophages/ DCs, and neutrophils (Di Carlo et al., 1998). IL-4 transfected C26/FRa tumor induces TH2 and Tc2 cells. Tc2 lymphocytes from immunized animals release IL-4 upon stimulation with tumor cells, and these cells confer rejection of lung metastasis. Interestingly, CD8 T cells from IL-4-vaccinated IFN-c knockout, but not from IL-4 knockout mice are able to cure lung metastases, thus indicating that IL-4 produced by Tc2 cells was critical for tumor rejection. The antitumor effect of adoptively transferred Tc2 lymphocytes required host CD8 T cells and AsGM1 leukocyte populations, and partially granulocytes (Rodolfo et al., 1999). IL-4 knockout mice are severely impaired in the development of tumor immunity to tumor lines that are rejected in wild-type animals. The lack of antitumor immunity in IL-4 knockout mice is associated with reduced IFN-c production, diminished levels of tumor-reactive serum IgG2a, and undetectable CTL activity, indicating a defective TH1 response in the absence of endogenous IL-4. When IL-4 is provided by gene-modified cells together with immunizing tumor cells, tumor immunity can be induced in IL-4-deficient mice, indicating that tumor immunity requires IL-4 in the priming phase for the generation of effector cells

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(Schüler et al., 1999). Anti-IL-4 antibody treatment in wild-type animals bearing the tumors indicates that IL-4 is critical in the induction phase, but not in the memory phase, suggesting the importance of IL-4 in activation of DCs. IL-4 expressed by CD8 T cells appears to be important in activation of DCs and thus CTL generation, as adoptive transfer of CD8 T cells from IL-4 (/) mice are not capable of inducing CTL response in the host (Schuler et al., 2001). The central nervous system (CNS) has been regarded as an at least partially immune privileged site. Although cytokine gene therapy for CNS tumors is an attractive approach, inducing considerable inflammation following direct transgene-derived cytokine gene expression in the CNS may cause lifethreatening brain edema or elevation of intracranial pressure. Peripheral immunization of CNS tumorbearing rats with syngeneic, IL-4-expressing 9L glioma cells are more effective than peripheral vaccination with 9L transfected with other cytokines, including IL-12, IFN-a and GM-CSF, or direct intracranial implantation of cytokine expressing glioma cells (Okada et al., 1999, 2001b). Anti-glioma immunity by peripheral vaccination requires both CD4 and CD8 cells, but not humoral responses (Giezeman-Smits et al., 2000). However, vaccinations with IL-4transfected glioma induces elevation of IgG1 antibodies in the sera of host animals. Enhanced humoral immune responses may lead to isolation of tumorassociated antigens, in turn providing insights into T cells recognized targets. Combining the IL-4 tumor vaccine with serological identification of antigens produced by recombinant cDNA expression libraries (SEREX) (Okada et al., 2001a) may be a promising approach. IL-4 transfected neuronal progenitor cells have been used as a vehicle to deliver IL-4 to the brain tumor sites (Benedetti et al., 2000). Since neural progenitor cells have the unique property of migrating toward brain tumors, this strategy may hold promise as an effective cytokine delivery system for invasive intracranial gliomas.

Clinical studies Clinical trials of recombinant IL-4 Recombinant human IL-4 (rhuIL-4) has been tested in various types of human tumors. The possible

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mechanisms of the anti-tumor effects of IL-4 include induction of immune responses, anti-angiogenenic effects, and induction of apoptotic death and/or direct growth inhibition of tumor cells. Phase I clinical trials with rhuIL-4 as a single antitumor agent began in 1988. In general, rhuIL-4 has been relatively well tolerated at lower doses, but dose-limiting toxicities have been reached with both the glycosylated and nonglycosylated forms (Lotze et al., 1990; Atkins et al., 1992; Gilleece et al., 1992; Prendiville et al., 1993). Side effects include fatigue, diarrhea, gastric ulceration, arthralgia, dyspnea, headache, sinus congestion and hyponatremia. The more common side effects are headache, nasal congestion, fever, fluid retention, anorexia, fatigue, nausea and vomiting, and diarrhea. With the exception of some inflammatory reactions at subcutaneous injection sites, no allergic reactions have been reported, and no antibodies to rhuIL-4 have been described. Pharmacokinetic studies indicate that IL-4 can be detected in the serum 8–12 h after subcutaneous administration, but it is rapidly cleared after i.v. administration with a t1/2 of less than 1 h (Prendiville et al., 1993). Elevated liver enzymes (e.g. alkaline phosphatase and transaminase) are frequently reported, especially in patients with hepatic disease. A study in 10 patients using relatively high doses (10 or 15 lg kg1, three times a day intravenous bolus injection) determined a dose of 10 lg kg1 of IL-4 to be the maximum-tolerated dose for this schedule, however, no clinical benefit was observed (Atkins et al., 1992). In a study with 62 patients with advanced nonsmall cell lung cancer (Vokes et al., 1998), a dose of 0.25 lg kg1 or 1.0 lg kg1 of recombinant IL-4 has been administered subcutaneously three times per week. Symptoms encountered in patients treated with 1.0 lg kg1 include fatigue (18/41), fever (14/41) and anorexia (12/41). Among 55 evaluable patients, there was one partial response of more than 5 years, and eight patients had stable disease of 106 to 350 days. E. coli-derived rhu IL-4 was found to be safe and well tolerated at doses up to 5 lg kg1 day1 and 10 lg kg1 when administered to animals three times a week (Leach et al., 1997a, 1997b). Diverse effects observed in primate studies have generally not been observed in human patients. A dose escalation study in a total of 12 patients with rapid intravenous infusion of rhIL-4 given as once a

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day, two cycles of 7 days separated by a week of rest reached a final dose of 30 lg kg1 without major toxicities, and minimal symptoms such as mild hypotension, peripheral swelling, nasal congestion and headache observed in a dose-dependent fashion. This trial defined an a phase of approximately 8 min and a b clearance of approximately 48 min using a biologic assay with the induction of CD23 on a malignant B cell line (Custer and Lotze, 1990). Essentially no responses were observed in the 48 patients receiving IL-4 alone. A phase II study with previously treated non-Hodgkin’s lymphoma patients demonstrated that the patients tolerated subcutaneous rhuIL-4 at a dose of 3 lg/kg given three times per week, but only rare objective responses were noted (Taylor et al., 2000).

Clinical trials of IL-4 gene therapy IL-4 expressed from genetically modified fibroblasts could indeed cause local endothelial activation and recruitment of immune effectors, such as DC and T cells, to the injected site in patients (Lotze, 1993). Tissue biopsies of multiple vaccination sites obtained from these patients demonstrated transgene expression in situ for up to 2 weeks (Suminami et al., 1995). A phase I trial for malignant glioma has been launched using IL-4 gene transfected autologous glioma cells or transfected fibroblasts admixed with irradiated autologous glioma cells as vaccines (Pollack et al., 2000). Although preparation of autologous gene transfected cells may be sometimes difficult due to the low-transfection efficacy, an alternative strategy may be the use of allogeneic genetransfected cells. Melanoma vaccines using HLA-A2 allogeneic cell line transduced with IL-2 or IL-4 (Belli et al., 1998) demonstrated regression of skin nodules in some patients, but no changes were observed in other lesions. The side effects were mild, including transient fever and erythema at the site of injection. Vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cells response against peptide Melan-A/MART-1(27–35) of autologous, untransduced tumor, in one of six patients’ samples examined (Arienti et al., 1999). It is likely that the complexity of immune reactivity will preclude the use of a single cytokine to elicit or maintain long-term effective immune reactivity to human tumors. How-

ever, the further testing of IL-4 as a gene therapy is certainly given credence by the currently available reports from this institution, as well as others.

IL-4 toxin therapy Since IL-4 receptors (IL-4R) are present on a wide variety of human cancer cells, the possibility of targeting IL-4R with chimeric proteins composed of human IL-4 and mutant forms of Pseudomonas exotoxin A (PE) has been investigated (Debinski et al., 1994). Administration of chimeric toxin to animals caused regression of established xenografts. A toxin-fused circularly permuted IL-4 was found to have several-fold higher affinity to IL-4R and to be at least several-fold more toxic to human renal cell carcinoma cells (RCC) (Puri et al., 1996b) and human malignant astrocytomas (Puri et al., 1996a) compared with the original form of non-permuted IL-4 toxin. Intratumoral administration of the IL-4 toxin into well-established human glioblastoma xenografts demonstrated complete remission of flank tumors in all animals (Husain et al., 1998). Immunohistological analyses of expression of IL-4Ra in surgical/biopsy samples of brain tumor tissues indicate that 15 of 18 glioblastoma multiforme (GBMs) tumors are moderately to intensely positive. In contrast, no IL-4R protein is detectable in brain tissue specimens. Accordingly IL-4Ra positive primary GBM explanT cells cultures are specifically sensitive to IL-4 cytotoxin (Joshi et al., 2001), providing a rationale for the use of IL-4 toxin in clinical studies.

CONCLUSIONS IL-4 is produced by and affects many cell types. The biological milieu provides signals for production of IL-4, and at the same time, the effects of IL-4 production depend on the timing and maturation/activation stage of many cell types. IL-4 influences the differentiation of effector cells at the level of antigen presentation and activation of T cells. The presence of other cytokines can also dramatically change the outcome of an immune response. For cancer patients, local delivery of IL-4 by gene vectors may hold more promise than recombinant IL-4 by achieving high local concentration of the cytokine at the site of induction

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of anti-tumor response. Development of more effective and regulatable gene delivery systems may allow us to design combination strategies of multiple cytokines based on a deeper understanding of how each cytokine plays a role in the sequence of immune events. Understanding the receptors and their expression has led to the development of toxin-conjugated IL-4 for the treatment of cancer. Future analyses of cancer specific receptor chains will enable design of more specific compounds. Soluble forms of the IL-4 receptor may be important for patients with allergic diseases. In diseases in which excess TH1 shifted balance is apparent, such as chronic inflammatory diseases, delivery of rIL-4 or IL-4 gene may be of therapeutic benefit. Understanding the signal transduction and messenger molecules, as well as structure of IL-4, IL-4R and their complexes may allow development of specific inhibitors or enhancers of the complex biologic properties of IL-4.

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11 Interleukin-5 Chee Choy Kok, Gretchen T. Schwenger, Ron I.W. Osmond, Debra L. Urwin and Colin J. Sanderson The Western Australian Institute of Medical Research and Curtin University of Technology, Perth, Western Australia

Our knowledge is a little island in a great ocean of non-knowledge. Isaac Bashevis Singer

INTRODUCTION Interleukin-5 (IL-5) is produced by T lymphocytes as a glycoprotein with an Mr of 40–45 kDa and is unusual among the T-cell-produced cytokines in being a disulphide-linked homodimer. It is the most highly conserved member of a group of evolutionally related cytokines, including IL-3, IL-4, IL-9, IL-13 and granulocyte–macrophage colony-stimulating factor (GM-CSF), which are closely linked on human chromosome 5. Historically, two lines of research converged when it was demonstrated that two very different biological activities were properties of this molecule. On the one hand, in the early 1970s, a number of different factors have emerged which showed activity on mouse B cells in vitro. These preparations were mixtures of cytokines, particularly IL-4 and IL-5, and the assays available did not distinguish between the two molecules. It was not until the early 1980s that a group of high-molecular weight activities emerged which were The Cytokine Handbook, 4th Edition, edited by Augus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

clearly based on IL-5, although in published reviews in 1984 the identity of the activities was not appreciated (Howard et al., 1984; Vitetta et al., 1984). Three main groups were working with this molecule: Takatsu in Japan who considered the activity to be various forms of T-cell-replacing factor (TRF) (Takatsu et al., 1988), Swain and Dutton in California, who used the term B-cell growth factor II (BCGFII) (Swain et al., 1988), and Vitetta in Texas, who called it B-cell differentiation factor l (BCDFl) (Vitetta et al., 1984). In 1985, Takatsu’s group purified TRF and showed that it was identical to BCGFII (Harada et al., 1985). On the other hand, and also in the early 1970s, work on the colony-stimulating factors (CSFs) by Metcalf’s group in Australia had demonstrated the production of eosinophilic colonies from mouse bone marrow in the presence of crude spleen-cell-conditioned medium (Metcalf et al., 1974). It is now clear that this medium contained IL-5 because it was shown to produce a selective stimulation of human eosinophil

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colonies (Metcalf et al., 1983) and IL-5 is the only eosinophil haematopoietic growth factor which cross-reacts between man and mouse. This line of work culminated in the identification of murine eosinophil differentiation factor in our laboratory in the mid-1980s. This was done using a liquid assay system, which in the mouse is a much more sensitive assay than the colony assay (Sanderson et al., 1985; Warren and Sanderson, 1985). However, as is discussed in detail in this review, IL-5 is a colonystimulating factor for which the name Eo-CSF would have been appropriate. These two lines of research came together in 1986, when our group purified eosinophil differentiation factor (EDF) and showed that it was identical to BCGFII (Sanderson et al., 1986). It is interesting to note that the observations on the identity of TRF, BCGFII and EDF were made before the cloning of IL-5, which thus confirmed the biochemical data. There are two intriguing aspects of these dual biological activities of IL-5. First, although there is a wellknown association between eosinophilia and IgE levels, IL-5 does not appear to be involved in the IgE response, where IL-4 is the major controlling cytokine. This raises the possibility of some common features in the control of IL-4 and IL-5 expression. Second, although the activity on murine B cells in vitro is well characterized (see below), hIL-5 is not active in assays on human B cells analogous to those used in the mouse system. The role of IL-5 in eosinophilia, coupled with a better understanding of the part played by eosinophils in the development of tissue damage in chronic allergy, suggests that IL-5 will be a major target for a new generation of antiallergy drugs.

GENE STRUCTURE AND EXPRESSION The coding sequence of the IL-5 gene forms four exons (Figure 11.1). The introns show areas of similarity between the mouse and human sequences, although the mouse has a considerable number of sequences (including repeat sequences) which are not present in the human gene. The mouse includes a 738-base pair fragment in the 3-untranslated region (also known as the Alu-like repeat) which is not pres-

Mouse IL-5 gene

Mouse IL-5 mRNA

Human IL-5 gene

Human IL-5 mRNA

738bp insert in mouse gene Coding sequences

FIGURE 11.1 Maps of the human and mouse IL-5 genomic genes and corresponding mRNA. Exons are shown as boxes. The shaded area indicates the insert in the 3-untranslated region of the mouse gene.

ent in the human gene, and thus the mouse messenger RNA (mRNA) is 1.6 kilobases (kb) while the human is 0.9 kb (Campbell et al., 1987). Each of the exons contains the codons for an exact number of amino acids.

Coexpressiona with other cytokines IL-5 is often found to be coexpressed with IL-4 and IL13 (Shimbara et al., 2000; Verheyen et al., 2000), and because they are closely linked in the same chromosome, many believe in the coordinate regulation of these genes (Loots et al., 2000; Takemoto et al., 2000). The coexpression of IL-5 and IL-4 could explain the frequent association of eosinophilia and IgE. However, the molecular mechanism of this remains unclear as alignment of the two promoter regions shows no significant areas of homology. Even the conserved lymphokine element (CLE0) element (see below) is only 56% homologous. In fact, many studies have indicated that different signals are required for the induction of these two genes. Anti-CD3 induces the expression of IL-4, IL-5 and GM-CSF mRNA in mouse T cells, whereas treatment with IL-2 induced

a

Coexpression is used here to denote simultaneous expression of genes in an organism, not necessarily in the same cells, and not due to coordinate genetic control. Coordinate expression implies the simultaneous (or sequential) expression of genes in the same cell controlled at the genetic level.

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IL-5 and IL-13 mRNA expression but did not induce detectable amounts of IL-4 and GM-CSF messengers (Bohjanen et al., 1990, Verheyen et al., 2000). Furthermore, IL-4 increases the IL-2-induced production of IL-5 and IL-13, while IL-12 acts as a suppressor. In the presence of antigen-presenting cells (APC), glucocorticoids induce T-cell populations synthesizing high levels of IL-10 but reduced amounts of IL-4, IL-5 and IL-13 (Richards et al., 2000). In humans, a T helpertype 2 (TH2)-like pattern of cytokine mRNA expression could be demonstrated in asthmatic patients (Robinson et al., 1993; Okudaira et al., 1995). T cells in the bronchoalveolar lavage of mild atopic asthmatics showed elevated expression of IL-4 and IL-5 (Robinson et al., 1993). However, T cells purified from peripheral blood of non-atopic asthmatics secreted raised amounts of IL-5, but not IL-4, as compared with normal controls, whereas those from atopic patients secreted elevated quantities of both IL-4 and IL-5 (Corrigan and Kay, 1992). These differences are emphasized by the observation that cyclic AMP and 4b-phorbol 12-myristate 13-acetate (PMA) have different effects on the induction of different cytokines in the mouse lymphoma EL4. PMA induces IL-2 and IL-4 but has only a low effect on IL-5 induction. While cAMP markedly enhances the effect of PMA on the induction of IL-5, it has an inhibitory effect on IL-2, IL-3, GM-CSF and IL-10, and no effect on IL-4 (Derigs et al., 1990; Lee et al., 1993; Chen and Rothenberg, 1994; Karlen et al., 1996). Similarly, pertussis toxin induces IL-4 but not IL-5 mRNA synthesis, whereas cyclophosphamide stimulates the transcription of IL-5 but not of IL-4 (Sewell and Mu, 1996). More recently, it has been shown that nonactin suppresses IL-5 synthesis by human T helper cells without affecting IL-2 or IL-4 (Mori et al., 2001). From these observations it appears that IL-4 and IL-5 expression is not coordinately regulated, suggesting that unique control mechanisms exist for these lymphokines. This raises two questions: (1) What is the basis for the coexpression of IL-5, IL-4 and IL-13 in certain allergic diseases? There is no clear answer at this point. (2) Are there control elements that are specific to these genes? By developing a reporter system in which IL-5 transcription is studied in its genomic context, we found evidence that regulatory sites exist which may be involved in the specific regulation of the gene.

-400GATA3

PRE2

-152GATA3

-240 Oct

-70GATA3

-110NFAT PRE1 CLE0 TATA

FIGURE 11.2 Diagram showing relative positions of elements in the human IL-5 promoter. CLE0 binds JunD, Fra-2, Oct-1 and Oct-2 proteins. PRE1 (palindromic regulatory element) binds YY1, Oct-1 and an Oct-like protein. PRE2 binds YY1 and NF-AT.

Promoter region The 5 flanking region of the IL-5 RNA initiation site contains several motifs involved in the transcription of the gene (Figure 11.2). There is a short sequence of the CLE0 between nucleotides (nt) 56 and 42 in the human (h)IL-5 promoter region. This element is essential for promoter activity (Naora et al., 1994). CLE0 is conserved among the regulatory regions of several other lymphokine genes such as IL-3, IL-4 and GM-CSF (Masuda et al., 1993). Although most studies using deletion and mutation analysis have shown CLE0 to be critical for IL-5 expression (Naora and Young, 1994; Bourke et al., 1995; Lee et al., 1995, 1998; Siegel et al., 1995; Mori et al., 1997), a few have indicated that it may not be essential (Stranick et al., 1995, 1997). The CLE0 element contains sequences similar to the binding sequences for activating protein (AP)-1 and nuclear factor (NF)–AT regulatory proteins, but only the AP-1 moiety has been demonstrated to be important for inducible complex formation in EL4 cells (Siegel et al., 1995; Karlen et al., 1996). Fos and Jun proteins have been found to bind to this sequence in phorbol myristate acetate (PMA)/cAMP-stimulated EL4 cells (Siegel et al., 1995; Karlen et al., 1996). These proteins have been specifically identified recently in EL4 cells and primary T cells as JunB and FosB (Salerno et al., 2001). Octamer (Oct)-1 and Oct-2 factors were also found to bind murine CLE0 in this study. Surprisingly, in another study, protein complexes binding to CLE0 in the mouse T-cell clone D10.G4.1, were found to be constitutive, and a consensus oligonucleotide for AP-1 was unable to inhibit complex formation (Stranick et al., 1995). It has been suggested that CLE0–binding protein 1 (CLEBP-1) and high-mobility group 1/2 (HMG1/2) proteins may play a role in facilitating expression of murine (m)IL-5 CLE0 (Marrugo et al., 1996).The AP-1 members

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binding to hIL-5 CLE0 in an inducible fashion have recently been identified in the human leukaemic T-cell line PER-117 as JunD and Fos-related antigen (Fra)-2. The same report also identified constitutive binding of Oct1 and inducible binding of Oct2 to hIL-5 CLE0. These octamer factors were shown to be involved in positive regulation of the hIL-5 gene (Thomas et al., 1999a). Blumenthal et al. (1999) have also reported binding of Ets factors to human IL-5 CLE0 in Jurkat cells. The CLE0 element is thought to work in concert with other activation elements in the IL-5 promoter. The binding of Oct (Gruart-Gouilleux et al., 1995) and GATA (Siegel et al., 1995; Yamagata et al., 1995; Zhang et al., 1997; Lee et al., 1998) proteins at two sites immediately upstream of the CLE0 element has been reported. Binding of proteins to the Oct element was found to be dependent on activation, whereas binding of GATA was constitutive. Mutations in the GATA element have been shown to abolish IL-5 expression (Zhang et al., 1997; Lee et al., 1998), whereas the functional role of the octamer motif in this region is not clear. GATA-3 has been reported to be involved in the TH2specific expression of the IL-5 gene (Lee et al., 1998). More recent results have identified this TH2-specific expression to be controlled via GATA-3-dependent remodelling of the IL-4/IL-5/IL-13 chromosomal locus during differentiation of naive cells to TH2 cells (Miyatake et al., 2000). Furthermore, ectopic expression of GATA-3 in developing and committed TH1 cells can induce TH2 cytokine expression and chromatin remodelling of the IL-4/IL-5/IL-13 locus (Ferber et al., 1999, Lee et al., 2000; Takemoto et al., 2000). An additional Oct element located at positions 244 to 237 has been identified (Gruart-Gouilleux et al., 1995). This element forms complexes with factors antigenically related to members of the Oct group, and is involved in positively regulating the IL-5 promoter. This element has recently been footprinted in the hIL-5 promoter with nuclear extracts from T-cell clones with protection extending from 249 to 217 bp (Cousins et al., 2000). The same article also reports footprint of a previously unknown Ets binding site extending from 274 to 264 bp. Two palindromic regulatory elements, mPRE1-IL-5 (79 to 90) and mPRE2-IL-5 (459 to 470) have been identified in the mIL-5 promoter (Schwenger

et al., 1998). Although these elements appear to bind proteins constitutively, mutations of specific motifs within these elements, which act to abolish protein binding, also significantly reduce IL-5 promoter activity. These results suggest that these elements are essential for enhancing mIL-5 gene expression. Stranick et al. also report the presence of a protected region, which corresponds to mPRE1-IL-5 around position 76 to 90, which is likely to be involved in positive regulation of the mIL-5 gene (Stranick et al., 1998). Recently, similar palindromic regulatory elements have been reported in the hIL-5 gene. We have identified a negative regulatory element between positions 79 and 90 within a protected region (binding region 3) in the hIL-5 gene. This negative regulatory element overlaps with the hPRE1-IL-5 element which is similar to mPRE1-IL-5 and binds Oct-1, octamer-like and ying yang (YY)1 nuclear factors (Mordvinov et al., 1999). A second palindromic element in the distal hIL-5 gene was identified by us as hPRE2-IL-5. This element is in a similar position to mPRE2-IL-5, has sequence homology with hPRE1IL-5 and contains overlapping binding sites for YY1 and NFAT (447 to 459). The hPRE2-IL-5 element also acts to repress expression of the hIL-5 gene, displaying a novel function for NFAT (Schwenger et al., 1999). It is interesting to note the difference in the function of the PRE-IL-5 elements between the human and mIL-5 genes when considering the conservation of sequence and position of these elements and the conservation of function of the IL-5 gene between species. A previously identified response element, RE-II, between position 123 and 92 has recently been identified to bind a unique mRNA initiated within a middle intron of WHSC1/MMSET. This protein was shown to suppress hIL-5 transcription and endogenous IL-5 production (Garlisi et al., 2001). NFAT, in conjunction with AP-1 family members, has been found to bind to the mIL-5 promoter sequence located at positions 117 to 92 in EL4 cells (Lee et al., 1995), and to a similiar position of the human hIL-5 promoter in the human T-cell clone SP-B21 (Stranick et al., 1997). The role of this site in IL-5 gene expression remains controversial, as mutation analysis in some studies has suggested that this site does play a critical role (Lee et al., 1995, 1998; Stranick et al., 1997), while others have shown little or

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no effect (Siegel et al., 1995; Zhang et al., 1997). The NFAT site has been found to cooperate with the downstream GATA consensus site to regulate the hIL-5 promoter in a mouse mast cell line (Prieschl et al., 1995). This site has recently been shown to be involved in positive regulation of hIL-5 in both PER-117 cells and peripheral blood lymphocytes (De Boer et al., 1999). Upstream of the NFAT site another GATA consensus site has been identified. Electrophoretic mobility shift assays (EMSA) with an oligonucleotide encompassing the hIL-5 promoter from positions 177 to 80 has suggested that GATA does bind here in IgE/antigenstimulated mouse mast cells (Prieschl et al., 1995). No functional significance of binding was determined in this study. A recent study by Schwenger et al. (2001) identified two putative GATA binding sites in this region at positions 128 and 152. These were identified by TF Search (Heinemeyer et al., 1998) but only the 152 site was confirmed by DNAse I protection assay in unstimulated and PMA/cAMP stimulation of PER117 cells. The 152 site was shown to bind GATA-3 and has a positive effect on the hIL-5 promoter regulation while the 128 site neither bound GATA-3 nor had any effect on IL-5 expression. The same study also identified a putative GATA binding site at 400 in the hIL-5 promoter. This site was protected in unstimulated and PMA/cAMPstimulated PER117 cells and shown to bind GATA-3. More importantly, this site acts as a powerful repressor of IL-5 transcription with mutagenesis of this site allowing high-level expression of IL-5 without activation of other factors normally required for IL-5 expression. This result suggests a unique role for GATA-3 in regulating transcription of hIL-5 rather than being only involved in regulation of the IL4/ IL-5/IL13 locus as a whole. Through deletion and mutation analysis, several distal promoter elements that may play a positive role in IL-5 gene expression have been identified. Mutations in the region designated IL-5A (948 to approximately 933) element were found to decrease IL-5 promoter activity in EL4 cells by 60% upon PMA/cAMP stimulation (Lee et al., 1995). Using stable transfectants of EL4 cells, deletion analysis revealed a positive element located between positions 1016 and 929 upon PMA stimulation (Bourke et al., 1995). Mutation of a CTF/NF1 consensus site within this region converted the stable transfectants to con-

stitutive expression, suggesting that this site may be important for inducible expression. Negative regulatory elements have also been identified in the IL-5 promoter. In the mIL-5 promoter, two negative regulatory elements, NREI and NREII, were mapped to the regions between positions 431 and 392, and 300 to 261, respectively (Stranick et al., 1995). In addition, investigations of the hIL-5 promoter in mouse T cells has demonstrated that two negative regulatory elements lie between positions 404 and 312 (Gruart-Gouilleux et al., 1995), and 172 to 127 (Stranick et al., 1997). The activity of these elements is dependent on activation of the cells since deletion of the regions containing these elements results in a marked increase in inducible promoter activity. Nuclear proteins that may interact with these negative regulatory elements in the IL-5 promoter have not yet been characterized. Recent work on understanding expression of the IL-5 gene has focused on examining coordinate regulation of the IL-4/IL-5/IL-13 locus. This has been carried out by cross-species analysis, transgene studies and allelic expression patterns. Findings point to the existence of a coordinate regulator in this locus, with both trans-activating and cis-regulatory elements underlying this coordinate regulation, and that these cytokines are expressed from one allele (Kelly and Locksley, 2000; Lacy et al., 2000; Loots et al., 2000).

Post transcriptional regulation While post-transcriptional regulation is emerging as an important control point in cytokine gene expression, no evidence was found to suggest that IL-5 is regulated at the level of mRNA stability or translation efficiency by either the 5 or 3UTR (Thomas et al., 1999b).

PROTEIN STRUCTURE IL-5 is a homodimeric glycoprotein consisting of two identical polypeptide chains that are 115 amino acid residues in length (113 in the mouse). Characterization of E. coli-expressed recombinant human IL-5 gives a molecular mass of 26 kDa (Graber et al., 1993), close to the theoretical molecular mass of 24 kDa.

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However, recombinant IL-5 expressed in mammalian systems has a molecular mass of over 45 kDa as estimated by SDS-PAGE analysis (Sanderson et al., 1985), thus a large proportion of the mass is carbohydrate. Human IL-5 has N-linked carbohydrate at position Asn-28 and O-linked carbohydrate at Thr-3 (Minamitake et al., 1990). MIL-5 is glycosylated at analogous sites, plus a further N-linked carbohydrate at Asn-55 (Kodama et al., 1992). A potential N-linked site at Asn-69/71 is not glycosylated in either species. Variation in carbohydrate attachment contributes to the wide variation in migration observed in SDSPAGE (45–60 kDa) and isoelectric focusing experiments (pI 4.5–8.0). However, removal of carbohydrate from IL-5 does not appear to affect its biological activity (Tominaga et al., 1990; Kodama et al., 1993), but does cause loss of thermostability and may be the cause of the hydrophobicity observed for recombinant bacterial protein. Determination of the three-dimensional structure of IL-5 by X-ray crystallography (Milburn et al., 1993) revealed that IL-5 belongs to a family of structurally related proteins, including IL-2, IL-4, macrophage colony-stimulating factor (M-CSF), GM-CSF and growth hormone. These proteins fold to produce a tertiary structure of four bundled a helices (designated A, B, C and D from the N-terminus), arranged with an up–up, down–down topology (Milburn et al., 1993). An unusual structural feature of the homodimeric IL-5 is that it forms two four-helical bundles, each containing three helices (A–C) from one chain and one from the other (D). Each bundle contains helices A and D paired in an antiparallel orientation to form one face of the molecule, and the B and C helices, also paired in an antiparallel orientation, to form the other face. The AD and BC helical pairs of each bundle are at an angle of 40º relative to each other, an unusual feature of four-helical bundle cytokines (Milburn et al., 1993). In addition, the structure features two short antiparallel b sheets on opposite sides of the molecule that are formed between residues 32–35 of chain-1 and 89–92 of chain-2 (Plate 11.3) (see Plate section). The dimeric structure is held together by two disulphide bridges between residues Cys-44 and Cys-86 of opposing chains (Minamitake et al., 1990; McKenzie et al., 1991; Proudfoot et al., 1991). Approximately 7000 Å2 of buried surface area between the two bun-

dles is also likely to contribute hydrophobic and electrostatic interactions that stabilize the dimer (Milburn et al., 1993). Despite formation of dimers in the native state, studies with artificially engineered IL-5 monomers suggest that all of the structural information required for activity are on a single chain (Dickason and Huston, 1996). When just the dimerbridging cysteines are mutated, thereby producing monomeric IL-5, only biologically inactive material is produced (McKenzie et al., 1991). However, when an extra eight amino acids are inserted into loop 3 of IL-5 to generate a loop analogous to that of GM-CSF, biologically active monomeric IL-5 is obtained (Dickason and Huston, 1996). A comparison of the structure of IL-5 with closely related cytokines revealed that loop 3 of the IL-5 is shorter, thus confirming that in the native material it is the shorter loop 3 that prevents monomeric folding. The receptor for IL-5 consists of two subunits; an a subunit (Ra) that specifically binds IL-5, and a signalling b subunit (bcom) that is also recognized by IL-3 and GM-CSF (Tavernier et al., 1991; Lopez et al., 1992). Binding studies show that IL-5 interacts with cells expressing the Ra subunit with a KD of approximately 1 nM, and with cells expressing both Ra and bcom with a KD of 250 pM. However, direct binding between IL-5 and bcom is yet to be demonstrated (Wu et al., 2000). The point of contact between IL-5 and its receptor chains has been investigated by mutation analysis, constructing species hybrid molecules and the mapping of neutralizing antibodies (McKenzie et al., 1991; Cornelis et al., 1995; Graber et al., 1995; Morton et al., 1995; Tavernier et al., 1995; Dickason et al., 1996; Li et al., 1996; Edgerton et al., 1997; Plugariu et al., 2000; Wu et al., 2000). These studies suggest that charged residues in the b sheet, centred on Arg-91 of b strand 2, are important for binding to the Ra chain. Central regions of the AD face are also implicated, in particular amino acid residues in the C-terminus centred around Glu-110. Although IL-5 has two potential sites for interaction with the receptor, only a 1:1 complex is formed (Devos et al., 1993). Studies of the contact point of IL-5 with bcom have highlighted Glu-13 (of helix A) as the critical residue involved, and an IL-5 mutant (Glu-13→Gln-13) has been shown to have antagonistic properties (Tavernier et al., 1995). In addition, recent evidence has suggested that charge of

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Glu-110 is also important in bcom activation (Plugariu et al., 2000; Wu et al., 2000).

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study of the control of haematopoiesis by the immune system.

Eosinophil production in vitro

EOSINOPHILIA T-cell dependence Eosinophilia is T-cell dependent and therefore it is not surprising that the controlling factor is a T-cellderived cytokine (Sanderson et al., 1985). It is characteristic of a limited number of disease states, most notably parasitic infections and allergy. Clearly, as eosinophilia is not characteristic of all immune responses, it is obvious that the factors controlling eosinophilia are not produced by all T cells. Similarly, as it is now clear that IL-5 is the main controlling cytokine for eosinophilia (see below), then if IL-5 has other biological activities, it is likely that these will coincide with the production of eosinophils.

Biological specificity One of the features of eosinophilia which has attracted the curiosity of haematologists for several decades is the apparent independence of eosinophil numbers on the numbers of other leukocytes. Thus eosinophils are present in low numbers in normal individuals but can increase dramatically and independently of the number of neutrophils. Such changes are common during the summer months in individuals with allergic rhinitis (hay fever), or in certain parasitic infections. Clearly, such conditions will result in more broadly based leukocytosis when complicated by other infections. Although this specificity has been known for many years, somewhat surprisingly it is not easy to find clear examples in the early literature. In experimental infection of volunteers with hookworms (Necator americanus), it was noted that an increase in eosinophils was the only significant change (Maxwell et al., 1987), and our own work with Mesocestoides corti in the mouse, demonstrated massive increases in eosinophils, independent of changes in neutrophils (Strath and Sanderson, 1986). This biological specificity suggests a mechanism of control that is independent of the control of other leukocytes. This, coupled with the normally low numbers of eosinophils, provides a useful model for the

In the mouse system, IL-5 induces the production of eosinophils in liquid bone marrow cultures and this is much more sensitive than the corresponding colony assay in semi-solid medium (Sanderson et al., 1985, 1988; Sanderson, 1990). In contrast, both IL-3 and GM-CSF induce eosinophils as well as other cell types, most notably neutrophils and macrophages in bone marrow cultures (Campbell et al., 1988). The production of eosinophils is considerably higher when the bone marrow is taken from mice infected with M. corti than it is from normal marrows suggesting that marrow from infected mice contains more eosinophil precursors than marrow from normal mice (Sanderson et al., 1985, 1988; Sanderson, 1993). Similarly, it has been reported that numbers of CD34/IL5Ra cells are increased in the bone marrow after allergen challenge of asthmatic subjects (Sehmi et al., 1997). In human bone marrow cultures both IL-3 and GMCSF, but not IL-5, appeared to amplify the number of eosinophil colony precursors (Clutterbuck and Sanderson, 1990). This led to the concept of IL-5 as a late-acting factor in eosinopoiesis. However, experiments in vivo have not substantiated this, and it seems likely that culture systems do not fully mimic the production of eosinophils in vivo. More recent studies of human umbilical cord blood-derived CD34 cells has demonstrated that IL-3 and GM-CSF can amplify the number of eosinophils present without the addition of exogenous IL-5. However, this effect has been shown to be at least partly dependent on IL-5, in that IL-3 and GM-CSF both induce endogenous IL-5 expression which itself up-regulates the transmembrane form of the IL-5 receptor (Tavernier et al., 2000). Interleukin-9 has also been shown to enhance the viability of cord blood-derived eosinophils. This increase in viability is associated with increased expression of the IL-5Ra chain thus encouraging IL-5-mediated differentiation and maturation (Gounni et al., 2000) A number of factors has suggested that IL-5 may be at least partly dependent on stromal cells, even in the late stage of eosinophil differentiation. For example,

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in the mouse system few eosinophil colonies form in semi-solid medium, whereas large numbers of eosinophils are produced in the adherent layer of stromal cells in liquid culture (Sanderson et al., 1985; Warren and Sanderson, 1985). Second, in human liquid bone marrow cultures more eosinophils are produced in round-bottomed vessels than in flatbottomed vessels, possibly due to better cell–cell interactions (Clutterbuck and Sanderson, 1988). A recent study has shown that bone marrow stromal cells are able to produce IL-5 which may account for some of these findings (Hogan et al., 2000). Studies have also shown that IL-5 binds strongly to certain proteoglycans, suggesting that the presentation of IL-5 on the extracellular matrix in the bone marrow could be important in its biological activity (Lipscombe et al., 1998).

Eosinophil production in vivo An approach to understanding the role of IL-5 in vivo is to alter the expression of IL-5 in transgenic mice. As IL-5 is normally a T-cell product and the gene is transcribed for only a relatively short period of time after antigen stimulation, transgenic mice in which IL-5 is constitutively expressed by all T cells have been produced (Dent et al., 1990). These mice have detectable levels of IL-5 in the serum. They display a profound and lifelong eosinophilia, with large numbers of eosinophils in the blood, spleen and bone marrow. This indicates that the expression of IL-5 is sufficient to induce the full pathway of eosinophil differentiation. If other cytokines are required for the development of eosinophilia, then either they must be expressed constitutively, or their expression is secondary to the expression of the IL-5 gene. This clear demonstration that the expression of the IL-5 gene in transgenic animals is sufficient for the production of eosinophilia, provides an explanation for the biological specificity of eosinophilia. It therefore seems likely that because eosinophilia can occur without a concomitant neutrophilia or monocytosis, a mechanism must exist by which IL-5 is the dominant haemopoietic cytokine produced by the T-cell system in natural eosinophilia. Another important aspect of these transgenic animals is that despite their massive long-lasting eosinophilia the mice remained normal. This illus-

trates that an increased number of eosinophils is not of itself harmful, and that the tissue damage seen in allergic reactions and other diseases must be due to agents that trigger the eosinophils to degranulate. Another important approach to the understanding of the biological role of IL-5 comes from the administration of neutralizing antibody. Mice infected with Trichinella spiralis develop eosinophilia and increased levels of IgE; however, when treated with an anti-IL-5 antibody, no eosinophils are observed (Coffman et al., 1989). Indeed, the number of eosinophils is lower than that seen in control animals. These experiments illustrate the unique role of IL-5 in the control of eosinophilia in this parasite infection. They also show that the apparent redundancy seen in vitro, where both IL-3 and GM-CSF are also able to induce eosinophil production, does not operate in these infections. Furthermore, IL-5 plays no role in the development of IgE antibody (this activity is controlled by IL-4), or in the development of the granuloma seen surrounding schistosomes in the tissues (Sher et al., 1990). The generation of mice with an inactive IL-5 gene (IL-5 knockout mice) has confirmed the key role of IL-5 in the control of eosinophilia (Foster et al., 1996; Kopf et al., 1996). No eosinophils were produced in response to either a parasite infection or aeroallergen sensitization with ovalbumin. In fact, the low background level of eosinophils seen in normal control mice was substantially reduced in non-sensitized knockout mice, to leave a very small number of eosinophils produced in the absence of IL-5. The lack of effect on other cell types or on antibody production confirmed the unique specificity of IL-5 for the eosinophil lineage, which had been proposed over a number of years (Sanderson, 1992). Knockout mice have provided an important animal model to test the biological role of IL-5 and eosinophils. Following the induction of eosinophils in the lung by the challenge of sensitized mice with an antigen aerosol, a high degree of lung inflamation develops. However, in knockout mice lung eosinophilia is not observed, and there is very little development of inflamation or lung damage (Foster et al., 1996). This provides an experimental rationale for the role of eosinophils in human asthma, as has been proposed over many years (Gleich, 1990; Corrigan and Kay, 1992).

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ACTIVATION OF EOSINOPHILS The ability of eosinophils to perform in functional assays can be increased markedly by incubation with a number of different agents, including IL-5. The phenomenon of activation is apparently independent of differentiation. It appears to have a counterpart in vivo, as eosinophils from different individuals vary in functional activity. It has been demonstrated that the ability of eosinophils to kill schistosomula increases in proportion to the degree of eosinophilia (David et al., 1980; Hagan et al., 1985). This is consistent with a common control mechanism for both the production and activation of eosinophils in these cases. The first observations on selective activation of human eosinophils by IL-5 showed that the ability of purified peripheral blood eosinophils to lyse antibody-coated tumour cells was increased when IL-5 was included in the assay medium (Lopez et al., 1986). Similarly, the phagocytic ability of these eosinophils towards serum-opsonized yeast particles was increased in the presence of IL-5. There was a 90% increase in surface C3bi complement receptors, as well as an approximately 50% increase in the granulocyte functional antigens GFA-1 and GFA-2. A possible marker for activated eosinophils, CD69 (Nishikawa et al., 1992), is expressed upon IL-5 stimulation (Hartnell et al., 1993; Urasaki et al., 2001). IL-5induced CD69 expression on blood cells is unique to eosinophils because the specific receptor for IL-5 is absent on neutrophils and rarely, if ever, expressed on other cells that express CD69 (Migita et al., 1991; Murata et al., 1992). Studies have demonstrated that IL-5 increases ‘polarization’, including membrane ruffling and pseudopod formation, which appear to reflect changes in the cytoskeletal system. IL-5 also induces a rapid increase in superoxide anion production by eosinophils (Lopez et al., 1988). In addition, IL-5 appears to be a specific survival factor for eosinophils within the human system (Bagley et al., 1997), and has been shown to induce Bcl-xL, an antiapoptotic gene (Dibbert et al., 1998). A further interesting observation in this context was the demonstration that IL-5 is a potent inducer of immunoglobulin-induced eosinophil degranulation, as measured by the release of eosinophil-derived neu-

rotoxin (EDN). IL-5 increased EDN release by 48% for secretory IgA and by 136% for IgG. This enhancing effect appeared by 15 min and reached a maximum by 4 h (Fujisawa et al., 1990). The finding that secretory IgA can induce eosinophil degranulation is particularly important because eosinophils are frequently found at mucosal surfaces where IgA is the most abundant immunoglobulin.

TISSUE LOCALIZATION Another aspect of the pathology of diseases characterized by eosinophilia, is the preferential accumulation of eosinophils in tissues. As the blood contains both eosinophils and neutrophils, a specific mechanism must exist that allows the eosinophils to pass preferentially from the blood vessels to the tissues. A number of factors, including IL-5, are reported to have a specific chemotactic activity for eosinophils (Yamaguchi et al., 1988; Wang et al., 1989). The different tissue distribution of eosinophils in the two transgenic mice systems probably results from the different tissue expression of IL-5. Using the metallothionein promoter, transgene expression was demonstrated in the liver and skeletal muscle, and eosinophils were observed in these tissues (Murata et al., 1992). In contrast, the CD2–IL-5 mice with IL-5 expression in T cells did not have eosinophils in the liver or skeletal muscle (Dent et al., 1990). This suggests that eosinophils migrate into tissues where IL-5 is expressed. While IL-5 is reported to be chemotactic for eosinophils (Yamaguchi et al., 1988; Wang et al., 1989), the activity is relatively weak. Thus, it probably plays a more important role as a cofactor to enhance eosinophil response to other chemotactic factors (Lampinen et al., 1999), and to eliminate the inhibitory effect of IL-2 on eosinophil migration (Lampinen et al., 2001). An alternative mechanism of extravasation of eosinophils is suggested by experiments in which IL-5 has been shown to up-regulate adhesion molecules. Thus, it was demonstrated that IL-5 increased the expression of the integrin CD11b on human eosinophils (Lopez et al., 1986), and this increased expression was accompanied by an increased adhesion to endothelial cells (Walsh et al., 1990). Adhesion was inhibited by antibody to CD11b or CD18,

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suggesting that the integrins are involved in eosinophil adhesion to endothelial cells (Walsh et al., 1990). More recently, it has been shown that eosinophils can use the integrin very late activation antigen-4 (VLA-4) (CD49d/CD29) in adherence to endothelial cells. In this case the ligand is vascular cell adhesion molecule-1 (VCAM-1). In contrast, neutrophils do not express VLA-4 and do not use this adherence mechanism (Walsh et al., 1991). Thus, while IL-5 appears to be involved in eosinophil localization, the identification of eotaxin, eotaxin-2 and eotaxin-3, members of the chemokine family, provides an important step in understanding the specificity of eosinophil localization. Like IL-5, eotaxins are specific for the eosinophil lineage, but are powerful chemoattractants and activators of eosinophils. Eotaxin and eotaxin-2 are important for eosinophil recruitment in the early phase of the late asthmatic response, while eotaxin-3 may be responsible for prolonged or late phase recruitment (Berkman et al., 2001). There is good evidence for a biological interaction between IL-5 and eotaxin (Collins et al., 1995).

ACTIVITIES ON OTHER CELL TYPES Basophils The most pronounced effect of IL-5 on cells other than eosinophils in humans, is the effect on basophils. While our studies suggested that IL-5 induces only eosinophils, a detailed study by electron microscopy of cells produced in human cord blood cultures revealed a small number of basophils (Dvorak et al., 1989). Other studies have shown that IL-5 primes basophils for increased histamine production and leukotriene generation (Bischoff et al., 1990; Hirai et al., 1990), and basophils in the blood clearly express the IL-5 receptor (Lopez et al., 1990). Thus, while the effect of IL-5 on the production of basophils may be minor, the priming effect on mature basophils may be of significance in the allergic response.

B cells As discussed in the Introduction, the characterization of the activities of IL-5 on mouse B cells developed

around several different in vitro assay systems. In the TRF assay, IL-5 induces specific antibody production by B cells primed with antigen in vivo (Takatsu et al., 1988). The BCGFII assay was based on the ability of IL-5 to induce DNA synthesis in normal splenic B cells in the presence of dextran sulphate, and later on the ability of IL-5 to increase DNA synthesis in the BCL1 cell (a mouse B-cell tumour) line (Swain et al., 1988). The BCDFl assay depends on the ability of IL-5 to induce BCL1 cells to secrete IgM (Vitetta et al., 1984). IL-5 is a late-acting factor in the differentiation of primary B cells, requiring a priming stimulus to make resting B cells responsive. This can be either polyclonal stimulants such as dextran sulphate, bacterial lipopolysaccharide (LPS), anti-immunoglobulin or specific antigen. Large splenic B cells, presumed to have been activated in vivo, when cultured with IL-5 for 7 days show markedly enhanced numbers of IgMand IgG-producing cells (O’Garra et al., 1986). IL-5 in combination with antigen is sufficient to induce growth and differentiation of B cells at the single cell level (Alderson et al., 1987). Combinations of IL-2, IL-4 and IL-5 appear to regulate the amount of IgG1 isotype secreted by B cells (McHeyzer-Williams, 1989; Purkerson and Isakson, 1992). Neutralizing antibody to IL-5 was found to inhibit the polyclonal antibody response induced by T-cell clones on B cells, suggesting a critical role for IL-5 in this system (Rasmussen et al., 1988). A possible role for IL-5 in the development of autoimmunity in mice was suggested by the observation that this cytokine stimulates B cells from NZB mice to produce high levels of IgM anti-DNA antibody (Howard et al., 1984). In another study the B cells from autoimmune NZB/W mice were found to be hyperresponsive to IL-5, whereas two other strains of mice which are prone to autoimmunity did not show this response. As NZB/W mice have elevated numbers of Ly-1–positive B cells, these were tested and found to show a higher response to IL-5 than the negative cells, suggesting that the increased responsiveness to IL-5 in these mice may be due to the increased numbers of Ly-1 B cells (Umland et al., 1989). In support of this, freshly isolated peritoneal Ly-1 B cells express high levels of IL-5 receptor, and IL-5 increases the frequency of cells that produce autoantibodies (Wetzel, 1989). As these effects mainly concern the production of IgM, whereas autoimmune disease mainly appears

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to be due to IgG, the significance of these findings for autoimmunity is unclear. However, these experiments point to the possible restriction of IL-5 activity to the Ly-1 subpopulation of B cells. A potentially interesting observation was the demonstration that IL-5 appears preferentially to enhance IgA production. When added to cultures in the presence of LPS, the highest increase over background occurs with the IgA-producing cells, with significant increases in IgM and IgG1 as well (Bond et al., 1987; Yokota et al., 1987). The interpretation of these experiments is not straightforward, as the LPS itself induces a large effect and the activity on IgA and IgG1 was small in comparison to the total levels of IgM produced. In a study of B cells from gut-associated lymphoid tissue (Peyer’s patches), IL-5 increased the production of IgA but maximum enhancement of IgA in these cultures requires IL-4 (Murray et al., 1987; Lebman and Coffman, 1988). This effect of IL-5 was shown to be due to the induction of a high rate of IgA synthesis in cells positive for surface IgA expression. No IgA secretion was induced in the surface IgAnegative cells (Murray et al., 1987; Harriman et al., 1988; Kunimoto et al., 1988). This suggests that IL-5 does not induce switching to IgA production, but acts after switching to enhance the production of IgA. In contrast to these studies, which suggest a key role for IL-5 in the production of IgA, more recent studies have indicated that its effect is minor compared with the activity of other cytokines, and that it may only augment these activities. For example, IL-5 was shown to enhance IgA secretion from B cells isolated from Peyer’s patches, but the effect was small compared with the effect of IL-6 (Beagley et al., 1989). The combination of IL-5 and IL-6 had a greater effect than either cytokine alone (Kunimoto et al., 1989). It has been shown that TGF has an important activity in the switching to IgA production in LPS-stimulated B cells, and while IL-5 enhances this effect it is less active than IL-2 (Sonoda et al., 1989). The action of IL-5 allows the cells to respond to IL-2 by amplification of J chain mRNA. Thus, IL-5 and IL-2 are both necessary for IgM secretion (Matsui et al., 1989). A possible mechanism for the effect of IL-2 on B cells is suggested by the observation that IL-5 increases the expression of the IL-2 receptor (Loughnan et al., 1988). The significance of these activities remains unclear.

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While transgenic mice expressing IL-5 on a metallothionein promoter were shown to develop autoimmunity (Tominaga et al., 1991), no effect on B cells or antibody levels was detected in transgenic mice expressing IL-5 under control of the CD2 locus control region (Sanderson, 1993). Similarly, treatment of mice with anti-IL-5 antibody completely blocked eosinophil production but had no effect on antibody levels (Finkelman et al., 1990). Despite this large body of research on IL-5 as a B-cell growth factor in vitro, IL-5 knockout mice show a surprising absence of effect on immunoglobulin levels or ability to mount an antibody response. In fact, the only detectable effect of IL-5 gene disruption was a transient decrease in Ly B cells in the first few weeks of life. This is in contrast to the major effects on eosinophil production discussed above. In view of the well-characterized activity of IL-5 on mouse B cells it was surprising that no activity could be demonstrated in a wide range of human B-cell assay systems (Clutterbuck et al., 1987). This lack of activity of human IL-5 has been confirmed in many different systems (Bende et al., 1992). Although two reports of low activity in some human assays has reopened this question (Bertolini et al., 1993; Huston et al., 1996), the failure to detect IL-5 receptor on the surface of human B cells and the absence of significant effects in IL-5 knockout mice probably dismiss the concept of IL-5 as an important cytokine in the generation of an antibody response.

Nervous system An intriguing observation that both IL-4 and IL-5 regulate nerve growth factor production by astrocytes suggests a possible role in the regulation of the neural system (Awatsuji et al., 1993).

Bone metabolism Osteogenic precursor cells are found in the bone marrow stroma (Pereira et al., 1995; Prockop, 1997) and have been found to interact with these cells (Lorenzo, 1991; Manolagas and Jilka, 1995). Interestingly, a study of transgenic mice overexpressing IL-5 has indicated a role in bone homeostasis, although it is not clear whether this is a direct effect or mediated through eosinophils or B cells (Macias et al., 2001).

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SOURCES OF IL-5

SUMMARY

All of the original reports on the characterization, purification and cloning of mIL-5 utilized T-cell lines or lymphomas as the source of material, suggesting that T cells are an important source of the cytokine. The demonstration that IL-5 as well as other cytokine mRNAs are produced by mast cell lines raises the possibility that these cells may serve to induce or amplify the development of eosinophilia (Burd et al., 1989; Plaut et al., 1989). Similarly, the observation that human Epstein–Barr virus-transformed B cells produce IL-5 raises the possibility that B cells may be an additional source of this cytokine (Paul et al., 1990). Furthermore, eosinophils themselves have been demonstrated to produce IL-5 (Broide et al., 1992), although they do not appear to produce enough to sustain their own survival. Other sources of IL-5 include natural killer cells (Warren et al., 1995; Walker et al., 1998), basophils (Ying et al., 1995), bone marrow microvascular endothelial cells (Mohle et al., 1997) and epithelial cells (Salvi et al., 1999). In a careful study of cells producing IL-5 in bronchial biopsies from asthmatic subjects it was concluded that T cells are the major source of IL-5. The apparent dominance of mast cells in some studies was attributed to the fact that mast cells store IL-5 in their granules, whereas T cells rapidly secrete IL-5 as it is synthesized. Thus, immunohistological staining for IL-5 underrepresents the number of T cells compared with in situ hybridization (Ying et al., 1994, 1997). It is not clear whether the non-T cell-derived IL-5 plays a significant biological role in the development of eosinophilia. Eosinophilia has been observed in a significant proportion of a wide range of human tumours. In many cases the presence of eosinophils has been found to be of positive prognostic significance (reviewed by Sanderson, 1992). Clearly, it is important to understand the mechanism of production of these eosinophils. In a study of Hodgkin’s disease with associated eosinophilia, all 16 cases produced a positive signal for IL-5 mRNA by in situ hybridization (Samoszuk and Nansen, 1990). This suggests that IL-5 may be responsible for the production of eosinophils in these cases, and raises the possibility that eosinophilia in other tumours may also be due to the production of IL-5 by the tumour cells.

Interleukin-5 is the most highly conserved member of a group of evolutionary related cytokines which are associated with eosinophilia. It plays an essential role in promoting growth, differentiation, survival and activation of eosinophils. Thus, it is important to understand the mechanisms of IL-5 regulation and its activities on eosinophils. To date, most studies have focused on the regulation of the IL-5 gene. While many findings have been made, the complete mechanism of IL-5 regulation has yet to be understood. More studies on the IL-5 gene, protein and receptor are needed to provide a better understanding of its functions and how it may be specifically regulated for therapeutic purposes.

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12 Interleukin-6 (IL-6) Tadamitsu Kishimoto Osaka University Graduate School of Medicine, Osaka, Japan

His absence is good company Scottish saying

INTRODUCTION Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities, is produced by both lymphoid and non-lymphoid cells and regulates immune reactivity, the acute-phase response, inflammation, oncogenesis and hematopoiesis (Kishimoto, 1989; Le and Vilcek, 1989; Sehgal et al., 1989; Heinrich et al., 1990; Hirano and Kishimoto, 1990, 1992; Van Snick, 1990; Hirano, 1992a). IL-6 was originally known by a variety of names, such as interferon-b2 (IFNb2) (Weissenbach et al., 1980; May et al., 1986; Zilberstein et al., 1986), T-cell replacing factor (TRF)-like factor (Yoshizaki et al., 1982), B-cell differentiation factor (BCDF) (Okada et al., 1983), 26-kDa protein (Haegeman et al., 1986), B-cell stimulatory factor-2 (BSF2) (Hirano et al., 1985, 1986), hybridoma– plasmacytoma growth factor (HPGF or IL-HP1)

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

(Aarden et al., 1985; Nordan and Potter, 1986; Van Damme et al., 1987a; Van Snick et al., 1988), hepatocyte-stimulating factor (HSF) (Andus et al., 1987; Gauldie et al., 1987), and monocyte–granulocyte inducer type 2 (MGI-2) (Shabo et al., 1988). However, molecular cloning of IFNb2 (May et al., 1986; Zilberstein et al., 1986), 26-kDa protein (Haegeman et al., 1986) and BSF-2 (Hirano et al., 1986) revealed that all these molecules are identical (Sehgal et al., 1987a), and it was proposed at the end of 1988 that this molecule be referred to as IL-6 (Kishimoto, 1989; Le and Vilcek, 1989; Sehgal et al., 1989; Van Snick, 1990; Hirano and Kishimoto, 1990; Heinrich et al., 1990). In the following sections, the structure and function of IL-6 and its receptor, the regulatory mechanisms governing IL-6 gene expression, signal transduction mechanisms and the possible involvement of IL-6 in a variety of diseases are described.

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BIOLOGICAL ACTIVITY Immune responses B cells stimulated with antigen proliferate and differentiate into antibody-forming cells under the control of various cytokines produced by T cells and macrophages (Kishimoto, 1985). IL-6 was identified as one of the factors acting on B cells in the culture supernatants of phytohemagglutinin (PHA)- or antigenstimulated peripheral mononuclear cells which induce immunoglobulin production in Epstein–Barr virus (EBV)-transformed B-cell lines (Kishimoto et al., 1978). Furthermore, it was demonstrated that IL-6 functions in the late phase of Staphylococcus aureus Cowan I (SAC) stimulation of normal B cells (Hirano et al., 1984a; Teranishi et al., 1984) or leukemic B cells (Yoshizaki et al., 1982), inducing immunoglobulin production when other factors such as IL-2 are available. Recombinant IL-6 acts on B-cell lines at the mRNA level and induces biosynthesis of secretorytype immunoglobulin (Kikutani et al., 1985). Transcriptional activation is the primary mechanism for the quantitative increase in mRNAs of secretory immunogloblin (Raynal et al., 1989). Furthermore, IL-6 was found to activate immunoglobulin heavy chain enhancer (El) in large but not unstimulated small B cells obtained from transgenic mice carrying the El and j light chain promoter-driving chloramphenicol acetyltransferase (CAT ) gene (Miller et al., 1992). IL-6 acts on B cells activated with SAC or pokeweed mitogen (PWM) to induce immunoglobulin (Ig)M, IgG and IgA production, but not on resting B cells (Muraguchi et al., 1988). Anti-IL-6 antibody inhibits PWM-induced immunoglobulin production, indicating that IL-6 is essential for this production (Muraguchi et al., 1988). An essential role of IL-6 was also demonstrated in IL-4-dependent IgE synthesis (Vercelli et al., 1989) and in polysaccharide-specific antibody production (Ambrosino et al., 1990) in human B cells, as well as in the influenza A virus-specific primary response in murine B cells (Hilbert et al., 1989). Anti-IL-6 antibody inhibits IL-4-driven IgE production, suggesting that endogenous IL-6 is essential for the IL-4dependent induction of IgE (Vercelli et al., 1989). Indeed, it has been demonstrated that IL-4 induces IL-6 production in normal human B cells (Smeland

( IL - 6 )

et al., 1989). A crucial role for IL-6 in antibody production has also been shown in IL-2-induced immunogloblin production in SAC-stimulated B cells (Xia et al., 1989). In this case, IL-2 does not induce IL-6 production but may induce the IL-6 responsiveness in SAC-activated B cells which produce IL-6 spontaneously. The dependence on IL-2 by the action of IL-6 in B cells was shown previously by utilizing partly purified IL-6 (Teranishi et al., 1984) and this dependence has been confirmed with recombinant IL-6 (Splawski et al., 1990), indicating that, in addition to antigenic stimulation, additional signals provided by growth factors such as IL-2 are required for B cells to acquire IL-6 responsiveness. The need for IL-6 was found to be different for antigen-specific antibody production by primary and by secondary murine B cells. The former response depends on IL-6 but the latter does not (Hilbert et al., 1989). IL-6 and IL-1 synergistically stimulate the growth and differentiation of murine B cells activated with anti-immunoglobulin or dextran sulphate (Vink et al., 1988). In addition, IL-6 increases IgA production in murine Peyer’s patch B cells (Beagley et al., 1989; Kunimoto et al., 1989) or in human appendix B cells expressing the IL-6 receptor (Fujihashi et al., 1991). That this effect of IL-6 is not the result of isotype switching was shown by the finding that membranebound IgA-negative B cells could not be induced to secrete IgA by IL-6 (Beagley et al., 1989). These results indicate that IL-6 plays a role in mucosal immune response (Fujihashi et al., 1992). IL-6 was also found to augment the in vivo production of anti-sheep red blood cell (SRBC) antibodies in mice (Takasuki et al., 1988). Consistent with this, IL-6 transgenic mice or mice bearing a retrovirus vector expressing IL-6 display massive plasmacytosis and hypergammaglobulinemia (Suematsu et al., 1989; Brandt et al., 1990). All these results show that IL-6 plays roles in immunoglobulin production in vivo. However, IL-6 may not be essential for immunoglobulin production and could be compensated for by other factors in vivo. IL-6-deficient mice showed a reduced IgG response, but no reduction in the IgM response to either soluble protein or vesicular stomatitis virus (VSV) antigen (Kopf et al., 1994). The mucosal IgA antibody response in IL-6-deficient mice was striking, causing a major reduction in the number of IgAproducing cells (Ramsay et al., 1994). This reduced

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IgA response was completely restored after intranasal infection with recombinant vaccinia viruses engineered to express IL-6. IL-6 is also involved in T-cell activation, growth and differentiation (see reviews by Van Snick, 1990; Houssiau and Van Snick, 1992). IL-6 induces IL-2 receptor (Tac antigen) expression in one T-cell line (Noma et al., 1987) and in thymocytes (Le et al., 1988), and functions as a second signal for IL-2 production by T cells (Garman et al., 1987). IL-6 promotes the growth of human T cells stimulated with PHA (Houssiau et al., 1988; Lotz et al., 1988) or mouse peripheral T cells (Uyttenhove et al., 1988), and also acts on murine thymocytes to induce proliferation (Helle et al., 1988; Le et al., 1988; Uyttenhove et al., 1988). The effects of IL-6 are synergistic with IL-1 and tumor necrosis factor (TNF) (Le et al., 1988). This cytokine enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate (Hodgkin et al., 1988). As IL-6 stimulates thymocyte proliferation and IL-1 can induce IL-6 production in thymocytes (Helle et al., 1989), the effect of IL-1 on thymocyte proliferation is possibly mediated by induced IL-6. After the removal of thymocytes with low buoyant density which are capable of producing IL-6 following stimulation with IL-1, IL-1 cannot induce cell proliferation but IL-6 or IL-2 is still comitogenic; the IL-1-induced proliferation of thymocytes thus seems to depend on endogenous IL-6 production (Helle et al., 1989). A part of the effect of IL-6 on T-cell growth is mediated by endogenously produced IL-2. Anti-IL-2Ra chain (Tac) antibody generally inhibits IL-6-induced T-cell proliferation (Garman et al., 1987; Le et al., 1988; Helle et al., 1989; Kawakami et al., 1989; Tosato et al., 1990). Furthermore, IL-1 and IL-6 synergistically induce IL-2 production (Holsti and Raulet, 1989; Houssiau et al., 1989) and IL-2Ra chain expression in T cells (Houssiau et al., 1989), while IL-6 also induces the differentiation of cytotoxic T lymphocytes (CTLs) in the presence of IL-2 from murine as well as from human thymocytes and splenic T cells (Okada et al., 1988; Takai et al., 1988; Uyttenhove et al., 1988). Purified murine T cells were used to demonstrate that both IL-1 and IL-6 are required for the generation of CTLs and, in this case, induction of the IL-2Ra chain and IL-2 production by IL-1 and IL-6 were critical for CTL generation (Renauld et al., 1989). Finally, IL-6 also induces serine

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esterase and perforin, which are required for mediating target cell lysis in the granules of CTLs (Takai et al., 1988; Liu et al., 1990), which suggests that IL-6 plays a part in the differentiation and expression of cytotoxic T-cell function. In IL-6-deficient mice, the generation of cytotoxic T cells against the vaccinia virus was three- to ten-fold reduced, although the action of CTL against lymphocytic choriomeningitis virus (LCMV) was not reduced (Kopf et al., 1994).

Hematopoiesis IL-6 and IL-3 synergistically induce the proliferation of murine pluripotential hematopoietic progenitors in vitro (Ikebuchi et al., 1987). The combination of IL-6 and IL-3 acts on blast cell colony-forming cells, causing them to exit the G0 stage earlier. IL-6 appears to trigger the entry into the cell cycle of the dormant progenitor cells whereas IL-3 can support continued proliferation of progenitors after they exit from the G0 phase (Ogawa, 1992). The colony-forming units in spleen (CFU-S) increased as the result of culturing bone marrow cells in the presence of both IL-6 and IL-3 (Bodine et al., 1989; Okana et al., 1989). A 6-day culture of these bone marrow cells produced a much higher capability to rescue lethally irradiated mice than could be attained in cells cultured with IL-3 alone. These data indicate that the combination of IL-6 and IL-3 stimulates hematopoietic stem cells in vitro and therefore could be applied to bone marrow transplantation. IL-6 synergizes with macrophage colony-stimulating factor (M-CSF) in the stimulation of colony-forming unit-macrophage (CFU-M) affecting both the number and size of macrophage colonies (Bot et al., 1989). IL-6 has also been found to act synergistically with granulocyte–macrophage colonystimulating factor (GM-CFS) (Caracciolo et al., 1989). Colony-forming units in culture (CFU-C) in the spleen and femur of mice which had been exposed to 750 rads and reconstituted with bone marrow cells increased when IL-6 was injected (Okana et al., 1989). Furthermore, the survival rate of lethally irradiated mice transplanted with 5  104 bone marrow cells increased as the result of IL-6 treatment from 20% to 75% on day 21. The effect of IL-6 was more pronounced if it was administered as a continuous perfusion via an osmotic minipump (Suzuki et al., 1989). An interesting study of IL-6 and the hematopoietic

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system mentions that the defect in hematopoietic differentiation in Fanconi anemia may be due to a deficiency in IL-6 production (Rosselli et al., 1992). Consistent with the possible role of IL-6 in hematopoietic stem cells, IL-6-deficient mice showed a decrease in the absolute number of CFU-Sd12 and pre-CFU-S progenitors and reduced functioning of the long-term repopulating stem cells (Bernad et al., 1994). In vitro megakaryopoiesis is supported by several hematopoietic CSFs. IL-6 was found to induce the maturation of megakaryocytes in synergism with IL-3 (Ishibashi et al., 1989a) and IL-6 promotes marked increments in megakaryocyte size and acetylcholinesterase activity. Furthermore, IL-6 induced a significant shift towards higher ploidy classes. These effects of IL-6 on megakaryocytes have been confirmed elsewhere (Lotem et al., 1989; Williams et al., 1990). The role of IL-6 in megakaryocyte development is further demonstrated by the fact that anti-mouse IL-6 monoclonal antibody inhibits megakaryocyte development in mouse bone marrow cultures in both the absence and presence of IL-3 (Lotem et al., 1989). Human megakaryocytes were seen to express IL-6 receptor and produce IL-6, suggesting that IL-6 may regulate terminal maturation of megakaryocytes in an autocrine manner (Hegyi et al., 1990). IL-6 can also function in vivo. The number of mature megakaryocytes in the bone marrow was increased in IL-6 transgenic mice (Suematsu et al., 1989). Moreover, it was found that administration of IL-6 increased the number of platelets in both mice (Ishibashi et al., 1989b) and monkeys (Asano et al., 1990). The additive or synergistic effect of IL-3 and IL-6 on megakaryocytopoiesis was also demonstrated in mice (Carrington et al., 1991) and monkeys (Geissler et al., 1992). The in vivo effect of IL-6 was consistent with the results obtained in IL-6-deficient mice, which showed a reduction in megakaryocyte progenitors (Bernad et al., 1994). Human and mouse myeloid leukemic cell lines, such as human histiocytic U937 cells and mouse myeloid M1 cells, can be induced to differentiate into macrophages and granulocytes in vitro by several synthetic and natural products. Several factors have been identified that can induce differentiation of leukemic cells, such as G-CSF (Nicola et al., 1983), macrophage–granulocyte inducer type 2 (MGI-2)

( IL - 6 )

(Sachs, 1987), which was found to be identical to IL-6 (Shabo et al., 1988), D-factor (Tomida et al., 1984), and leukemia inhibitory factor (LIF) (Gearing et al., 1987). IL-6 itself can also induce growth inhibition and macrophage differentiation of several human and murine myeloid leukemic cell lines, which suggests that IL-6 may play a part in the final maturation of cells of the granulocyte–monocyte lineage (Miyaura et al., 1988; Shabo et al., 1988; Onozaki et al., 1989; Oritani et al., 1992; Revel, 1992). Consistent with this finding, the predominant cell type of GM-CFU colonies was monoblast–myeloblast in IL-6-deficient mice and macrophage in wild-type mice (Bernad et al., 1994).

Acute-phase response The biosynthesis of acute-phase proteins by hepatocytes is regulated by several factors, including IL-1, TNF and HSF. It was found that recombinant IL-6 can function as HSF (Gauldie et al., 1987) and that the activity of crude HSF can be neutralized by anti-IL-6 (Andus et al., 1987), indicating that HSF activity is exerted by the IL-6 molecule (see reviews by Heinrich et al., 1990; Gauldie et al., 1992). IL-6 can induce a variety of acute-phase proteins, such as fibrinogen, a1-antichymotrypsin, a1-acid glycoprotein and haptoglobin, in the human hepatoma cell line HepG2. In addition to these proteins, it induces serum amyloid A, C-reactive protein (CRP) and a1-antitrypsin in human primary hepatocytes (Castell et al., 1988). The proteins induced in rats by IL-6 comprise fibrinogen, cysteine proteinase inhibitor, a2-macrogloblin and a1-acid glycoprotein (Andus et al., 1987; Gauldie et al., 1987; Heinrich et al., 1990). In vivo administration of IL-6 in rats induces typical acute-phase reactions similar to those induced by turpentine, with the IL-6induced expression of mRNAs for acute-phase proteins being more rapid than that induced by turpentine (Geiger et al., 1988). These results confirm the in vivo effect of IL-6 in the acute-phase reaction. It has also been reported that serum levels of IL-6 correlate well with those of CRP and with fever in patients with severe burns (Nijstein et al., 1987). Furthermore, an increase in serum IL-6 concentration has been observed before an increase in serum CRP levels in patients undergoing surgical operations (Nishimoto et al., 1989; Shenkin et al., 1989). This supports the

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BIOLOGICAL ACTIVITY

concept of a causal role for IL-6 in the acute-phase response. In fact, IL-6-deficient mice show a severely defective inflammatory acute-phase response after tissue damage or infection (Kopf et al., 1994). It is also likely that different patterns of cytokines are involved in systemic and localized tissue damage; IL-6 is an essential mediator of the inflammatory response to a localized inflammation, such as that induced by turpentine, but not to a systemic inflammation as induced by lipopolysaccharide (LPS) (Fattori et al., 1994).

Other activities IL-1 stimulation of glioblastoma or astrocytoma cells was found to induce the expression of IL-6 mRNA (Yasukawa et al., 1987). In addition, virus-infected microglial cells and astrocytes were seen to produce IL-6 (Frei et al., 1989), indicating the possible involvement of IL-6 in nerve cell functions. In fact, IL-6 induces neurite outgrowth of PC12 cells into neural cells (Satoh et al., 1988; Ihara et al., 1996; Wu and Bradshaw, 1996). Furthermore, IL-6 can support the survival of cultured cholinergic neurons (Hama et al., 1989). IL-6 stimulates the secretion of the adrenocorticotrophic hormones either through a corticotrophinreleasing hormone (Naitoh et al., 1988) or directly (Fukata et al., 1989). IL-6 also stimulates the release of a variety of anterior pituitary hormones, such as prolactin, growth hormone and luteinizing hormone (Spangelo et al., 1989). Anterior pituitary cells produce IL-6 spontaneously (Spangelo et al., 1990) as do trophoblasts. The biological significance of IL-6 in the placenta is, however, unknown (Kameda et al., 1990). Because IL-6 stimulates hepatic lipogenesis in mice, and IL-6 is induced by TNF, the lipogenic effects of TNF may in part be mediated by IL-6 (Grunfeld et al., 1990). IL-6 is produced by vascular smooth muscle cells (Loppnow and Libby, 1990) and may induce their growth (Nabata et al., 1990), suggesting the possible involvement of IL-6 in arteriosclerosis. IL-6 is expressed both in adipose tissue and centrally in hypothalamic nuclei with its receptor that involve regulation of body fat. Like leptin, the serum level of IL-6 is known to correlate with body-mass index (BMI) (Mohamed et al., 1997; Fried et al., 1998; Vgontzas et al., 2000). Similarly, it also correlates with

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metabolic parameters other than BMI, such as insulin sensitivity (Bastard et al., 2000). Additionally, IL-6 can enhance the serum level of triglycerides and glucose, and also stimulate the hypothalamic–pituitary– adrenal (HPA) axis, which activity may be important for obesity-associated morbidity (Friedman and Halaas, 1998; Bjorntorp, 1997). These findings suggest high levels of IL-6 in serum may exert pathogenic actions on age-related diseases, such as obesity and atherosclerosis. Taken together, it is conceivable that suppression of IL-6 exhibits favorable effects on these diseases in humans (Staels et al., 1998; McCarty, 1999; Yudkin et al., 1999; Ershler and Keller, 2000). In fact, it has been demonstrated that IL-6-deficient mice develop mature-onset obesity, which is partly reversed by IL-6 replacement, and that these obese mice disturb carbohydrate and lipid metabolism, increased leptin levels and decreased responsiveness to leptin treatment (Wallenius et al., 2002). These findings suggest that IL-6 has anti-obesity effects on rodents. IL-6 may directly or indirectly affect osteoclast development and play a role in postmenopausal osteoporosis, because osteoclast development was enhanced in mice subjected to ovariectomy in order to eliminate the estrogen-induced suppression of IL-6 gene expression. Furthermore, enhancement of osteoclast development in ovariectomized mice was prevented by administration of anti-IL-6 antibody (Jilka et al., 1992), while estrogen was found to inhibit the IL-1- and TNFa-induced production of IL-6 (Girasole et al., 1992). The involvement of IL-6 in ovarectomy-induced osteoporosis has now become evident because ovariectomy in IL-6-deficient mice does not induce any change in either bone mass or bone remodeling rates (Poli et al., 1994). Intraperitoneal injections of either LPS or IL-1b failed to evoke a febrile response in IL-6-deficient mice and this response was restored by the intracerebroventricular injection of recombinant human IL-6, but not of IL-1, demonstrating that IL-6 is an essential component of the febrile response to both IL-1 and LPS (Chai et al., 1996). IL-6 acts as a growth factor for various cells, including plasmacytoma, myeloma, hybridoma, renal cell carcinoma, Kaposi’s sarcoma and keratinocyte (see below). The involvment of IL-6 is essential for the regeneration of hepatocytes because IL-6-deficient mice display impaired liver

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regeneration characterized by liver necrosis and failure (Cressman et al., 1996). On the other hand, IL-6 acts as a growth inhibitor for a number of carcinoma and leukemia cell lines, including breast carcinoma, ovarian carcinoma and myeloleukemic cell lines (Revel, 1992). Inability to eliminate Listeria monocytogenes has been observed in IL-6-deficient mice (Kopf et al., 1994; Dalrymple et al., 1995). This inability is most likely due to that of neutrophils to function in IL-6deficient mice, which suggests that IL-6 plays a critical role in listeriosis via the stimulation of neutrophils (Dalrymple et al., 1995). IL-6-deficient mice show an increased susceptibility to Escherichia coli infection and are unable to induce neutrophilia following a challenge with E. coli (Dalrymple et al., 1996). Furthermore, IL-6-deficient mice are more susceptible than wild-type mice to virulent Candida albicans. Impairment of macrophages, neutrophils and T helper 1 (TH1)-associated protective immunity has been observed in IL-6-deficient mice (Romani et al., 1996). These findings indicate that IL-6 plays a part in the function of macrophages and neutrophils in vivo.

STRUCTURE OF THE IL-6 PROTEIN AND ITS GENE IL-6 is a glycoprotein with a molecular mass ranging from 21 to 28 kDa. Posttranslation modifications include N- and O-linked glycosylations and phosphorylations (May et al., 1988, Santhanam et al., 1989). Human IL-6 consists of 212 amino acids including a hydrophobic signal sequence of 28 amino acids (Hirano et al., 1986), and shows homology with IL-6 from the mouse and rat by 65% and 68% at the DNA level and 42% and 58% at the protein level, respectively (Van Snick et al., 1988; Northemann et al., 1989). The mouse and rat protein sequences are 93% identical (Van Snick et al., 1988; Northemann et al., 1989). Both the C- and the N-terminus play a critical role in its biological function (Brakenhoff et al., 1989; Ida et al., 1989; Kruttegen et al., 1990). A computer-aided structural analysis predicts that IL-6 consists of four antiparallel helices with two long and one short loop connections, similar to the model of other cytokines, including growth hormone, prolactin, erythropoietin, IL-2, IL-4, G-CSF and GM-CSF, LIF, oncostatin M

( IL - 6 )

(OSM) and ciliary neurotrophic factor (CNTF) (Bazan, 1992). The evidence suggests an evolutionary relationship among these molecules acting in the immune, hematopoietic, endocrine and nervous systems. The human and mouse IL-6 gene are approximately 5 and 7 kilobases in length, and both consist of five exons and four introns (Yasukawa et al., 1987; Tanabe et al., 1988). The genomic gene for human IL-6 is mapped to chromosome 7p21 and its murine counterpart to chromosome 5 (Sehgal et al., 1986; Bowcock et al., 1988; Mock et al., 1989). The sequence similarity in the coding region of human and mouse IL-6 genes is about 60%, whereas the 3 untranslated region and the first 300 base pairs of the 5 flanking region are highly conserved (80%) (Tanabe et al., 1988). The production of IL-6 is regulated by a variety of stimuli. IL-6 production is induced in T cells or T-cell clones by T-cell mitogens or antigenic stimulation (Van Snick et al., 1987; Hodgkin et al., 1988; Horii et al., 1988; Espevik et al., 1990). LPS enhances IL-6 production in monocytes and fibroblasts, whereas glucocorticoids inhibit it (Helfgott et al., 1987; Sehgal, 1992). Various viruses induce IL-6 production in fibroblasts (Sehgal et al., 1988; Van Damme et al., 1989) or in the central nervous system (Frei et al., 1988). Human immunodeficiency virus also induces IL-6 production (Nakajima et al., 1989; Breen et al., 1990; Emilie et al., 1990). A variety of peptide factors, such as IL-1, TNF, IL-2, IFNb and platelet-derived growth factor (PDGF) (Content et al., 1985; May et al., 1986; Wong and Goeddel, 1986; Zilberstein et al., 1986; Kohase et al., 1987; Van Damme et al., 1987a,b; Kasid et al., 1989), protein kinase C (Sehgal et al., 1987b), calcium ionophore A23187 (Sehgal et al., 1987b) and various agents, which cause an increase in intracellular cyclic AMP levels (Zhang et al., 1988a,b), also induce IL-6 production. In contrast, IL-4 and IL-13 inhibit IL-6 production in monocytes (Gibbons et al., 1990; Velde et al., 1990; Minty et al., 1993). Several potential transcriptional control elements, such as glucocorticoid-responsive elements (GRFs), an activating protein-1 (AP-1) binding site, a c-fos serum-responsive element (c-fos SRE) homology site, a c-fos retinoblastoma control element (RCE) homology site, a cAMP-responsive element (CRE) site and a nuclear factor (NF)jB binding site have been identified within the conserved region of the IL-6 promoter

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STRUCTURE OF THE IL - 6 PROTEIN AND ITS GENE

(Ray et al., 1988, 1989; Tanabe et al., 1988; Sehgal, 1992), as shown in Plate 12.1. Of these elements, c-fos SRE and AP-1-like elements appear to contain the major cis-acting regulatory factors that induce responsiveness to several reagents (including serum, forskolin and phorbol ester) upon the heterologous herpes virus thymidine kinase (TK) promoter (Ray et al., 1989). The 23-bp oligonucleotide designated as multiple response element (MRE) within the IL-6 enhancer region (173 to 151) contains a CGTCA motif which binds nuclear proteins. A single copy of MRE inserted upstream of the herpes virus TK promoter renders this heterologous promoter inducible by IL-1a, TNF and serum, as well as by activators of protein kinase A (forskolin) and protein kinase C (phorbol ester). The IL-1-responsive element was also mapped within the region from 180 to 111 bp of the IL-6 gene and a nuclear factor, NF-IL6 CCAAT/enhancer binding protein b (C/EBP-b), was identified which binds specifically to a 14-bp palindrome (Akira et al., 1990; Isshiki et al., 1990). Shimizu et al. (1990) showed that the NFjB binding motif located between 73 and 63 bp relative to the mRNA cap site is required for IL-1/ TNFa-induced expression of the IL-6 gene. Libermann and Baltimore (1990) and Zhang et al. (1990) also demonstrated the involvement of an NFjB-like molecule in IL-6 gene expression. In fact, the antisense oligonucleotide of NFjB inhibits the expression of IL-6 mRNA in tumor cells derived from human T-lymphotropic virus-1 tax transgenic mice (Kitajima et al., 1992) and p40 tax induces IL-6 mRNA through the NFjB binding site and simultaneously induces the NFjB binding protein (Muraoka et al., 1993). The involvement of NFjB is also implicated in IL-6 gene induction by non-structural regulatory protein 1 (NS-1) of human parvovirus B19 (Moffatt et al., 1996). In monocytic cell lines, the NFjB site is crucial for LPS-induced IL-6 gene expression (Dendorfer et al., 1994; Sanceau et al., 1995). Synergistic induction of the IL-6 gene in monocytic cells by IFNc and TNFa, involves cooperation between the interferon regulatory factor-1 (IRF-1) and NFjB p65 homodimers in association with simultaneous removal of the negative effects of the retinoblastoma control element (Sanceau et al., 1995). NFjB is also involved in CD40-mediated IL-6 gene expression (Hess et al., 1995). Although the NFjB site functions as a potent IL-1/TNF-responsive element in non-lymphoid cells,

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its activity is repressed in lymphoid cells while NFjB binding factor containing c-Rel is thought to act as a repressor in lymphoid cells (Nakayama et al., 1992). p53 and retinoblastoma (RB) also repress the IL-6 gene promoter, although the biological significance of this finding remains to be evaluated (Sehgal, 1992).

The IL-6 cytokine receptor superfamily The IL-6 receptor consists of two molecules, an 80-kDa IL-6 binding protein (a chain) and a 130-kDa signal transducer, gp130 (b chain) (Yamasaki et al., 1988; Taga et al., 1989; Hibi et al., 1990) (Plate 12.2). Although IL-6 cannot directly bind to gp130, it can bind to IL-6R to generate the high-affinity complex of IL-6/IL-6R/gp130. Many cytokine receptors, including the receptors for growth hormone, CNTF, IL-2, erythropoietein, G-CSF and IL-5, are similar in structure to IL-6Ra and constitute the type I cytokine receptor superfamily (Bazan, 1990, 1992). The most striking features of these receptors are the conservation of four cysteine residues and a tryptophan–serine–X–tryptophan–serine (W–S–X–W–S) motif (WS motif) located just outside the transmembrane domain. The cytoplasmic domain of IL-6Ra is not required for IL-6-mediated signal transduction (Taga et al., 1989; Hibi et al., 1990). Insertion of the intracisternal A particle gene-long terminal repeat (IAP-LTR) in the cytoplasmic domain of murine IL-6Ra is found in a mouse plasmacytoma, which abundantly expresses an abnormal but functional IL-6Ra whose cytoplasmic domain is replaced with an IAP sequence (Sugita et al., 1990). Furthermore, even the complex of IL-6 and soluble IL-6Ra can generate IL-6-mediated signal transduction (Taga et al., 1989; Hibi et al., 1990). The binding of IL-6 to IL-6Ra and gp130 induces the formation of a hexamer composed of two members each (Plate 12.3) (Paonessa et al., 1995). IL-6 has three topologically distinct receptor binding sites: site 1 around Arg-179 on helix D is the binding site for IL-6Ra (Savino et al., 1993); site 2, composed of residues from helix A and C, is the binding site for one gp130; and site 3, centred around the N-terminal end of helix D, is the binding site for another gp130 (Brakenhoff et al., 1994; Savino et al., 1994a,b) (Plate 12.3). Furthermore, residues around Asn-230 and His-280 of IL-6Ra are involved in the interaction

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between IL-6Ra and gp130 (Yawata et al., 1993; Salvati et al., 1995). Actually, substitutions at Asn-230, His-280 and Asp-281 were found to impair the capability of soluble IL-6Ra to associate with gp130, without affecting its affinity for IL-6, and to antagonize IL-6 bioactivity (Salvati et al., 1995). As both sites 2 and 3 of IL-6 are required for the dimerization of gp130, which is essential for signal transduction, IL-6 variants carrying amino acid substitutions in either site 2 or site 3, or both, function as inhibitors of IL-6 (Sporeno et al., 1996). Redundancy of activity is another feature of cytokines. For example, IL-6, LIF and OSM all induce macrophage differentiation in the myeloid leukemia cell line, M1 (Miyaura et al., 1988; Shabo et al., 1988; Metcalf, 1989; Rose and Bruce, 1991; Oritani et al., 1992) and acute-phase protein synthesis in hepatocytes (Andus et al., 1987; Baumann et al., 1987; Gauldie et al., 1987; Baumann and Wong, 1989, Richards et al., 1992). One of the important findings regarding cytokine receptors is that one constituent of a certain cytokine receptor is shared by several other cytokine receptors, as was demonstrated for GM-CSF, IL-3 and IL-5 receptor systems (Miyajima et al., 1992). Another example is gp130, which is shared by the receptors for CNTF, LIF, OSM, IL-11 and CT-1, as illustrated in Plate 12.4 (Ip et al., 1992; Kishimoto et al., 1995). Furthermore, the b chain of the IL-2 receptor (IL-2Rbc–c) is shared by the IL-15 receptor, and the c chain of the IL-2 receptor (cc) is shared by the IL-4, IL-7, IL-9, IL-15 and IL-21 receptors (Sugamura et al., 1995; Taniguchi, 1995). Thus, the molecular mechanisms of redundancy in cytokine activity can be explained, at least in part, by the sharing of receptor subunits among several cytokine receptors.

Mechanism for generating cytokine diversity Investigations of the IL-6R system have provided evidence that a complex of IL-6 and a soluble form of IL-6Ra can act on cells that express gp130 but not IL-6Ra (Fig.5). Another molecule that acts in a similar manner on soluble IL-6Ra–IL-6 complexes is IL-12, which consists of a disulphide heterodimer of 40-kDa (p40) and 36-kDa (p35) subunits (Wolf et al., 1991). p35 itself is a helical cytokine and p40 shows similarity to the soluble form of IL-6Ra (Gearing et al., 1991).

( IL - 6 )

IL-12 is therefore a complex of a cytokine and a soluble form of its presumed receptor, which implies that the p40–p35 heterodimer can act through IL-12R, which is most closely related to gp130 (Chua et al., 1994). Another example is CNTFRa, which is anchored to the cell membrane by a glycosylphosphatidyl inositol (GPI) linkage (Davis et al., 1991), while the complex of soluble CNTFRa and CNTF acts on the cells that express LIFRb and gp130 (Davis et al., 1993). Potential physiological roles for the soluble CNTFRa are suggested by the presence of the soluble form of the a chain in cerebrospinal fluid and its release from skeletal muscle in response to peripheral nerve injury. On the basis of these findings, Kishimoto et al. (1995) proposed a novel mechanism by which the cytokine system generates functional diversity (Plate 12.5). A complex consisting of a soluble cytokine receptor and its corresponding cytokine acquires different target specificity from the original cytokine and, therefore, it should express different functions from those of the original cytokine. In fact, double transgenic mice expressing human IL-6 and IL-6Ra showed myocardial hypertrophy (Hirota et al., 1995), indicating that the complex of IL-6 and the soluble form of IL-6Ra acts on heart muscle cells that express gp130, an action which IL-6 alone cannot exert. This action leads to the induction of cardiac hypertrophy, so that the effect is similar to that of cardiotrophin-1 (CT-1). This model could also be applied to the glial cell line-derived neurotrophic factor (GDNF) receptor system, which consists of a GDNF-specific binding molecule, GDNFRa, a GPI-anchored membrane molecule and a signal transducing GDNFR, Ret, which is a receptor for tyrosine kinase (Jing et al., 1996; Treanor et al., 1996). In addition, this novel mechanism may function in a wide range of other receptor systems. It may contribute to the generation of the functional diversity of cytokines and may also play a pathological role in various diseases, in which an increase in the serum-soluble form of various cytokine receptors has been reported.

THE CYTOKINES AND CHEMOKINES

JAK – STAT SIGNAL TRANSDUCTION PATHWAY THROUGH THE IL - 6 RECEPTOR

JAK–STAT SIGNAL TRANSDUCTION PATHWAY THROUGH THE IL-6 RECEPTOR As the cytoplasmic domain of most cytokine receptors, including gp130, does not have an intrinsic catalytic domain, one of the most controversial issues until 1993 was the identification of catalytic molecules that associate with cytokine receptors. This issue was resolved by the finding that several Janus family tyrosine kinases (JAK1, JAK2, JAK3, Tyk-2) are involved in the signal transduction of cytokines and hormones. Furthermore, the signal transducer and activator of transcription (STAT) was found to play a central role in a variety of cytokine signal transduction pathways (Darnell et al., 1994; Ihle et al., 1994; Schindler and Darnell, 1995). JAK1, JAK2 and Tyk-2 associate constitutively with gp130 and are tyrosine phosphorylated in response to IL-6, CNTF, LIF or OSM (Lutticken et al., 1993; Stahl et al., 1993; Matsuda et al., 1994). Furthermore, IL-6 activates STAT3, STAT1 and STAT5 (Akira et al., 1994; Zhong et al., 1994; Lai et al., 1995; Nakajima et al., 1996). In the absence of JAK1, the activation of transcriptional factor STATs following stimulation by IL-6 is not effective, although both JAK2 and Tyk-2 are activated. This suggests that there is a hierarchy among gp130-associated JAKs (Guschin et al., 1995). Two types of IL-6-responsive element (RE) have been identified in the genes encoding acute-phase proteins. The presence of type I IL-6 RE, which is a binding site for NF–IL-6/IL-6DBP/LAP/C/EBPb (Akira et al., 1990; Poli et al., 1990; Cao et al., 1991; De Groot et al., 1991), has been comfirmed in the CRP, hemopexin A and haptoglobin genes. The binding activity of NF–IL-6 is probably induced by IL-6 via the increased expression of the NF–IL-6 gene, rather than through its posttranslational modification (Baumann et al., 1992, 1993). Type II IL-6 RE is contained in the fibrinogen, a2-macroglobin, a1–acid glycoprotein and haptoglobin genes. IL-6 triggers the rapid activation of a nuclear factor, known as acute-phase response factor (APRF), which binds to type II IL-6 RE (Wegenka et al., 1993). The purification and molecular cloning of APRF revealed that it is identical to STAT3 (Akira et al., 1994; Zhong et al., 1994).

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In parallel with the results of these studies, Nakajima et al. (1993) identified the IL-6-responsive element of the junB gene (JRE–IL-6), which consists of a putative Ets binding site (JEBS) and a CRE-like site. The IL-6-inducible JEBS binding protein contains mainly STAT3, although the JEBS is a low-affinity binding site for STAT3 (Nakajima et al., 1996). IL-6 induces the formation of a complex consisting of STAT3 and p36-CRE-like site binding molecules on the JRE–IL-6 and on the IL-6/IFNc RE in the IRF-1 promoter (Kojima et al., 1996). Such binding complex formations seem to be important for STAT to act on low-affinity binding sites, such as the JEBS, and may contribute to the diversity of target genes of STAT proteins. In addition to the tyrosine phosphorylation of STAT3 by JAK tyrosine kinase, an H7-sensitive pathway, most likely a serine/threonine kinase, is required for STAT3 to become transcriptionally active on both the JRE–IL-6 and type II IL-6 RE (Nakajima et al., 1993). In certain cell lines, STAT3 requires phosphorylation on serine to form a STAT3–STAT3 homodimer (Zhang et al., 1995). Maximal activation of transcription by STAT1 and STAT3 requires both tyrosine and serine phosphorylation in line with the involvement of a serine/threonine kinase in the STAT signal pathway (Wen et al., 1995).

Multiple signal transduction pathways through gp130 involved in cell growth, differentiation and survival Human gp130 has 277 amino acid residues in its cytoplasmic domain, which contains two motifs conserved among the cytokine receptor family, termed Box1 and Box2 (Plate 12.2) (Hibi et al., 1990; Fukunaga et al., 1991; Murakami et al., 1991). The membraneproximal region containing Box1 and Box2 is sufficient for the activation of JAK through gp130 (Narazaki et al., 1994). Human gp130 has six tyrosine residues in its cytoplasmic domain, and the tyrosine phosphorylation of Src homology 2 protein tyrosine phosphatase-2 (SHP-2) (also called protein tyrosine phosphatase-1D (PTP1-D), SHPTP-2, PTP2C and Syp), a phosphotyrosine phosphatase, and that of STAT3 depend on the second tyrosine from the membrane (Y2), and any one of the four tyrosines (Y3, Y4, Y5, Y6) in the carboxy terminus that have a glutamine residue at the third position behind tyrosine

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(Y–X–X–Q) (Plate 12.6) (Stahl et al., 1995; Yamanaka et al., 1996). Since STAT3 is involved in the activation of type II acute-phase genes, the membrane-proximal region of gp130, containing 133 amino acids and Y3, is necessary for the activation of IL-6 responsive acutephase genes (Baumann et al., 1994). IL-6 induces growth arrest and macrophage differentiation in the murine myeloid leukemic cell lines M1 and Y6 (Miyaura et al., 1988; Shabo et al., 1988; Oritani et al., 1992). The membrane-proximal region of gp130, consisting of 133 amino acids, is sufficient for the generation of the signals for growth arrest, macrophage differentiation, down-regulation of cmyc and c-myb, induction of junB and IRF-1, and the activation of STAT3 (Yamanaka et al., 1996). The region between amino acids 108 and 133 contains two tyrosine residues (Plates 12.2 and 12.6): one (Y3) at amino acid position 126 with the YXXQ motif, and the other (Y2) without the motif at amino acid position 118. Y2 is essential for gp130-mediated egr-1 gene induction (Yamanaka et al., 1996), whereas Y3 is critical for generating the signals not only for STAT3 activation but also for growth arrest and differentiation associated with the down-regulation of c-myc and c-myb and the immediate–early induction of junB and IRF-1. These results show the close correlation between STAT3 activation and the signals for growth arrest and differentiation. In fact, dominant-negative forms of STAT3 inhibit both IL-6-induced growth arrest and macrophage differentiation in M1 transformants (Nakajima et al., 1996). Blocking STAT3 activation inhibits IL-6-induced repression of c-myb and c-myc, but not egr-1 induction. Furthermore, IL-6 enhances the growth of M1 cells when STAT3 is suppressed. Thus, IL-6 generates both growth-enhancing signals and growth arrest and differentiationinducing signals at the same time, but the former is apparent only when STAT3 activation is suppressed. The essential role of STAT3 in the IL-6-induced macrophage differentiation of M1 cells has also been demonstrated (Minami et al., 1996). For the growth signal, it was shown that a 65 amino acid region proximal to the transmembrane domain is sufficient for generating a growth response by using gp130 transfectants of an IL-3-dependent pro B-cell line BAF/BO3 (Murakami et al., 1991; Kishimoto et al., 1995). However, the membrane-proximal region of 68 amino acids is not sufficient for the induction of

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tritium thymidine (3H-Tdr) uptake when cells are starved of IL-3. The membrane-proximal region containing 133 amino acid residues is both required and sufficient for cell growth (Fukada et al., 1996). Furthermore, at least two distinct signals are required for gp130-induced cell growth: a cell cycle progression signal dependent on the second tyrosine residue, Y2, and possibly mediated by SHP-2; and an antiapoptotic signal dependent on the third tyrosine residue, Y3, and mediated by STAT3 through Bc1-2 induction. However, a recent study using mice with STAT3 deficient in a T cell-specific manner has revealed that STAT3 activation is involved in IL-6dependent T-cell proliferation through prevention of apoptosis without the need for Bcl-2 induction (Takeda et al., 1998). Thus, STAT3 plays pivotal roles in gp130-mediated signal transduction regulating cell growth, differentiation and survival. In addition to the JAK–STAT signal transduction pathway, the Ras– mitogen-activated protein (MAP) kinase pathway is also activated through SHP-2 (Fukada et al., 1996) or Shc (Kumar et al., 1994). Furthermore, non-receptor tyrosine kinases, such as Btk, Tec, Fes and Hck (Ernst et al., 1994; Matsuda et al., 1995a,b) are activated through the IL-6 receptor, as well as through a variety of other cytokine receptors (Taniguchi, 1995), although the biological significance of these signal transduction pathways remains to be clarified. As summarized in Plate 12.6, several distinct signal transduction pathways are generated through different regions of the cytoplasmic domain of gp130. The expression pattern of these signaling molecules determines which set of signaling pathways is activated in a given cell. Furthermore, these signaling pathways may interact with each other and contribute to a variety of biological activaties. In fact, a recent study has reported that knock-in mutation mice lacking SHP-2 signal showed sustained gp130-induced STAT3 activation, which indicates a negative regulatory role of SHP-2 for STAT3 activation (Ohtani et al., 2000) (Plate 12.7). Moreover, these knock-in mice displayed splenomegaly and lymphoadenopathy and an acutephase reaction. In contrast, all known STAT3 binding site-deficient mice, like the gp130-deficient mice, died perinatally (Ohtani et al., 2000). However, it has also been reported that the STAT3 signal-deficient mice displayed a severe joint disease in association with mitogenic hyperresponsiveness of the synovial

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cells to the IL-6 family cytokines as a result of sustained gp130-mediated SHP-2 activation due to a lack of the SHP-2 inhibitor induced by STAT3 (Ernst et al., 2001).

New inhibitors of IL-6 signaling Cytokine signaling including IL-6 is negatively regulated with respect to both magnitude and duration. Recently, it has become clear that at least two new families of inhibitors contribute to the negative regulation of IL-6 signaling: the suppressor of cytokine signaling (SOCS) and the protein inhibitors of activated STATs (PIAS). SOCS-1, also known as SSI-1 (STATinduced STAT inhibitor-1) or JAB-1 (JAK binding protein-1), was described in 1997 as a negative regulatory molecule of IL-6 signaling on the basis of its binding to JAK (Starr et al., 1997; Naka et al., 1997; Endo et al., 1997). Database searches have shown that the SOCS family now includes eight members (CIS and SOCS1–SOCS7), all of which are characterized by a central SH2 domain flanked by an N-terminal region containing a conserved motif known as a SOCS box (Starr et al., 1997; Minamoto et al., 1997; Masuhara et al., 1997; Hilton et al., 1998). The mRNAs of SOCS-1, SOCS-2 and SOCS-3 are induced by various cytokines including IL-4, IL-6, IFNc, G-CSF and several other members, and they have been shown to inhibit cytokine-activated JAK/STAT signal pathways (Naka et al., 1999; Yasukawa et al., 2000; Krebs and Hilton, 2001). However, the factors that induce mRNA of other SOCS families, such as SOCS-4, SOCS-5, SOCS-6 and SOCS-7, have not been clarified and their functions have not been thoroughly characterized. SOCS-1 and SOCS-3 are especially well known as inhibitors of IL-6 signaling (Nicholson et al., 1999). However, they act as inhibitors of IL-6 signaling through different mechanisms (Plate 12.8). SOCS-1 directly interacts with JAKs, and thus inhibits their catalytic activity (Naka et al., 1997; Endo et al., 1997; Narazaki et al., 1998; Nicholson et al., 1999; Yasukawa et al., 1999; Fujimoto et al., 2000). SOCS-3 also inhibits JAK activity, but, compared with SOCS-1, it only partially inhibits JAK activity although its effect is augmented in the presence of receptors, which suggests that SOCS-3 inhibits cytokine signaling by binding to the receptor complex (Hansen et al., 1999). In the IL-6 signal pathway, an SHP-2 interaction site of gp130 is

also a SOCS-3 contact site, so that SOCS-3 may compete for the SHP-2–gp130 interaction site (Nicholson et al., 1999; Schmitz et al., 2000). SOCS-1-deficient mice have an intact IL-6 signaling pathway (Alexander et al., 1999), suggesting that SOCS-3 may act as a crucial inhibitor of IL-6 signaling. Unlike the SOCS family, PIAS constitute a family of constitutively expressed negative regulators of STATs (Plate 12.8). Five members of this family have been identified with the yeast two-hybrid method and by searching the expressed sequence tag (EST) database: PIAS-1, PIAS3, PIAS-Xa, PIAS-Xb and PIAS-Y (Chung et al., 1997; Liu et al., 1998). They all share homology and contain several highly conserved domains, including a putative zinc-binding motif and a highly acidic region. PIAS-1 and PIAS-3 have been identified as specific inhibitors of STAT signaling pathways (Chung et al., 1997; Liu et al., 1998). Overexpression studies have shown that PIAS-1 associates only with activated STAT1 dimers and inhibits their DNA-binding activity, but that no monomeric forms of STAT1 are present (Liu et al., 1998). Similarly, PIAS-3 associates specifically with activated STAT3 but not STAT1, resulting in the blocking of all STAT3-mediated gene transcriptions (Chung et al., 1997). PIAS-3 is especially well known as an inhibitor of IL-6 signaling in M1 cell lines (Chung et al., 1997). The constitutive expression of PIAS proteins implies that their physiological role differs from that of SOCS proteins, which are induced by cytokine stimulation. To date, however, the differences in the physiological roles of these two family proteins are not well known.

DISEASE The possible involvement of the deregulated expression of the IL-6 gene in polyclonal B-cell abnormalities was first demonstrated in patients with cardiac myxoma (Hirano et al., 1987). Since then, much evidence has been accumulated indicating that the deregulated production of IL-6 could be involved in a variety of diseases, including inflammation, autoimmune disease and malignancy. Considering the possible involvement of IL-6 in such diseases, it seems worth noting that IL-6 was first identified as virusinduced IFNb2 (Weissenbach et al., 1980) as well as a factor found in the culture supernatants of cells

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infiltrating the pleural effusion of patients with tuberculous pleurisy, capable of inducing immunoglobulin production in activated human B cells (Teranishi et al., 1982).

Interleukin-6 and B-cell abnormalities in chronic inflammation Patients with cardiac myxoma display a variety of autoimmune symptoms, such as hypergammaglobulinemia, presence of autoantibodies and an increase in acute-phase proteins, all of which disappear following resection of the tumor cells. Involvement of IL-6 in B-cell abnormalities was first suggested in patients with cardiac myxoma (Hirano et al., 1987; Jourdan et al., 1990). Previous to this, it was demonstrated that pleural effusion cells of patients with pulmonary tuberculosis, when stimulated with purified protein derivative (PPD), produced a large amount of factors capable of inducing immunoglobulin production in activated normal B cells (Teranishi et al., 1982). One of these factors was identified as IL-6 (previously called either BCDF-II or BSF-2) (Teranishi et al., 1982; Yoshizaki et al., 1982). It is worth noting that patients with pulmonary tuberculosis often have a wide range of autoantibodies (Shoenfeld and Isenberg, 1988), and in certain cases significant diffuse hypergammaglobulinemia has been reported (Sela et al., 1987). All this evidence indicates that overproduction of IL-6 may play a critical role in autoimmune disease (Hirano et al., 1987). Abnormal IL-6 production was also observed in patients with Castleman’s disease (Yoshizaki et al., 1989) and rheumatoid arthritis (Hirano et al., 1988; Houssiau et al., 1988; Bhardwaj et al., 1989). Furthermore, IL-6 production was seen to occur in type II collagen-induced arthritis in mice (Takai et al., 1989) and MRL/1pr mice (Tang et al., 1991), which develop autoimmune disease with proliferative glomerulonephritis and arthritis. IL-6 was also found to be produced by islet b cells and the thyroid (Bendtzen et al., 1989; Campbell et al., 1989), suggesting that, by enhancing the response of autoreactive T cells, IL-6 may be involved in type I diabetes (Campbell and Harrison, 1990). This evidence suggests that IL-6 plays a role in autoimmune disease, although IL-6 alone may not be sufficient for its generation

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(Hirano, 1992b). The observation that the anti-IL-6 antibody inhibits the development of insulindependent diabetes in NOD/WEHI mice seems to support the concept that IL-6 plays a role in autoimmune disease (Campbell et al., 1991). Other interesting evidence is that a striking increase in the prevalence of agalactosy1 IgG has been observed in a variety of autoimmune and/or IL-6-related diseases, such as pulmonary tuberculosis, rheumatoid arthritis, Crohn disease, sarcoidosis, leprosy, Castleman’s disease, Takayasu’s arteritis, multiple myeloma and pristane-induced arthritis (Nakao et al., 1991; Rook et al., 1991; Rook and Stanford, 1992). In accordance with these facts, IL-6 transgenic mice showed an increase in agalactosyl IgG activity (Rook et al., 1991), providing further evidence of the close relationship between IL-6 and certain autoimmune diseases.

Chronic inflammatory proliferative disease Glomerulonephritis is commonly accompanied by a variety of autoimmune diseases, and several growth factors have been suggested as candidates that may induce the pathological growth of mesangial cells. IL-6 is a possible autocrine growth factor for rat mesangial cells (Horii et al., 1989; Ruef et al., 1990). It is produced by renal mesangial cells in patients with mesangial proliferative glomerulonephritis (Horii et al., 1989). IL-6 is detected in urine samples from patients with mesangial proliferative glomerulonephritis, but not from those with other types of glomerulonephritis. A correlation has been established between the levels of IL-6 in urine and the progressive stage of mesangial proliferative glomerulonephritis. Other chronic proliferative diseases that may be related to IL-6 are psoriasis (Grossman et al., 1989) and Kaposi’s sarcoma (Miles et al., 1990), with IL-6 being considered as one of the growth factors for keratinocytes and Kaposi’s sarcoma cells. Because mesangial cell proliferative glomerulonephritis, psoriasis and Kaposi’s sarcoma are diseases in which abnormal cell growth and inflammatory and/or immunological reactions are active, they may be categorized as chronic inflammatory proliferative diseases. From this point of view, rheumatoid arthritis

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(RA) is also included in this disease category, because one of its prominent features is chronic expansion of synovial cells. RA is a major immune-mediated disease of unknown cause. Nevertheless, previous evidence suggests that cytokines are important mediators for the pathology of RA (Feldmann et al., 1996), especially the so-called proinflammatory cytokines, such as IL-1, TNFa, and IL-6, which play a pivotal role in the pathology of RA. Ohshima et al. (1998) demonstrated by using IL-6 deficient mice, that IL-6 is essential for the development of antigen-induced arthritis (AIA), which is an experimental autoimmune arthritis model histologically resembling RA. Furthermore, Crohn disease (CD) is also a chronic inflammatory disease of the gastrointestinal tract characterized by increased resistance of mucosal T cells to apoptosis. Proinflamatory cytokines have been demonstrated to play a crucial role in the pathogenesis of this disease (Fuss et al., 1996), with enhanced expression of IL-6 having been repeatedly observed (Gross et al., 1992; Mitsuyama et al., 1995). A neutralizing antibody for IL-6R could cause suppression by inducing apoptosis of lamina propria T cells, an established experimental colitis in an animal model of CD mediated by TH1 cells (Yamamoto et al., 2000; Atreya et al, 2000).

Plasma cell neoplasia IL-6 is a potent growth factor for murine plasmacytoma cells (Aarden et al., 1985; Van Damme et al., 1987a; Van Snick et al., 1988) and human myeloma cells (Kawano et al., 1988), which suggests the possible involvement of IL-6 in the generation of plasmacytoma or myeloma (Hirano, 1991). A significant association has been found between the occurrence of plasma cell neoplasias and chronic inflammation (Isobe and Osserman, 1971; Isomaki et al., 1978). Plasmacytomas can be induced in BALB/c mice by mineral oil or pristane, both of which are potent inducers of chronic inflammation and IL-6 biosynthesis (Potter and Boyce, 1962; Nordan and Potter, 1986). The in vitro growth of the primary mouse plasmacytoma thus developed was found to be dependent on IL-6 (Namba and Hanaoka, 1972). IL-6 was also identified as a possible autocrine growth factor for human myeloma cells (Kawano et al., 1988). Freeman et al. (1989) demonstrated that

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myelomas and plasma cell leukemias express IL-6 mRNA, while cytoplasmic IL-6 was detected in myeloma cells of the bone marrow by light and electron microscopy (Ohtake et al., 1990). It was reported that the growth-inducing activity of IL-1 or TNF on freshly isolated myeloma cells could be due to an IL-6-mediated autocrine mechanism (Carter et al., 1990). Rearrangement of the IL-6 gene has been reported in some myeloma cells that express the IL-6 gene (Fiedler et al., 1990). Constitutive IL-6 production in a murine plasmacytoma cell line resulting from the insertion of an IAP retrotransposon in the IL-6 gene has also been reported (Blankenstein et al., 1990). Expression of the IL-6 gene in an IL-6-dependent murine plasmacytoma cell line caused the cells to proliferate in an autocrine manner (Tohyama et al., 1990; Vink et al., 1990). These cells displayed greatly enhanced tumorigenicity. Monoclonal antibodies capable of blocking the binding of IL-6 to its receptor inhibited their growth in vivo (Vink et al., 1990). IL-6 was also found to be an autocrine growth factor for EBV-transformed B-cell lines (Yokoi et al., 1990) and expression of an exogenous IL-6 gene in these B-cell lines conferred growth advantage and in vivo tumorigenicity (Scala et al., 1990). However, there is no consensus on whether all myeloma cells produce IL-6 because only some myeloma cell lines have been found to produce it (Kawano et al., 1988; Klein et al., 1989; Shimizu et al., 1989; Hata et al., 1990). Bone marrow adherent cells but not bone marrow non-adherent cell populations containing myeloma cells were demonstrated to be major producers of IL-6 (Klein et al., 1989). Evidence suggests that IL-6 plays an important role in the in vivo growth of myeloma cells and generation of plasma cell neoplasia in an autocrine or paracrine manner. This concept has been supported by the following findings. The in vitro IL-6 responsiveness of myeloma cells obtained from patients with multiple myeloma was found to correlate directly with the in vivo labeling index of these tumors (Zhang et al., 1989) and an increase in serum IL-6 levels showed good correlation with disease severity in multiple myeloma and plasma cell leukemia (Bataille et al., 1989). Finally, administration of anti-IL-6 antibodies resulted in the suppression of myeloma cell growth (Klein et al., 1991). However, IL-6 alone is not sufficient for the

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generation of plasmacytoma, since plasma cells generated in IL-6 transgenic mice were not transplantable to syngeneic animals. This indicates that additional factors may be required for malignant transformation. An interesting finding is that C57BL/6 IL-6 transgenic mice, when backcrossed to BALB/c mice, showed progression from polyclonal plasmacytosis to fully transformed monoclonal plasmacytoma which contained chromosomal translocations combined with c-myc gene rearrangement (Suematsu et al., 1992). This strongly supports the hypothesis that deregulated expression of the IL-6 gene can trigger polyclonal plasmacytosis, resulting in the generation of malignant monoclonal plasmacytoma (Hirano, 1991). Further support is provided by the finding that in IL-6-deficient mice pristane cannot induce plasmacytoma (Hilbert et al., 1995).

Clinical trials of anti-IL-6 receptor antibody As mentioned previously, continuous overproduction of IL-6 is observed in patients with some chronic inflammatory diseases such as RA and Castleman’s disease, which are frequently associated with similar abnormalities as those seen in IL-6 transgenic mice (Suematsu et al., 1989). Anti-IL-6 antibody has been used to block IL-6 signal transduction as a therapeutic strategy in animal models. As mentioned before, IL-6 transgenic mice displayed hypergammaglobulinemia with massive plasma cell infiltration in the lymph nodes and spleen, lymphocytic interstitial pneumonia with lymphocyte infiltration in lung and mesangial proliferative glomerulonephritis with proteinuria and hematuria. Treatment with a rat anti-mouse IL-6 receptor antibody known as MR16-1 completely prevented these lesions in many organs. Similarly, all the other abnormalities observed in the IL-6 transgenic mice, such as increase in serum fibrinogen, serum amiloyd A (SAA) and platelet counts and decreases in serum albumin were ameliorated by the administration of MR16-1 (Nishimoto et al., 2000a). Administration of MR16-1 also prevented collagen-induced arthritis (CIA) in DBA/1J mouse (Takagi et al., 1998). These findings suggest that anti-IL-6 receptor antibody may be a therapeutic agent for the treatment of human diseases caused by overproduction of IL-6 such asRA,Castleman’sdiseaseandmultiple myeloma.

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Humanized anti-IL-6 receptor antibody as therapeutic agent Castleman’s disease is an atypical lymphoproliferative disorder with benign hyperplastic lymph nodes characterized histologically by follicular hyperplasia and capillary proliferation associated with endothelial cell hyperplasia. It also displays chronic inflammatory features such as fever, general fatigue, increased levels of CRP and fibrinogen, and immunological abnormalities such as polyclonal hypergammaglobulinemia and the presence of auto-antibodies. These findings can all be explained by overproduction of IL-6. Indeed, constitutive overproduction of IL-6 from the lymph nodes and serum of a patient with Castleman’s disease has been observed (Yoshizaki et al., 1989). Seven patients with Castleman’s disease were treated with the humanized anti-IL-6 receptor antibody, known as MRA (Nishimoto et al., 2000b). Therapy with MRA rescued all patients from fever, fatigue and anemia as well as abnormalities detected in the serum, such as CRP, SAA, fibrinogen, albumin and polyclonal hypergammaglobulinemia. Similarly, histological examination of affected lymph nodes also showed remarkable improvement after treatment with MRA. MRA therapy was applied to eleven patients with refractory RA (Yoshizaki et al., 1998). The symptoms associated with RA improved remarkably in the eight patients who were able to receive the full course of MRA therapy. These results indicate that MRA therapy is relatively safe and may be useful for treatment for patients with Castleman’s disease and RA.

CONCLUSIONS IL-6 plays a central role in defense mechanism(s), immune response, hematopoiesis and acute-phase reactions. On the other hand, deregulated expression of the IL-6 gene has been implicated in the pathogenesis of a variety of diseases, especially autoimmune diseases, plasmacytoma–myeloma and several chronic inflammatory proliferative diseases. Future studies on the regulation of IL-6 gene expression and the mechanisms of IL-6 action through its receptor, as well as development of inhibitors of IL-6 action, should provide information critical for a better understanding of the molecular mechanisms of disease and the development of new therapeutic methods.

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REFERENCES

ACKNOWLEDGEMENTS This chapter was revised on the basis of the third edition (The Cytokine Handbook, 1998) described by Dr Hirano.

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Stucture–function analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6binding and for IL-6 signal transduction through gp130. EMBO J. 12, 1705–1712. Yokoi, T., Miyawaki, T., Yachie, A. et al. (1990). Epstein–Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor. Immunology 70, 100–105. Yoshizaki, K., Nakagawa, T., Kaieda, T. et al. (1982). Induction of proliferation and Igs-production in human B leukemic cells by anti-immunoglobulins and T cell factors. J. Immunol. 128, 1296–1301. Yoshizaki, K., Matsuda, T., Nishimoto, N. et al. (1989). Pathogenic significance of interleukin-6 (IL-6/BSF-2) in Castleman’s disease. Blood 74, 1360–1367. Yoshizaki, K., Nishimoto, N., Mihara, M. and Kishimoto, T. (1998). Therapy of RA by blocking IL-6 signal transduction with humanized anti-IL-6 receptor antibody. Spring. Semin. Immunopathol. 20, 247–259. Yudkin, J.S., Kumari, M., Humphries, S.E. and Mohamed-Ali, V. (1999). Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link? Atherosclerosis 148, 209–214. Zhang, X., Blenis, J., Li, H.C. et al. (1995). Requirement of serine phosphorylation for fomation of STAT-promoter complexes. Science 267, 1990–1994.

( IL - 6 )

Zhang, Y., Lin, J.-X. and Vilcek, J. (1988a). Synthesis of interleukin 6 (interferon-b2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP. J. Biol. Chem 263, 6177–6182. Zhang, Y., Lin, J.-X. Yip, Y.K. and Vilcek, J. (1988b). Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6. Proc. Natl Acad. Sci. USA 85, 6802–6805. Zhang, X.-G., Klein, B. and Bataille, R. (1989). Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma. Blood 74, 11–13. Zhang, Y., Lin, J.-X. and Vilcek, J. (1990). Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a jB-like sequence. Mol. Cell Biol. 10, 3818–3823. Zhong, Z., Wen, Z. and Darnell, J.E. (1994). Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264, 95–98. Zilberstein, A., Ruggieri, R., Korn, J.H. and Revel, M. (1986). Structure and expression of cDNA and genes for human interferon-b2, a distinct species inducible by growthstimulatory cytokines. EMBO J. 5, 2529–2537.

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H

A

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T

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13 INTERLEUKIN-7 Peter K.E. Trinder 1 and Markus J. Maeurer 2 1

Nemod Immuntherapie AG, Berlin, Germany 2

University of Mainz, Mainz, Germany

What is not fully understood is not possessed Johann Wolfgang von Goethe

INTRODUCTION Interleukin-7 (IL-7) is an exceptional cytokine, as it mediates lymphopoiesis in mice in a non-redundant fashion. In contrast, targeted gene deletion of other cytokines, including IL-2, IL-4 or IL-10 (Schorle et al., 1991; Kuhn et al., 1991, 1993), revealed that these cytokines are not essential for development and proper function of either B or T lymphocytes. IL-7 is secreted by both immune and non-immune cells and appears not only to be involved in the development of an effective immune system, but also in the generation and maintenance of strong and effective cellular immune responses. IL-7 serves as the major growth and differentiation factor for both thymic and extrathymic development of cd T lymphocytes. IL-7 promotes immune effector functions in T lymphocytes, natural killer (NK) cells and monocytes– macrophages, and modulates the quantity and quality of immune responses in vitro and in vivo. The availability of IL-7-targeted gene-deleted mice or IL-7 The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

transgenic animals has allowed a more detailed study of the physiology and pathophysiology of paracrine and systemic effects of IL-7 which represents a key regulator of T-cell homeostasis. The implementation of IL-7 in the treatment of different diseases, including immunodeficiency disorders and malignancy, suggests that IL-7 may facilitate a number of therapeutic endeavors including bone marrow and organ transplantation, cancer immunotherapy, the treatment of infectious diseases and, in general, ‘immunereconstitution’. Despite the substantial work on IL-7 in development and differentiation of B and T cells, novel co-factors have been identified: the pre-pro B cell growth-stimulating factor (PPBSF), the variant beta chain of the hepatocyte growth factor, which is covalently bound to IL-7. In addition, an ‘IL-7-like’ factor has been debunked as thymic stromal lymphopoietin (TSLP) which interacts with the respective TSLP-receptor, but also with the IL-7 receptor alpha chain. The identification of IL-7-like molecules and IL-7-hybrid cytokines, as well as soluble IL-7

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receptors suggests that IL-7-mediated effects are finely tuned and tightly controlled. To date, IL-7 represents the single non-redundant cytokine responsible for shaping the humoral as well the cellular immune system.

CLONING, PURIFICATION AND STRUCTURE Following the development of culture techniques for studying in vitro bone marrow cultures it became apparent that B-cell maturation occurred in the presence of bone marrow stromal cells, suggesting the existence of a growth and/or maturation enhancing cytokine (Hunt et al., 1987). Namen and coworkers subsequently demonstrated that conditioned medium from stromal cell cultures stimulated the growth of B-cell precursors.They immortalized a stromal cell line by transfecting it with the plasmid pSV3neo (large and smallT antigens of SV40) and isolated a clone (IN/A6) which produced a factor initially called lymphopoietin1 (LP-1) that stimulated the growth of B-cell precursors. Conditioned medium from the growth of this clone was then purified. High-performance liquid chromatography (HPLC) column fractions containing LP-1 bioactivity were isolated. A single unit of LP-1 activity is that causing half-maximal 3HTdR incorporation in a culture of precursor B cells (LP-1 bioassay). At this stage of purification it was clear that several proteins were present in the fraction that could account for the biologic activity. Additional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing conditions associated bioactivity with a protein of 25  103 Da, which was substantiated by 125 I-labeled LP-1-binding experiments. The purified protein exhibits a specific activity of approximately 4  106 U/mg of protein and is active at a half-maximal concentration of 10 13 M (Namen et al., 1988). The same murine stromal cell clone provided a cDNA library which was screened for LP-1 activity following expression in COS-7 cells. A clone (1046) was identified that was associated with high biological activity. The sequence contains a 548 bp 5 non-coding region which may be involved in expression regulation. The sequence includes a 462 bp open reading frame and a 579 bp 3 non-coding region containing a consensus polyadenylation signal and terminating in 15 adenine

residues. Purified protein was subjected to N-terminal analysis, which suggested that the nucleotide sequence from clone 1046 codes for the same protein, the protein was designated IL-7.The mature protein has a 25 amino acid leader sequence followed by 129 amino acids with two N-linked glycosylation sites and six cysteine residues leading to intramolecular disulfide bond formation. The importance of disulfide bond formation is suggested by loss of activity following treatment with 2-mercaptoethanol breaking disulfide-bond formation. The disparity between calculated molecular weight and that predicted by migration of the native protein may be accounted for by glycosylation (Cosenza et al., 1997; Namen et al., 1988). Two such N-linked glycosylation site in murine IL-7 are located at amino acid residues 69 and 90 (Namen et al., 1988). IL-7 mRNA has been detected in murine thymus spleen, kidney and liver by Northern blot analysis. Interestingly, although message was present in thymus and spleen, no biological activity could be detected in these tissues. Goodwin and colleagues characterized human IL-7 by nucleic acid hybridization of cDNA prepared from a hepatocarcinoma cell line (SK-HEP-1, ATCC HTB 52) with the murine IL-7 probe. There is considerable homology between the two IL-7 nucleotide sequences (81% in the coding region) and up to 60% amino acid homology with all six cysteine residues being conserved (Goodwin et al., 1989). The human IL-7 gene contains 6 exons over 33 kb (Lupton et al., 1990). The human IL-7 cDNA is composed of 534 nucleotides encoding a protein of 177 amino acid residues (17.518 Da) with a signal sequence of 25 amino acid residues and three potential N-linked glycosylation sites (Goodwin et al., 1989). There is a 19 amino acid insert for human IL-7 (coded for by exon 5 in the human genome) which does not exist in murine IL-7 (Plate 13.1) and appears not be essential for biological IL-7 activity using a proliferation assay of progenitor B cells (Goodwin et al., 1989). Additionally, an apparently alternatively spliced human IL-7 mRNA lacking the entire exon 4 (44 amino acid residues) results in loss of ability to stimulate proliferation of murine progenitor B cells isolated from the SK-HEP-1 line. Alternatively spliced IL-7 isoforms have also been identified in different tissues, or human cell lines. Human small intestinal epithelial cells, which secrete IL-7, express two alternatively spliced mRNA tran-

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scripts (Madrigal-Estebas et al., 1997). Alternatively spliced IL-7 mRNA has also been identified in human malignant hematopoietic cells obtained from children with acute lymphoblastic leukemia (ALL). Three IL-7 splice variants have been identified which lack either exon 3, exon 5 (like the original cDNA clone identified by Goodwin and coworkers), or both exons 3 and 5 in combination with exon 4 (Korte et al., 1999). In addition to these in-frame IL-7 variants, several out-of-frame variants have also been identified. The role of these isoforms is still enigmatic. Differentially spliced IL-7 may act as a natural antagonist, a situation which has already been observed for IL-4 (Atamas et al., 1996). In addition, the readout system, e.g. proliferation of progenitor B cells, may not be able to detect alternate functions by such IL-7 variants. Human recombinant IL-7 is active on murine and human B-cell progenitors. In contrast, murine IL-7 acts only on murine, but not on human cells. To date, there is only a limited source of biological assays available to detect IL-7 bioactivity and the readout is based on proliferation of a murine precursor B-cell line. However, these assays detect only a single facet out of a magnitude of IL-7 activities, e.g. induction of V(D)J recombination, proliferation of mature T cells, maintenance of T-cell survival or activation of monocytes. Thus, different biological readout systems may be advisable if either mutant IL-7 forms or differentially spliced IL-7 isoforms are scrutinized for biological activity. To date, most studies have used the cell line IN/2b (Park et al., 1990) or the pre-B cell line 2E8, which was initially maintained on stromal cells and then in IL-7-conditioned medium (Pietrangeli et al., 1988) to define IL-7 bioactivity. Recently, a limited number of biological assay systems has been added to the panel of IL-7-responsive cells, e.g. the acute myeloid leukemia (AML)-derived cell line MUTZ-3 proliferates in response to IL-7 and to thymic stromal lymphopoietin (TSLP) (Quentmeier et al., 2001). Alternatively, the murine pre-B-cell line PB-1 is dependent on the presence of IL-7 and can be used to detect IL-7 in both plasma and serum samples with a sensitivity in the range of 50 pg/ml IL-7 (Mire-Sluis et al., 2000). This bioassay appears to be more sensitive for detecting IL-7 as compared with the IL-7dependent 2E8 cell line and it shows no response to cytokines (e.g. TGFb and IL-13) which may interfere with 2E8 proliferation (Mire-Sluis et al., 2000). A stable

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pre-pro B-cell line (YS-PPB), derived from AA4.1 yolk sac cells from day 10 mouse embryos may also be implemented to study the role of IL-7 in B-cell differentiation and expansion (Lu and Auerbach, 1998). Human IL-7 is predicted to conform to the ‘small hematopoietin’ subclass members, forming a fourhelix bundle structure. Human or murine IL-7 has not yet been crystallized. However, using a combination of theoretical sequence structure recognition prediction and disulfide bond assignments (human IL-7 exhibits six cysteine residues, see Plate 13.1), three-dimensional models of IL-7 have been constructed (Cosenza et al., 2000) by superimposing IL-7 helices into the IL-4 template (Walter et al., 1992a, 1992b) which has been defined by X-ray structure crystallized IL-4 (Bajorath et al., 1993), incorporating the disulphide bond assignments (Cys3–Cys142, Cys35–Cys130, Cys48–Cys93) into the model. The disulphide bridges have been determined by matrixassisted laser desorption/ionisation (MALDI) mass spectroscopy and by cysteine to serine mutational analysis (Cosenza et al., 1997). No biological activity was retained, using a murine pre-B-cell precursor assay, if all six cysteine residues were substituted with serine (Cosenza et al., 1997), which is in keeping with earlier results that b-mercaptoethanol treatment abolishes bioactivity (Henney, 1989). Cysteine residues were reintroduced into the mutant IL-7 proteins and assayed for bioactivity. Apparently, only a single disulfide-bond forming IL-7 mutant protein is able to form a tertiary structure capable of stimulating precursor B-cell proliferation. These data were used to define the potential IL-7 structures involved in receptor binding. This is not only of interest in the context of structural analysis, but also pertaining to the development of ‘designer’ cytokine molecules which either act as antagonists or as ‘superagonists’ as compared with the wild-type cytokine protein. Circular dichroism analysis suggested that human IL-7 is primarily approximately 35% a helical, 31% random coil, 23% b sheet and 11% b turn. The threedimensional IL-7 structure has been postulated based on data obtained from IL-4 and GM-CSF: IL-7 belongs to the group of short chain four a helical bundle cytokine molecules defined by four (lefthanded) a helices in an up-up–down-down position. These helices are designed with A to D (Bazan, 1990). In general, amino acid residues of helices A to C bind

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the individual cytokine (i.e. IL-7Ra) receptor and residues located on helix D bind the to common c chain (cc) shared with different cytokines of the same family (see Plate 13.1). Of practical interest, these structural analyses provided the means to identify potential IL-7 molecules endowed with antagonistic effects, at least in regard to precursor B-cell proliferation. Two approaches have been reported. First, mutational analysis results in the construction of IL-7 variant proteins with amino acid substitutions at different positions (i.e. K121–A, L136–A, K140–A, W143–A), which ablated proliferation of murine precursor B cells. It is worth noting that the mutant hIL-7 (W143–A) is able to replace the wild-type IL-7 from the receptor and acts as an antagonistic molecule (Cosenza et al., 2000). A second approach has been reported by Kroemer and coworkers. Three-dimensional IL-7 models were altered based on the observation of alternatively spliced IL-7 isoforms and analyzed for IL-7 receptor binding by computer modelling (Kroemer et al., 1998). IL-7 isoforms with altered disulfide bonding patterns may show a different three-dimensional structure and also act as antagonists, at least in the murine precursor B-cell assay. However, other readout systems may help in better defining the biological impact of such IL-7 protein variants. The human IL-7 gene appears to represent a single copy gene located on the chromosome 8q12-13 in proximity to the p53/p56lyn gene, a member of the Src tyrosine kinases and the HYRC gene which is potentially involved in VDJ recombination located at 8q11 (Corey and Shapiro, 1994; Seckinger and Fougereau, 1994). The murine IL-7 gene lacks transcription regulatory elements which have been commonly identified in eukaryotic promoters, e.g. the TATA box, CAAT sequences, SP1 or GC-rich regions. Only one SP1 binding site has been identified in the human IL-7 gene. Additionally, a potential binding site for E12 is conserved in both murine and human IL-7 genes, as well as other sequences confirming to ‘helix–loop–helix’ class of DNA-binding proteins (Lupton et al., 1990). Of note, several IL-7 mRNA species of 1.5, 1.7, 2.6 and 2.9 kb have been identified in murine tissues. However, all four transcripts appeared to be present in the thymus. In contrast, only the 2.6- and the 2.9-kb IL-7 mRNA transcripts could be detected in kidney

(Namen et al., 1988). Regulation of murine IL-7 mRNA expression has been addressed by examining IL-7 RNA resolution in murine PAM 212 keratinocytes (Ariizumi et al., 1995). Treatment with IFNc yields preferential expression of the 1.5- and 2.6-kb mRNA species in addition to the constitutively expressed 2.9- and 1.7-kb mRNAs by the use of alternative transcription initiation sites. The 1.5- and 2.6-kb mRNAs are transcribed within 250 bp from the coding sequence. In contrast, the 1.7- and 2.9-kb mRNA species contained 400 bp in the 5 untranslated region. IFNc promotes conversion to 1.5- and 2.5-kb mRNA expression through the IFN-stimulated response element (ISRE) located 270 bp upstream from the coding sequence (GAAACTGAAAGT). This ISRE is immediately followed by a non-TAT-type transcription ‘initiator’ element (CTTACTCTTG). It appears that IFNc-induced transcription of IL-7 may be controlled through the ISRE–control complex, whereas other ‘initiator sequences’ may be responsible for the base-line IL-7 transcriptional activity in certain cell types (Ariizumi et al., 1995). It has been suggested that the IFNc-inducible IL-7 transcripts may be more translationally active as compared with the conventional ‘base-line’ 1.7- and 2.9-kb transcripts, a concept which may impact on the molecular definition of the cellular interaction of keratinocytes, a source of IL-7 secretion, and IFNc-producing cells in skin (see below). IL-7 has also been cloned from different species, for instance from swine (Ueha et al., 2001), rat (Visse et al., 1999) or bovine origin. IL-7 cDNA from a bovine leukemia virus-induced B-cell lymphosarcoma was characterized constituting a protein of 176 amino acids and showing 75% homology with human IL-7 and 65% homology with murine IL-7 (Cludts et al., 1992; Barcham et al., 1995) (Table 13.1).

Cytokines similar to IL-7 and IL-7-associated cytokines A number of studies have addressed the nature of stromal-derived growth factors capable of regulating pro-B and pre-B cell development (for review see (Baird et al., 1999).Only a limited number of studies address the factors that are involved in pre-pro B-cell differentiation. Using a long-term bone marrow culture system, a 30 kDa pre-pro B-cell factor (PPBSF)

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TABLE 13.1 IL-7 expression (mRNA or protein) in cell lines and tissues Tissue/Cell Type

Detection of mRNA

Detection of protein

Reference

Bone marrow stromal cells

(h,m)

(h, m)

Spleen

(h, m)

n.d.

Kidney Kidney allograft Renal cell cancer (RCC) tissue sections, or RCC-cell lines Fetal and adult thymus

(h,m) (h) (h)a

n.d. n.d. (h)

Namen et al., 1988; Sudo et al., 1989; Witte et al., 1993 Namen et al., 1988; Goodwin et al., 1989 Namen et al., 1988 Strehlau et al., 1997 Trinder et al., 1999

(h, m)

(h, m)

Thymic stromal cells

(m)



Hassall’s corpuscles Keratinocytes

(h) (h.m.)

n.d. (h, m)

Intestinal epithelial cells, epithelial goblet cells Colorectal cancer cells Cervical cancer cells Uterus Brain Head and neck squamous cancer Adult liver Hepatocarcinoma Activated/mature dendritic cells (CD80, CD83, CD86, CD40) EBV B-cell lines Burkitt’s lymphoma cells Chronic B-lymphocytic leukemia cells

(h)a

(h)

(h)a (h)a (m) (h) n.d. (r) (h)a (h)

(h) (h) n.d. n.d. (h) n.d. n.d. (h)

(h) (h) (h)a

(h) (h) (h)

(h) (h)

n.d. n.d.

(h)a (m) (h) (h) (h)

(h)

(h)b

n.d.

(h) n.d.

(h) (h)

Bladder cancer Inflammatory malignant fibrous histiocytoma Follicular dendritic cells Fibroblasts Oral mucosa vascular endothelial cells Hodgkin’s cell line, nodularsclerosing type Sezary lymphoma cells Lesions from tuberculoid lepra Lymph nodes from HIV patients

(h) (h) n.d.

a

Namen et al., 1988; Goodwin et al., 1989; Montgomery and Dallman, 1991; Sakata et al., 1990; Wiles et al., 1992 Sakata et al., 1990; Gutierrez and Palacios, 1991 He et al., 1995 Heufler et al., 1993; Matsue et al., 1993a, 1993b Watanabe et al., 1995; Madrigal-Estebas et al., 1997 Maeurer et al., 1997 our unpublished observations Appasamy, 1997 Appasamy, 1997; Paleri et al., 2001 Appasamy, 1997 Goodwin et al., 1989 Sorg et al., 1998 Benjamin et al., 1994 Benjamin et al., 1994 Frishman et al., 1993; Long et al., 1995 Kaashoek et al., 1991 Melhem et al., 1993 Kroncke et al., 1996 Aiba et al., 1994 Kroncke et al., 1996 Kroncke et al., 1996 Bargou et al., 1993 Foss et al., 1994; Asadullah et al., 1996)b Sieling et al., 1995 Napolitano et al., 2001

cloning and sequence analysis of mRNA exhibits alternatively spliced forms(s) of IL-7. IL-7 mRNA did not appear to be overexpressed as determined by semiquantitative analysis in skin biopsies from patients with mycosis fungoides, or with pleomorphic T-cell lymphoma when compared with biopsies obtained from normal skin, psoriatic lesions or atopic dermatitis. n.d., not determined. h, human; m, mouse, r, rat. b

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has been identified which formed a heterodimer with IL-7 (Lai et al., 1998; McKenna et al., 1998). The PPBSF cofactor (PPBSF-coF) is produced in stromal cells obtained from IL-7/ mice (McKenna and Goldschneider, 1993; McKenna et al., 1998). PPBSFcoF alone (i.e. without IL-7) maintains pre-pro B-cell viability (Lai et al., 1998). More recently, the PPBSFcoF has been identified as the free mitogenic b chain of the hepatocyte growth factor (HGF)/scatter factor (Lai and Goldschneider, 2001), which represents a heparin-binding, stromal-derived cytokine closely related to plasminogen (van der Voort et al., 2000). Interestingly, the b chain of HGF had not been reported to be independently produced of the HGF a chain. IL-7/HGFb represents a new member of ‘hybrid’ cytokines which are composed of the bioactive forms of cytokines which are independently produced. IL-7 complexes with HGFb in the presence of low molecule weight heparin-sulfate to the heterodimer (PPBSF) which may enable IL-7 to participate in stromal interactions and to trigger effectively the IL-7 receptor. IL-7 appears to be present almost exclusively as the PPBSF (IL-7/HGFb) heterodimer in pre-B cell culture systems (Lai et al., 1998). In contrast to the generation of IL-7 heterodimeric molecules, ‘IL-7-like’ cytokine(s) have also been described, the thymic stromal lymphopoietin (TSLP), which was originally identified in conditioned medium of a thymic stromal cell line that supported the development of IgM B cells from fetal liver hematopoietic progenitor cells. Both murine (Sims et al., 2000) and human TSLP (Quentmeier et al., 2001) have been cloned and characterized (Sims et al., 2000). The TLSP activities partially overlap IL-7-mediated functions. Both cytokines facilitate B lymphopoiesis in fetal liver cultures, bone marrow lymphocyte precursors and both cytokines are able to stimulate thymocytes and mature T cells (Friend et al., 1994). The TLSP receptor has recently been identified and shares high homology with the cc chain molecule (Pandey et al., 2000; Park et al., 2000). Binding of TSLP to the respective TSLP receptor results in a low-affinity interaction. Binding of TSLP to the TSLP-R is markedly increased in the presence of the IL-7Ra chain, which explains why IL-7Ra gene-deleted mice show a different phenotype as compared with IL-7/ animals, as TSLP is still able to interact with the IL-7Ra (Pandey et al., 2000; Park et al., 2000). Thus, TLSP binds to its indi-

vidual receptor with low affinity and does not bind to the cc receptor molecule. Both the IL-7Ra and the TLSP receptor are required for high-affinity binding of TSLP (see Plate 13.2b). Thus, IL-7 is able to form heterodimeric molecules and is ‘mimicked’ by other proteins, e.g. TSLP. However, IL-7/ mice clearly demonstrated the non-redundant nature of the IL-7 protein. Loss of IL-7 results in a severe reduction of ab T cells and the entire loss of cd T cells.

THE IL-7 RECEPTOR A cell line absolutely dependent on IL-7 for growth (IN /2b) (Park et al., 1990) was used to characterize the IL-7 receptor (IL-7R) designated as CD127 (Schlossman et al., 1994; Kishimoto et al., 1997). The IL-7R complex, exhibiting both high-affinity (~Kd 100 pM) and low-affinity (~Kd 1nM) binding sites (Page et al., 1993) is composed of at least two subunits: the IL-7Ra chain, identified by a direct expression cloning strategy (Goodwin et al., 1990), maps to the human chromosome 5p13 (Lynch et al., 1992) and the common c chain (cc) shared with receptors for IL-2, IL-4, IL-9 and IL-15 (Noguchi et al., 1993; Kondo et al., 1993, 1994; Russell et al., 1993, 1994; Giri et al., 1994). The cc chain appears to be constitutively expressed (Taniguchi and Minami, 1993). The IL-7Ra receptor subunit forms a heterodimer with the cc chain which is required for the high-affinity IL-7 binding (Noguchi et al., 1993; Kondo et al., 1994). It has been suggested that IL-7 receptors expressed on some cells may contain an as yet poorly defined subunit, since IL-7 binds (albeit at low affinity) to COS cells in the absence of transfected IL-7Ra and cc chains (Goodwin et al., 1990; Noguchi et al., 1993; Kondo et al., 1994). Alternatively, since COS cells represent primate kidney cells, endogenously expressed IL-7R may provide IL-7 binding sites. Six extracellular and four intracellular cysteine residues are present in human as well as in the murine IL-7Ra coding for a 439 amino acid protein with a calculated molecular weight of 49.5 kDa (Plate 13.2a). The IL-7Ra domain exhibits the characteristic features of the cytokine receptor superfamily: the cytoplasmic IL-7Ra domain, composed of 195 amino acid residues, does not exhibit consensus protein kinase sequences (reviewed in Sugamura et al., 1996). A

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differential splicing event results in mRNA encoding for the secreted form of the IL-7 receptor capable of binding IL-7 in solution (Plate 13.2a), a form that may be important for binding circulating IL-7 (Goodwin et al., 1990; Park et al., 1990; Mehrotra et al., 1995). IL-7Ra has been detected on pre-B cells, thymocytes, some T-lineage cells (Park et al., 1990; Rich et al., 1993), on human intestinal cells (Reinecker and Podolsky, 1995), colorectal cancer cells, renal cell cancer cells (our unpublished observations), and on a bone marrow-derived macrophage but not on mature B cells (Foxwell et al., 1992), on cutaneous T-cell lymphomas (Bagot et al., 1996), and thymic NK1.1 T cells (Miyaji et al., 1996). The human IL-7Ra (CD127) is expressed on both naive or activated memory CD4 or CD8 T cells (Plate 13.3). Similarly to the alternatively spliced IL-7 transcripts, the IL-7Ra not only codes for the ‘canonical’ IL-7Ra or the soluble form thereof (see above), but also for differentially spliced IL-7Ra isoforms (Korte et al., 2000). These transcripts have been identified in human acute lymphoblastic leukemia. These isoforms apparently lack a part of the cytoplasmic domain, but are still capable of binding IL-7 protein, similar to the intracellularly truncated erythropoietin receptor that fails to mediate proliferation and is capable of inhibiting the functions of the wild-type receptor in a dominant negative fashion (Nakamura and Nakauchi, 1994). The total number of IL-7 binding sites on individual cells may range from 1  104 up to 5  105/cell (Armitage et al., 1992b). Interestingly, IL-2 and IL-7 reciprocally induce IL-2Ra and IL-7Ra receptor expression on cd T lymphocytes, which may be important for proliferation and T-cell response to locally produced cytokines of intraepithelial lymphocytes (iIEL). However, there are two different subsets of such iIEL: cd T cell receptor (TCR) dim cells (cd TCRdim) are responsive to IL-2 (presumably provided in situ by ab T cells) and IL-7 (presumably provided in situ by epithelial cells or macrophages). In contrast, cd TCRbright cells did not respond by up-regulation of their IL-2 and IL-7 receptors (Fujihashi et al., 1996). Additionally, IL-7 binding to the IL-7R leads to expression of the transferrin receptor and the 4F2 antigen. Sequence analysis of the 5 flanking region of the murine IL-7Ra revealed that it contains CAATT and TATA sequences, potential glucocorticoid receptor binding sites, as well as a potential ISRE element.

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Using DNA microarray technology in order to analyze gene expression in peripheral blood cells obtained from healthy individuals upon exposure to glucocorticoids, IL-7Ra has been identified to be induced as the prominent gene by glucocorticoids both on the mRNA as well as on the protein level (Franchimont et al., 2002). Conversely, TCR signaling decreases IL-7Ra expression and the fine balance between IL-7Ra down-regulation and induction by glucocorticosteroid determines the final IL-7Ra level expression on T cells. Enhanced IL-7 expression has been shown to be associated with enhanced IL-7-mediated signaling and immune effector functions in responder cells (Franchimont et al., 2002). Interestingly, activation of PBMCs with anti-CD3 results in a four-fold downregulation of IL-7 receptors (high and low affinity) (Foxwell et al., 1992). These findings have recently been substantiated by the observation that IL7Ra– cc chain complexes are detectable in activated, but not in resting T cells, independent of total cell surface cc chain expression. Thus, stimulation of T cells may lead to assembly of IL7Ra–cc chain complexes which correlates with JAK3 expression. However, the work by Franchimont and coworkers suggests that expression of the IL-7Ra on T cells not only reflects T-cell activation status, but also the response to ‘stress’ mediated by glucocortocoids at high levels, either by exogenous application or by endogenous production. Thus, triggering the TCR may lead to activation-induced cell death (apoptosis) and the same may be true for apoptosis induced by stress hormones (Boumpas et al., 1993). Triggering the TCR and glucocorticoids may be able to prevent apoptosis and promote T-cell survival. Since IL-2 or IL-4 gene-deleted mice do not exhibit severe defects in T-cell differentiation, such as those observed in either IL-7 or IL-7Ra gene-deleted mice, IL-7 may account for most of the immunological defects observed in murine models of the X-SCID defect associated with defects of the common cc chain receptor unit (Takeshita et al., 1992; Noguchi et al., 1993; Leonard et al., 1994; DiSanto et al., 1995). The X-SCID defects can also be observed in humans (Lai et al., 1997). In human unstimulated T cells, the IL-7Ra is constitutively associated with the Src kinase enzymes p59fyn and p56lck (Plate 13.2b) (Page et al., 1993). IL-7 binding the IL-7R leads to both increased p59fyn and p56lck levels in stimulated and unstimulated T cells (Page et al.,

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1993). Signaling via the IL-7R also leads to increased activity of the Src kinases in stimulated and unstimulated T cells, suggesting that activation of p59fyn and p56lck is not exclusively responsible for IL-7-driven T-cell proliferation and that other signaling events (e.g. mediated through the cc chain) are required (Page et al., 1993). However, targeted gene deletion for p59fyn in mice did not show a major impact on lymphopoiesis (Stein et al., 1992; Grabstein et al., 1993; Sudo et al., 1993). In contrast, in p56lck gene-deleted mice, a thymocyte maturation block at the double negative state could be observed (Molina et al., 1992). However, similar effects could not be detected in CD4, CD8 or IL-2 gene-deficient deleted mice. These observations suggest that p56lck is also involved in the signaling pathways (Fung-Leung et al., 1991; Schorle et al., 1991). Thus, the observed effects of p56lck on lymphopoiesis may be attributed to the lack of IL-7-driven p56lck-mediated cellular responses. IL-7mediated phosphatidylinositol-3 (PI-3) kinase activation induced by tyrosine phosphorylation of the PI-3 kinase p85 subunit appears to be essential to the IL-7 proliferative signal (Sharfe et al., 1995). A different protein tyrosine kinase, termed pim-1, may also be involved in IL-7-mediated signaling, as IL-7mediated pre-B-cell expansion is decreased in pim-1deficient mice (Domen et al., 1993). IL-7 activates members of the Janus (JAK) family of non-receptor tyrosine kinase, JAK1 and JAK3 (Russell et al., 1994; Zeng et al., 1994; Musso et al., 1995), which are both activated by cc chain-sharing cytokines including IL-2, IL-4 and IL-9. These kinases may serve as the signal transduction pathway to the nucleus by phosphorylation and activation of signal transducers and activators of transcription (STATs) (Plate 13.2b). IL-7 has been shown to activate STAT-1, -3 and -5 (Zeng et al., 1994; Lin et al., 1995; van der Plas et al., 1996; Perumal et al., 1997) by interacting with an area spanning the tyrosine residue 409 at the C-terminal end of the IL-7 receptor (Lin et al., 1995). Thus, at least several alternate signal transduction pathways (e.g. p56lck, p59fyn, JAKs, STATs) may be operational in IL-7responsive cells (e.g. T cells or epithelial cells). It is possible that IL-7 may exert its functions in a cell- or tissue specific manner dependent on differential activation of the IL-7R signaling transduction pathway(s). For instance, the IL-7 receptor complex delivers signals of different quality to lymphoid progenitor cells

during rearrangement of the antigen receptors (reviewed in Candeias et al., 1997b). IL-7-mediated effects through the IL-7 receptor are also dependent on separate IL-7 receptor domains. These IL-7Ra regions have been mapped by transgenic expression of the mutant IL-7 receptor genes in the thymus of IL-7 Ra/ mice (Porter et al., 2001). The IL-7 receptor controls different individual properties during thymic development. The IL-7Ra tyrosine-containing carboxy-terminal T domain is crucial for restoring thymic cellularity in IL-7 Ra/ animals, important in pro/pre T-cell progression and T-cell survival. The functional differentiation of TCR ab T cells and cd T cells are partially independent of this IL-7 receptor region. Both PI-3 kinases and STAT-5 have been demonstrated to signal through the T-domain (Lin et al., 1995; Corcoran et al., 1996). Gene-deleted mice for either IL-7 (von Freeden-Jeffry et al., 1995), IL-7Ra (Peschon et al., 1994), cc (DiSanto et al., 1995), JAK1 (Rodig et al., 1998) or JAK3 (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995) exhibited a similar block in the early stage of T-cell development. Of interest in the context of IL-7-induced T-cell proliferation, JAK3 is intimately associated with the T-cell receptor (TCR) signalling complex: JAK3 is phosphorylated upon TCR triggering. This event is independent of the cc receptor, but dependent on the TCR-associated signaling molecules Lck and ZAP-70 and physically associated with the TCR f chain, one of the key components of mediating downstream TCRmediated signaling events (Tomita et al., 2001). To date, the steps associated with these signaling molecules have not yet been clearly defined. Although IL-7 stimulation does activate the PI-3 kinase, the src kinases and the transcription factor STAT5, genedeleted mice with loss of each of these molecules have not indicated a critical role for these components in the IL-7 downstream effects (Appleby et al., 1995; Yasue et al., 1997; Teglund et al., 1998; Terauchi et al., 1999). In contrast, the Pyk2 (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995; Yu et al., 1996), a protein-tyrosine kinase related to focal adhesion kinase is critically involved in IL-7-mediated effects (Benbernou et al., 2000). Pyk2 appears to be physically associated with JAK1 prior to IL-7 stimulation and critically involved in cell survival, since antisense Pyk2 resulted in accelerated cell death. In addition, Pyk2 can be activated independently of IL-7

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through muscarinic and nicotinic receptors: either carbochol or ionomycin/phorbol myristate-activated Pyk2 promotes cell survival in the absence of IL-7. Thus, Pyk2 appears to be one of the essential cofactor(s) associated with the trophic effects mediated by IL-7. Of note, Pyk2 has not only been shown to be involved in T-cell survival, but also in mediating IL-7 effects in neurons, e.g. by affecting the potassium channel KV1.2 (Lev et al., 1995). Thus, the IL-7Ra mediates a ‘trophic’ or a ‘maintenance’ effect regarding cell viability during gene rearrangement. Earlier studies showed that immature thymocytes undergo apoptosis when separated from the thymus. IL-7 is capable of sustaining these cells without inducing significant cell proliferation (Watson et al., 1989). These anti-apoptotic effects delivered by the IL-7Ra can also be observed in mature lymphoid cells (Komschlies et al., 1994) and may be attributed to the induction of Bcl-2 members (Hernandez-Caselles et al., 1995; Lee et al., 1996; Vella et al., 1997). However, other Bcl-2-related proteins, inducing Bcl-xL or Bcl-w, or other anti-apoptotic factors, may also be involved, since bcl-2 knock-out (/) mice exhibit a different picture concerning T-cell development as compared with alterations identified in IL-7Ra/ mice (Veis et al., 1993; Matsuzaki et al., 1997). The IL-7Ra may also deliver ‘mechanistic’ signals required for gene rearrangement. IL-7Ra/ mice exhibit impaired c gene rearrangement (Maki et al., 1996; Candeias et al., 1997b). The same was found to be true for IL-7Ra-mediated signals, required for immunoglobulin (Ig) heavy chain and TCRb-chain rearrangement (Corcoran et al., 1996; Crompton et al., 1997). The TCRd rearrangement may not be exclusively IL-7 dependent, as IL-7Ra/ mice exhibit d-chain rearrangements in vivo (Peschon et al., 1994; Corcoran et al., 1996; Oosterwegel et al., 1997; Candeias et al., 1997b). Such effects may derive from several factors. First, IL-7 induces RAG1 and RAG2 expression (Muegge et al., 1993). IL-7Ra/ mice exhibit decreased RAG expression in double-negative, but not in double-positive, cells (Crompton et al., 1997). Therefore, decreased recombinase activity may affect recombinational events in distinct thymic cells. Second, IL-7Ra-mediated signals may be required to prevent untimely apoptosis in thymocytes. It has been suggested that IL-7Ra-mediated signals may

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unmask genes associated with proliferation and antiapoptotic properties (Peschon et al., 1998). This is substantiated by the observation that peripheral T cells in IL-7Ra/ mice undergo apoptosis upon stimulation (Maraskovsky et al., 1996). Recent studies addressed these points in detail (Schlissel et al., 2000; Carvalho et al., 2001; Huang et al., 2001; Huang and Muegge, 2001; Ye et al., 2001), particularly the dissection of IL-7-mediated effects on lymphocyte progenitors, i.e. survival, proliferation and ‘recombination’. IL-7Ra-mediated effects (Ye et al., 2001), i.e. proliferation of lymphocyte precursors, are transmitted through the PI-3 (Corcoran et al., 1996; Pallard et al., 1999). Different signaling molecules are crucial for recombination of the T-cell receptor and immunoglobulin genes. In general, different ‘conserved’ recombination signals as well the RAG1/RAG2 recombinases are crucial for T- and B-cell development. It has been postulated that specific mechanisms should exist which make the appropriate gene locus accessible to recombinases according to the developmental stage of B- or T-cell differentiation (see Figure 13.4). IL-7 transmits an ‘early’ signal in lymphocyte development when V(D)J recombination takes place. The ‘recombination’ signal promoting variable (V), diversity (D), joining (J), recombination in the IgH and TCRc locus is also transmitted through the IL-7Ra. IL-7R-induced signaling leads to germline transcription and DNA rearrangement in D-distal variable segments in pro-B cells (see Figure 13.4) (Corcoran et al., 1996); the variable–joining recombination and germline transcription of TCRc genes is severely impaired in IL-7Ra-deficient animals (Durum et al., 1998; Ye et al., 1999). It is evident that the IL-7 receptor is required for TCRc accessibility (Schlissel et al., 2000). The STAT5 proteins (signal transducers and activators or transcription), activated through the IL-7R (see Plate 13.2b), interact with consensus motifs in the 5 regions of Jc segments leading to germline transcription (Ye et al., 1999). Thus, STAT5 is able to induce germline TCR transcription, V–J recombination of the TCRc genes and is also able to ‘rescue’ cdT-cell development in IL-7Ra/ mice. These STAT5–mediated actions are transmitted through recruitment of transcriptional coactivators to the TCR Jc germline promotor and by controlling accessibility of the TCRc locus via histone acetylation (Ye et al., 2001).

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FIGURE 13.4 B- and T-cell differentiation. Cell surface marker expression, V(D)J rearrangement, and IL-7 responsiveness according to the following (Hardy et al., 1991; Hardy and Hayakawa, 1991; Kitamura et al., 1991, 1992; Peschon et al., 1994; von Freeden-Jeffry et al., 1995; Li et al., 1996). All blood cells are derived from hematopoietic stem cells (HSC). Antigen-receptor rearrangements as well as phenotypic cell surface analysis help in segregating the differentiation status of cells of the B, T or NK lineage. Six developmental subsets of B cells have been identified. T cells can be segregated based on receptor expression (CD3 CD4 CD8, triple negative (TN); CD4 CD8, double positive (DP); and CD4/CD8, single positive (SP)). TN cells are divided into four subsets based on CD44/CD25 expression (for review see Baird et al., 1999; Schatz and Malissen, 2002). HPC, hematopoietic progenitor cell; CLP, common lymphoid progenitor; SLC, surrogate light chain.

IL-7 and B lymphocytes All lymphocytes derive from hematopoietic stem cells (HSC). Apparently, the IL-7 receptor mediates nonredundant signals for T as well as for B cells originating from HSC. Not only animal models with target gene deletion, but also examination of the IL-7associated signaling pathways in the thymus as well as in bone marrow indicated that the up-regulation of the IL-7 receptor starts at the stage of the clonogenic

common lymphoid progenitor, which is capable of developing into T, B or NK cells (reviewed in Akashi et al., 1998; Killeen et al., 1998). Detailed analysis of the fate of either B or T cells underlined the central role for IL-7 in reconstituting an functional immune repertoire which is not only crucial for a better understanding of immune cell development and differentiation, but also in the context of designing novel strategies for immune reconstitution (see Figure 13.4). In addition, novel data suggest that central par-

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adigms, e.g. T-cell lineage models, may have to be altered: T-cell development/differentiation may not be as ‘static’ as previously postulated. For instance, one of the central paradigms in T-cell development is that CD4CD8 (double-positive, DP) thymocytes develop into single (CD4 or CD8)-positive T cells by terminating transcription of the respective coreceptor molecule. In contrast to this model (see for overview Figure 13.4), DP thymocytes initially terminate CD8 transcription when differentiated into CD8(CD4–) T cells (Brugnera et al., 2000). CD8 thymocytes terminate CD8 transcription, even if differentiation into CD8 T cells has already been started. IL-7 is capable of mediating to ‘re-start’ CD8 transcription by silencing CD4 transcription in thymocytes which selectively terminate CD8 transcription, an observation coined as ‘coreceptor reversal’ which sheds new light on IL-7 in modulating the ultimate fate of CD4/CD8 T lymphocytes (Brugnera et al., 2000). A different example indicating that IL-7 substantially shapes the immune cell repertoire is that IL-7 also may act on the neonatal T-cell pool. Earlier models postulated that IL-7 and the IL-7Ra are critical for delivering signals during the early phases of thymic development (i.e. at the stage of CD4CD8 precursor evolution) but not necessarily in intrathymic expansion of positively selected lymphocytes. However, recent data show that intrathymic positive selection is associated with up-regulation of the IL-7Ra and that the MHC class II epithelial cells, driving postselection proliferation, express IL-7 mRNA. In addition, detailed examination of thymic tissue from IL-7Ra/ mice also showed that postselection expansion is reduced if signaling through the IL-7Ra does not occur (Hare et al., 2000). Thus, IL-7 may also be critical for driving the antigen-independent proliferation of either CD4 or CD8 T cells after positive selection, which impacts on the peripheral Tcell pool and immune receptor repertoire. This is a crucial observation helpful for designing new ways for effective immune reconstitution either in patients with HIV infection or in bone marrow transplant recipients. Most of the data pertaining to the role of IL-7-mediated effects on T- or B-cell development and differentiation is derived from animal models; data from humans are scant. ‘Experiments of nature’ reflecting gene defects in humans are examples of the importance of Il-7-derived signals. Severe combined immunodeficiency (SCID) can be derived from a

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number of different defects (Leonard, 1996; Buckley et al., 1997; Fischer et al., 1997). The most common form of SCID is the lack of the cytokine receptor c chain shared among IL-2, IL-4, IL-7, IL-9 and IL-15 receptors (Noguchi et al., 1993). The defective IL-7Ra chain leads to a TBNKSCID phenotype in humans (Puel et al., 1998), a phenotype which is likely to be expected in patients who lack functional IL-7 protein. The most compelling evidence that IL-7 represents an important lymphopoietin and possibly one with clinical importance comes from a number of in vivo investigations. IL-7 administration to normal mice (5 mg) twice daily for 4–7 days results in a two- to fivefold increase in the number of peripheral and splenic white cells without significant change in bone marrow cellularity. Analysis of the bone marrow showed an increase in B-cell precursors (B220, sIg) with a concurrent decease in 8C5 and MAC-1 cells (myelomonocytic markers) (Damia et al., 1992). In addition, injection of murine or human IL-7 into mice resulted in an increase of B-cell precursor (pro-B and pre-B cell) production (Valenzona et al., 1998). A general scheme for B-cell maturation is outlined in Figure 13.4. For the purpose of clarity, we have adapted the nomenclature of Hardy and coworkers defining early stages of differentiation of murine B cells. These cells can be identified in liver or in bone marrow and are divided into distinct classes (A–F) based on marker cell surface expression (Hardy et al., 1991; Li et al., 1993). Adult stem cells develop into ‘conventional’ B2 cells. Fetal liver stem cells are capable of differentiating into B1 cells, which persist in adult animals, reside primarily in the peritoneal cavity and stain positive for the CD5 antigen. The role of B1 and B2 cells in the context of IL-7 is further discussed below in the section titled ‘IL-7 and microbial immune responses’. The early stages of B-cell development will occur in the bone marrow in response to stromal cell contact and cytokines. Hematopoietic stem cells (HSC) of the adult bone marrow have been characterized by cell surface marker analysis. HSC can be derived from murine BM using the CD34 (sialomucin) antibody, other cell surface markers include the antigens CD4, MHC class I, ER-MP12, and AA4.1 (Katz et al., 1985; Berenson et al., 1988; Szilvassy et al., 1989; Wineman et al., 1992; Orlic et al., 1993; Slieker et al., 1993; Szilvassy and Cory, 1993). Additionally, B-cell differentiation may be defined by D–J,

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or V–D–J rearrangement (Hardy et al., 1991). The antigen receptor of B (and T cells) are encoded in the germline by individual DNA segments, termed V, D and J, which are joined during lymphocyte differentiation. This process (VDJ recombination ) is initiated by the RAG1 and RAG2 proteins which act together at the junctions between the coding segments and the recombination signal sequence in order to produce two types of DNA ends: a signal end, terminating in a blunt double-strand break, and a coding end, which terminates in a DNA hairpin. The involvement of double-strand DNA cleavage has suggested that this process is linked to the cell cycle: several lines of evidence indicate that the initiation of VDJ recombination takes place at the G0/G1 phase (Oettinger et al., 1990; Lewis, 1994). IL-7 appears to sustain expression of the RAG1 and RAG2 genes (Muegge et al., 1993). IL-7 does not alter the RAG mRNA levels, but rather affects posttranscriptional regulatory mechanisms. In order to discriminate progenitor cells from cells which are already committed to the B-cell lineage, Hardy and coworkers have recently investigated bone marrow stromal cells for expression of the B-cell lineage marker B220 and HSA in combination with the CD4 and AA4.1 markers (Li et al., 1996). The latter marker is expressed on HSC, B-cell/myeloid progenitors, and early B-cell lineage cells (McKearn et al., 1985; Loken et al., 1988; Cumano and Paige, 1992). About 50% of the B220, CD43 and HSA cells (formerly termed A) stained positive for AA4.1 expression (Li et al., 1996). This cell population was capable of proliferating on a stromal cell layer, indicating that it may indeed represent B-cell lineage precursors. Thus, the earlier designation of fraction A B cells had to be revised. Two AA4.1 fractions (A1 and A2) appear to represent the earliest stages of B-cell lineage development. The B220–, AA4.1, CD4low fraction has been designated as A0 cells and appears to represent yet uncommitted progenitor cells. However, these ‘earliest’ stages identified in B-cell development will have to be characterized for activity of B-cell differentiation factors such as IL-7, kit-ligand (Flanagan and Leder, 1990; Williams et al., 1990), and flk2/flk3 (Rosnet et al., 1991). IL-7 does not support in vitro growth of cells of the granulocytic–monocytic or erythroid lineage but does stimulate eosinophil colony formation. This activity can be abolished by anti-IL-5 antibody treatment which suggests that IL-7 acts by stimulating

release of IL-5 or that potentially IL-5 represents an obligate cofactor (Vellenga et al., 1992). More recent data pertaining to transcription factor expression at different B-cell differentiation stages (Nutt et al., 1999; Rolink et al., 1999) also help in better understanding the role of IL-7 in concert with different bone marrow-derived growth factors in B-cell development (Cory, 1999); for instance, the commitment to B-cell production as associated with the paired box transcription factor 5 (Pax5) which is identical to the B-cell-specific activator protein (Adams et al., 1992). Pax-deficient B-lymphoid cells stop at the pre-B1 stage (Figure 13.4). In general, B cells can be cultured in the presence of stromal cells and IL-7. If IL-7 is withdrawn, the Ig receptor genes are rearranged. Not surprisingly, pre-B1 cells which are Pax-5 deficient are not able to differentiate into B cells in the absence of IL-7 unless IL-7 is restored. Surprising was the fact that IL-7-starved Pax-5/ cells exhibit a wide range of developmental potential, e.g. giving rise to T cells or antigen-presenting cells, depending on the environmental factors including stromal cells or cytokines (Nutt et al., 1999; Rolink et al., 1999). The growth factor combination of IL-11 and MGF (mast cell growth factor) supports bipotential progenitor cells to commit either to the B or to the macrophage lineage (Kee and Paige, 1996). Single cell cloning assays suggested that IL-7 does not act directly on the decision of whether cells commit to the B cell or macrophage lineage. However, bipotential cells responded to IL-7 by an increase in number and IL-7 added to the IL-11/MGF mixture promoted expression of mRNA transcripts coding for B-cellspecific genes (Kee and Paige, 1996). Furthermore, the growth factor combination of IL-11/flt3/IL-7 appears to maintain the potential of bipotential precursors (Ray et al., 1996). However, in a different report, uncommitted Lin-SCA-1 bone marrow progenitor cells have been demonstrated to differentiate into B220, CD43, HSA B cells (without expressing cytoplasmic l heavy chain or sIgM) using a combination of Flt3 and IL-7, which proved to be superior in regard to driving B-cell differentiation as compared with the combination of stem cell factor and IL-7; the latter combination leading to production of mature granulocytes (Veiby et al., 1996a, 1996b). Concerning already committed B cells, early pro-B cells require a combination of IL-7 and factors provided by stromal

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cell layers; late pro-B cells are capable of proliferating in IL-7 without stromal cell support. The same was found to be true for early pre-B cells, but probably not for late-pre-B cells (Hardy et al., 1991). IL-7-mediated effects in B-cell differentiation may in part be mediated by regulation of the G1/S transition of the cell cycle (Yasunaga et al., 1995). Rearrangement of j light chains and sIgM expression correlates with IL-7Ra down-regulation and therefore IL-7 unresponsiveness (Cumano et al., 1990; Park et al., 1990; Era et al., 1991; Henderson et al., 1992). However, the most precise data concerning the role of IL-7 in B-cell development are provided by IL-7/ or IL-7Ra/ mice. Evaluation of B lymphopoiesis in bone marrow appeared to be blocked at the transition to pre-B cells (see Figure 13.4). IL-7/ mice were blocked in the transition between the pro-B (fractions B/C B220/IgM/ S7/HSA) to the pre-B-cell population (fraction D, B220, IgM, S7, HSA). Thus, differentiation and maturation of B/C fraction B cells to fraction D appears to be IL-7 dependent (von Freeden-Jeffry et al., 1995, Moore et al., 1996; Peschon et al., 1998). IL-7Ra gene-deleted mice showed a block in B-cell development at the transition of pre-proB cells (formerly fraction A) to pro-B cells (fraction B) (Peschon et al., 1994). This may be due to the action of other growth factors, potentially the thymic stromalderived lymphopoietin (TSLP) (Friend et al., 1994; Peschon et al., 1994) or flt3 ligand (Namikawa et al., 1996). Indeed, recent studies addressing TSLP and its receptor showed that these differences in B-cell development are linked to the TSLP receptor: TSLP has been demonstrated to impact on B-cell development in vitro (Levin et al., 1999). Ultimately, the study of TSLP/ or TSLP receptor/ mice will provide the answers pertaining to signaling pathways of TSLP and the involvement in lymphocyte development. In addition, more recent data suggest an IL-7independent pathway in B-lymphocyte differentiation. In IL-7/ mice, B-lymphocyte production takes place exclusively during fetal and perinatal life and stops 7 weeks after birth (Carvalho et al., 2001). However, the B-cell pool in the periphery of these mice appears to be stable during life and is exclusively constituted of B1 cells and cells which belong to the marginal zone (MZ) compartments. These ‘MZ cells’ are characterized by high IgM and CD21 and low IgD and CD23 cell surface expression. So-called ‘multi-

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reactive’ B cells with ‘sticky’ antigen receptors are recruited into MZ and respond to LPS or CD40 ligation. As discussed above, B1 cells are enriched in the peritoneal cavity and derived from early B-cell precursors (Hayakawa et al., 1985; Hayakawa and Hardy, 2000). These B1 cells show less N region diversity as compared with conventional B2 cells and are thought to be involved in autoantibody production (Gu et al., 1990; Lalor and Morahan, 1990; Tornberg and Holmberg, 1995). Of interest, serum immunoglobulins are increased up to five-fold in these mice. Thus, there appears to exist an IL-7-independent pathway of B1 and MZ B-cell generation which is active in early life: TSLP/ mice will reveal whether TLSP is able to drive development of this distinct B-cell subpopulation. Application of IL-7 neutralizing monoclonal antibodies of mice resulted in a similar B-cell maturation blockade compared with those observed in IL-7Ra knockout animals, but not to the B-cell maturation blockade observed in the IL-7 gene-deleted animals (Grabstein et al., 1993; Peschon et al., 1994). One potential explanation is that other cytokines (e.g. TSLP) may utilize the IL-7 receptor as well. Other cytokines, e.g. TLSP, stem cell factor (SCF)/c-kit, or flk2/flk3 may synergize with IL-7 to regulate B-cell development (Veiby et al., 1996b). The stem cell factor SCF/kit ligand which represents a growth factor for myeloid and erythroid progenitor cells synergizes with IL-7 in stimulating B-cell precursor cells (McNiece et al., 1991, Billips et al., 1992; Funk et al., 1993). However, some cytokines appear to counteract the IL-7-mediated effects. For instance, IL-1 (Suda et al., 1989), IFNc (Garvy and Riley, 1994) and TGFb (Lee et al., 1989) are able to inhibit IL-7-mediated B-cell precursor growth. Additionally, a number of genes involved in B-cell development may be up-regulated by IL-7, including n-myc, c-myc (Morrow et al., 1992), CD19 (Wolf et al., 1993), the precursor lymphocyte-specific regulatory light chain (PLRLC) (Oltz et al., 1992) and the aminopeptidase BP-1/6C3. Incubation with IL-7 is associated with an increase in 6C3Ag expression by pre-B cells, but not mature B cells. The BP-1/6C3 molecule is expressed by early B-lineage cells and some stromal cells and represents a type II integral membrane glycoprotein that belongs to the zinc family of metallopeptidases (Sherwood and Weissman, 1990).

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In humans, IL-7 did not stimulate proliferation of B-cell lineage cells expressing CD24. Human pro-B cells but not pre-B cells respond to IL-7 (Ryan et al., 1994; Dubinett et al., 1995); this is in contrast to the data for murine cells which suggests that speciesspecific differences in mode of action exist between humans and mice (Tushinski et al., 1991). This human–rodent dichotomy exists for other cytokines – perhaps most notably IL-4. In general, human HSC commitment and differentiation has not been extensively characterized as compared with that of the murine system. However, recent data suggest that certain stages of human B-cell development may not necessarily depend on the presence of IL-7. Using a human bone marrow stromal cell culture system, human HSC CD34 cells underwent commitment, differentiation and expansion into the B-cell lineage as defined by loss of CD34, increased CD19 cell surface expression and appearance of Ig receptor-expressing immature B cells. This was not significantly influenced either by exogenously added IL-7 or by addition of anti-IL-7 neutralizing antibody (Prieyl and LeBien, 1996). The implementation of the flt3 ligand in combination with IL-7 or IL-3 using human fetal bone marrow-derived CD34 CD19 pro-B cells in a stromal cell-independent and serum-deprived culture system revealed that flt3, like IL-3, synergizes with IL-7 in promoting B-cell growth and differentiation of the majority of cells into CD43, CD19, cIgM, sIgM pre-B cells; a minority of pro-B cells matured into sIgM B cells (Namikawa et al., 1996). After the productive rearrangement of Ig heavy chains, pre-B cells undergo a number of divisions in response to IL-7 (Smart and Venkitaraman, 2000). This stage of B-cell expansion is constrained by an inhibitory signal initiated by receptor assembly (Smart and Venkitaraman, 2000). In mice, immature B cells divide in response to IL-7 according to their developmental program. Pro-B cells, which have not yet completed IgH chain rearrangement, require stromal contact and IL-7 (Hayashi et al., 1990; Hardy et al., 1991; Ray et al., 1998). After productive IgH rearrangement, pre-B cells need only IL-7 for proliferation (Namen et al., 1988; Lee et al., 1989; Sudo et al., 1989). These B cells undergo limited proliferation until they finally develop into IL-7-unresponsive mature B cells (Suda et al., 1989; Decker et al., 1991). The loss of IL-7 responsiveness appears to be mediated through an as

yet ill-defined molecule which interacts with the tyrosine residue (tyr 410) in the cytoplasmic tail of the IL-7 receptor (Smart and Venkitaraman, 2000). The future characterization of this interaction will be essential in order to define mechanisms which are involved in gauging peripheral B-cell numbers.

IL-7 and T lymphocytes IL-7 added to murine fetal thymic organ cultures (day 13) causes a preferential expansion of immature cells exhibiting the CD4, CD8CD3, CD2, SCA-1 phenotype. Cells expressing cd TCR are increased and the number of ab TCR are decreased. Neutralizing anti-IL-7 antibody inhibits growth of fetal thymocytes (Leclercq et al., 1992; Plum et al., 1993). In vitro culture of human fetal thymocytes in rIL-7 results in the proliferation of CD4 and CD8 thymocytes and partial differentiation of thymocytes with preferential expansion of the CD4 CD8 population (Uckun et al., 1991). IL-7 promotes the growth of pre-T cells from fetal liver at day 14 and promotes the expression of TCR c, a and b genes. Culture of fetal liver cells in IL-7 is associated with the appearance of Thy-1 and Pgp-1 Ag phenotypes as occurs in day 14 fetal thymus (Appasamy, 1992). IL-7 mRNA can be detected in the fetal thymus as early as day 12 and peaks at day 15 (Wiles et al., 1992). IL-7 stimulates the generation of CD3 cells from human bone marrow cultures with the production of both CD4 and CD8 populations (Tushinski et al., 1991). These results suggest that IL-7 may be produced locally in the thymic and bone marrow microenvironments and that IL-7 plays a role in the proliferation and potentially differentiation of immature T cells (Watanabe et al., 1992). Similar studies indicated that IL-7 induces proliferation and maintenance of T-lymphocyte numbers, but not T-cell differentiation. However, with the advent of IL-7, or IL-7Ra gene-deleted mice, several central questions concerning the role of IL-7 in lymphopoiesis could be addressed in more detail. The macroscopic examination of IL-7/ mice indicated apparently normal development of both fertile sexes. The lymphatic organs or tissues, including thymus and spleen, were dramatically reduced in size and the peripheral lymph nodes and immune cells within the Peyer’s patches were not detectable (von Freeden-Jeffry et al., 1995). Accordingly, the reduced

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white blood count in IL-7 gene-deleted mice was caused by an absolute reduction in lymphocytes, the normal ratio as well as the absolute numbers of granulocytes and monocytes were decreased. Overall, the massive lymphocytic reduction in these animals was due to decreased B- and T-cell numbers (von Freeden-Jeffry et al., 1995; Moore et al., 1996) reflecting the inefficient thymic development of IL-7deficient mice. Only 5% of normal thymocyte numbers and only 15% of splenic cell numbers could be detected in IL-7 gene-deleted mice (von FreedenJeffry et al., 1995; Moore et al., 1996). However, the immune responses of the remaining cells appeared to be similar to those observed in normal mice with regard to their function as defined by testing B cells in response to LPS, splenic T cells to Con A , or proliferation of thymocytes to a mixture of Con A  IL-2 (von Freeden-Jeffry et al., 1995). Similar T-cell abnormalities to those observed in IL-7-gene deleted mice have been identified in IL-2Rc receptor chain knockout mice (Takeshita et al., 1992; Noguchi et al., 1993; DiSanto et al., 1995). As discussed above, the common cc chain is shared by several other cytokines, including IL-2, IL-4, IL-9 and IL-15 (Takeshita et al., 1992; Kondo et al., 1993; Giri et al., 1994). Since IL-2 or IL-4 gene-deleted mice do not exhibit defects in T-cell development, IL-7, but not other cytokines, appears to account for most of the lymphocyte defects observed in murine models of X-SCID associated with abnormalities of the cc chain receptor (Takeshita et al., 1992; Kondo et al., 1993; Noguchi et al., 1993; DiSanto et al., 1995). Thymic T-cell development has been segregated into sequential stages based upon expression of distinct cell surface markers (see Figure 13.4). Thymic IL-7 is produced primarily during fetal development (Chantry et al., 1989; Conlon et al., 1989; Okazaki et al., 1989). CD4CD8 fetal and adult immature thymocytes proliferate well in response to IL-7. In contrast, CD4CD8 thymocytes respond rather poorly. The capability to respond to IL-7 correlates with expression of the IL-7 receptor a chain (IL-7Ra) expressed by CD4CD8, CD4CD8 and CD4CD8, but not by CD4CD8 thymocytes (Chantry et al., 1989; Conlon et al., 1989; Okazaki et al., 1989; Everson et al., 1990; Suda and Zlotnik, 1991). Earlier studies indicated that IL-7 mediates effects on TCR rearrangement. T-cell precursors from thymus or fetal

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liver cultured in IL-7 express rearranged b or c-chain transcripts (Appasamy, 1992; Appasamy et al., 1993; Muegge et al., 1993). IL-7, sustaining expression of the RAG genes (Muegge et al., 1993) induces rearrangement of Vc2 and Vc4, but not Vc3 or Vc5, TCR chains in mice (Appasamy et al., 1993). Further evaluation of IL-7 gene-deleted mice showed reduced numbers of total T lymphocytes with preservation of the normal CD4/CD8 ratio and increased percentage of ab T cells compared with cd T cells (von Freeden-Jeffry et al., 1995; Moore et al., 1996). However, more recent data indicate that IL-7 may also be involved in T-cell differentiation, since the IL-7Ra controls chromatin accessibility (see above, section IL-7 receptor) due to histone acetylation (Huang and Muegge, 2001). Again, the IL-7-mediated effects on T cells are two-fold: trophic (survival signal) and ‘mechanistic’ (reviewed in Candeias et al., 1997a; Hofmeister et al., 1999). Thus, cellular immune deficiencies observed in IL-7Ra/ mice can, in part, be restored by reintroducing bcl2 as a transgene (restoring the trophic function), or alternatively, a rearranged TCR (Crompton et al., 1997). Most of the signaling molecules associated with IL-7Ra triggering in thymic development have been identified; however, recent studies showed that the Pim1 proto-oncogene may be critical for the IL-7 signaling pathway, since Pim1 has been demonstrated to reconstitute cellularity in IL-7/ and (cc/ mice. Of interest, Pim-1 transgenic, but Rag-deficient mice are able to expand CD4CD8 thymocytes: Pim-1 appears to bypass the pre-TCR controlled checkpoint in T-cell development (Jacobs et al., 1999). Immature thymocytes have been divided into four distinct phenotypes based on differential expression of the cell surface markers CD25, CD44 and CD117 (c-kit). CD4low (CD44, CD25, CD117, CD3CD8) and pro-T cells (CD44, CD25, CD117), representing the early stages of thymic differentiation, are present in IL7/ mice. In contrast, transition of pro-T cells to pre-T cells (CD44CD25, CD117) and post-preT cells (CD44, CD25, CD117) could not be detected in IL-7 gene-deleted mice (Moore and Zlotnik, 1995; Moore et al., 1996;). In the absence of signals mediated by the IL-7Ra, T-cell precursor cells are not able to initiate cleavage at the TCRc locus, as the chromatin structure is not accessible for RAG-mediated cleavage (Tsuda et al., 1996; Oosterwegel et al., 1997). Interestingly, IL-7Ra-mediated signals can be

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replaced by Trichostatin A, which is a specific inhibitor of histone deacetylases, which suggested a role for histone acetylation in chromatin opening associated with IL-7. However, these actions were global and not specific for the TCRc locus: IL-7 specifically modulates chromatin accessibility by targeting the histone acetylation at the TCRc locus (Huang et al., 2001). The failure of IL-7/ mice to rearrange the TCRc has been shown to represent a failure to initiate cleavage, but as a failure to relegate broken DNA ends (Schlissel et al., 2000). The detailed examination of IL-7Ra/ mice in the context of V(D)J recombination also resulted in novel insights pertaining to T-cell development: V(D)J recombination was thought to start at the pro-T2 stage (see Figure 13.4) after the arrest of IL-7Ra/ thymocytes at the pro-T1 stage. However, novel studies showed (Schlissel et al., 2000) that both TCRb and c recombination takes place in normal T1 cells. Exclusively TCRb recombination intermediates were detected in IL-7R/ mice underlining the role for IL-7Ra-mediated signals in TCRc recombination. More recent studies have scrutinized the role of IL-7 in the development of cd T lymphocytes. IL-7/ showed a profound reduction of CD4CD8 cd T cells to approximately 1% of normal levels (von Freeden-Jeffry et al., 1995; Moore et al., 1996). A substantial body of evidence supports the notion that IL-7 preferentially promotes development of cd TCR thymocytes as compared with ab thymocytes, due to differential IL-7Ra expression on cd thymocytes compared with ab TCR thymocytes. This notion is supported by the fact that cd T cells are absent in thymus, gut, liver and spleen in IL-7Ra/ mice. cd T cells are not only strictly dependent on IL-7, but also on the source of IL-7. Development of intrathymic cd T cells is dependent on intrathymic IL-7, while ‘peripheral’ IL-7 is sufficient to drive extrathymic cd T cell development (Laky et al., 1998). In contrast, ab TCR lymphocytes, and NK cells appeared to be reduced in number, but to develop normally (He and Malek, 1996; Maki et al., 1996). However, NK1 T cells can be detected in thymus, liver and spleen of IL-7Ra/ mice. Recent data have suggested that differentiation of these NK1 cells is dependent on signaling via the cc chain and expansion on IL-7Ra-mediated signals (Boesteanu et al., 1997). These results provide reasonable evidence that

signal transduction mediated by the IL-7 receptor is a prerequisite for cd T-cell development in both thymic and extrathymic pathways. Expression of the RAG-1 and RAG-2 genes is also significantly reduced in the thymus of IL-7Ra/ mice, but restored in doublepositive thymocytes observed in TCR transgenic IL7Ra/ mice (Crompton et al., 1997). Thus, signaling through the IL-7Ra appears be necessary for RAG expression and initiation of VDJ rearrangement, as described for VDJ recombinatorial events in B-cell differentiation (see above). VDJ rearrangement may impact on organ-specific immunity. For instance, pulmonary cells with the canonical fetal-type Vc6 chain are missing in nude mice owing to a preferred thymic pathway of TCR gene rearrangement, but not to thymic selection. These cells can be restored in vitro and in vivo by administration of IL-7 (Hayes et al., 1996). In murine fetal development, T-cell production can be detected at day 15 of gestation. T cells at this stage express the invariant TCR complex composed of Vc3 and Vd1 chains. Maturation of thymocytes is accompanied with differential expression of the CD24 (heatstable antigen) expression. First, immature Vc3 cells exhibit a TCR Vc3low and CD24 phenotype and progress to mature Vc3high and CD24 cells. These cd T cells may populate the epidermis, or potentially other epithelial sites, and represent the dendritic epidermal T cells (DTEC). Alternatively, cd T cells may also mature extrathymically. Interestingly, IL-7/ mice characteristically exhibit a block of maturation of Vc3low, CD24 T cells to Vc3high CD24low T cells (Moore et al. 1996). This observation provides another piece of evidence that IL-7 does not serve exclusively as a ‘maintenance’ factor for thymocytes, but may also be involved in T-cell maturation and differentiation. In recent years, characterization of T lymphocytes residing primarily in the intestine (intestinal intraepithelial lymphocytes; iIEL) has revealed a distinct phenotype as well as a different functional activity of such immune cells compared with ‘conventional’ ab T cells in the periphery (Van Kerckhove et al., 1992; Boismenu and Havran, 1994; Guy-Grand et al., 1994; Havran and Boismenu, 1994; Rocha et al., 1994). Of note, thymic and intestinal epithelial cells share the same embryologic origin as they are both derived from entoderm and may both be capable of secreting IL-7 in situ (Namen et al., 1988; Heufler et al., 1993; Matsue et al., 1993a, 1993b; Ariizumi et al., 1995;

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Watanabe et al., 1995; Maeurer et al., 1997). Thus, given that fact that IL-7/ mice (Moore and Zlotnik, 1995; von Freeden-Jeffry et al., 1995; Moore et al., 1996), cc chain knockout mice (Takeshita et al., 1992; Kondo et al., 1993; Noguchi et al., 1993; DiSanto et al., 1995) as well as JAK3-deficient mice (Nosaka et al., 1995; Park et al., 1995) lack cd T cells, IL-7 appears to represent the major growth/differentiation factor required for thymic and extrathymic development of cd T cells. Of note, ab TCR iIEL are detectable in IL-7/ mice, but not in cc or in JAK3-deficient mice, suggesting that other cytokines may be critical for generation of ab TCR iIEL, but not necessarily for cd TCR iIEL (Takeshita et al., 1992; Noguchi et al., 1993; DiSanto et al., 1995; Moore and Zlotnik, 1995; Park et al., 1995; von Freeden-Jeffry et al., 1995; Moore et al., 1996). To date, there is strong experimental evidence that TCR iIEL may develop in situ. Such immune cells are present in both congenitally athymic nude mice, as well as in athymic radiation chimeras (for review see Poussier and Julius, 1994; Klein, 1996). Much more controversy surrounds the origin of the various subsets of iIEL which show a limited TCR repertoire (Van Kerckhove et al., 1992; Guy-Grand et al., 1994; Poussier and Julius, 1994). IL-7 gene-deleted mice may help to define the impact of IL-7 in the generation and TCR composition of ab TCR lymphocytes at different anatomic sites, preferentially in the intestine. Recently, clusters of lymphocytes located in crypt lamina propria (designated cryptopatches) have been characterized within the murine small and large intestinal mucosa. Such lymphoid cells are characterized by cell surface expression of CD117 (c-kit), IL-7Ra, Thy1 and the absence of markers for CD3, ab TCR, cd TCR, sIgM and B220. It has been proposed that immune cell population, first detected on days 14–17 after birth, may represent the lymphohematopoietic progenitors for T and B cells in the intestine. The prominent role of IL-7 in lymphopoietic development is further underscored by the observation that such cryptopatch-associated lymphoid cells are virtually absent in IL-7Ra-deficient mice. Earlier studies showed that intestinal epithelial cells express stem cell factor (Puddington et al., 1994; Laky et al., 1997) and IL-7 (Fujihashi et al., 1996; Murray et al., 1998; Watanabe et al., 1998), thereby creating an environment that facilitates immune cell development. In order to define the role of Il-7 expressed by intestinal

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epithelial cells, IL-7 expression was restored in situ using the tissue-specific intestinal fatty acid binding protein (iFABP) promotor (Laky et al., 2000). T-cell development in bone marrow as well as in the thymus was similar to that in IL-7/ mice, but Il-7 elaborated by enterocytes was sufficient for extrathymic cd T-cell devlopment and crucial for development of mucosal lymphoid tissue (Laky et al., 2000). IL-7Ra CD3 cells have been postulated to act as direct induces of Peyer’s patches (Yoshida et al., 1999).

Tissue-specific immune responses and IL-7 The fact that IL-7 has been found to be synthesized in such a large variety of tissues is confirmation of the cytokine’s role in promoting local immune responses. IL-7 is produced by human and murine keratinocytes (Heufler et al., 1993; Matsue et al., 1993a, 1993b; Ariizumi et al., 1995) and is a major growth factor for dendritic epidermal T cells (DTEC) which express the cd TCR (Matsue et al., 1993a, 1993b). The mouse epidermis harbors a T-cell population characterized by expression of CD3, asialo-GM1, CD2, Thy-1, Ly48, and E-cadherin, but not CD4 or CD8 phenotypic markers (Steiner et al., 1988). These DETC express the cd TCR composed of the Vc3 and Vd1 chains without junctional diversity (Matsue et al., 1993a, 1993b; Moore et al., 1996; Steiner et al., 1988) and may play a role in monitoring stressed keratinocytes, or recognize class Ib antigens, such as TIa, or Qa (for review see Haday, 1995). Keratinocytes constitutively express IL-7 mRNA and secrete in vitro biologically meaningful amounts of IL-7 protein. In addition to being the principle growth factor for DETC, IL-7 also prevents apoptosis in DETC exposed to ultraviolet B radiation, or corticosteroid treatment (Takashima et al., 1995). This agrees with earlier observations that IL-7 is better than IL-2 at maintaining viability and responsiveness in antigen-specific T-cell lines. Interestingly it is IFNc, secreted by cd T cells, that modulates the growth of murine keratinocytes (Takashima and Bergstresser, 1996). IL-7 augments LFA-1 and VLA-4 expression in human PMA and Ionomycin-ca stimulated PBL, thus enhancing the capacity of these cells to adhere to parenchymal cell monolayers (Fratazzi and Carini, 1996). IL-7 also induces cell surface expression of the costimulatory molecule B7 as well as ICAM-1 (CD54)

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on pre-B cells, this being biologically relevant if B cells act as antigen-presenting cells (Dennig and O’Reilly, 1994). The involvement of IL-7 in cd T-cell homing to the epidermis is fundamental to the evolution of contact sensitivity to trinitrochlorbenzene, which can be abrogated by administering mAbs to cd T cells in vivo. cd T cells invading the site of antigen challenge typically exhibit a CD8a, CD8b, Vc3 phenotype, and proliferate in response to IL-7, but not to IL-2 or IL-4. Furthermore, in vivo application of IL-7 neutralizing antibodies inhibits accumulation of Vc3 T cells in the skin, as well as in the regional lymph nodes adjacent to the site of application (Dieli et al., 1997). Other cell types such as fibroblasts within the epidermis may also provide biologically meaningful quantities of IL-7 in vivo (Aiba et al., 1994). Some of the strongest evidence supporting IL-7’s role as the major growth factor for intra-epithelial lymphocytes is provided by Williams and Kupper (Williams et al., 1997), who showed that epidermal densities of DETC increase substantially in keratin 14 promotor-driven IL-7 transgenic mice, in which ectopic IL-7 is produced exclusively by keratinocytes (personal communication, Takashima and Bergstresser, 1996). Overexpression of IL-7 in transgenic mouse keratinocytes results in a lymphoproliferative skin disease in which mice develop dermal and epidermal T cell infiltrates associated with alopecia (Williams et al., 1997). Elevated IL-7 levels have also been observed in sera from patients with psoriasis, although no correlation with disease intensity was observed (Szepietowski et al., 2000). The role of IL-7 in skin immune reactions is supported by the observation that IL-7 mRNA is upregulated in mite allergen patch test reactions in patients with atopic dermatitis (Yamada et al., 1996). The site of positive patch reaction is also a site of eosinophilic infiltration, and IL-7 also up-regulates the low-affinity receptor for IgE (CD23) in activated PBL (Fratazzi and Carini, 1996). Studies have also indicated that IL-7 is involved in bullous pemphigoid, and IL-7 levels both in the blister fluid and in serum appear to correlate with disease intensity (D’Auria et al., 1999). In addition to creating an interactive environment between keratinocytes and cd T cells, IL-7 may also be involved in the germinal center reaction (Kroncke et al., 1996a). Both IL-7 mRNA and protein have been detected in human follicular dendritic cells (FDCs) obtained from tonsils. However, mature peripheral

IgM B cells appear to be unresponsive to IL-7, whereas antistimulated tonsilar B cells proliferate in response to IL-7 without secreting immunoglobulins, indicating that IL-7 may well regulate B-cell responses in the periphery. In addition to skin and tonsils, IL-7 mRNA and protein have also been detected in human intestinal cells (Reinecker and Podolsky, 1995; Watanabe et al., 1995) and overexpression of IL-7 is believed to play a role in chronic colitis. IL-7 transgenic mice developed a colitis histopathologically similar to human ulcerative colitis. This, together with the observation that IL-7 eliminates activated lymphocytes in the inflamed mucosa of ulcerative colitis in humans while stimulating the proliferation of inactivated mucosal lymphocytes, indicates that disregulation of IL-7 expression in epithelial cells results in chronic inflammation of the colonic mucosa (Watanabe et al., 1999). Additionally, IL-7 mRNA and protein have also been detected in human colorectal tumor cells (Maeurer et al., 1997), in normal kidney (Goodwin et al., 1989) and in human renal cell cancer cell lines (Trinder et al., 1999). These cells produce significant amounts of IL-7 protein in vitro. Of interest, elevated IL-7 mRNA expression appears to represent one of the most sensitive markers of graft rejection in patients after kidney transplantion (Strehlau et al., 1997). In a recent study it was shown that administration of IL-7 following allogeneic bone marrow transplantation enhanced lymphoid reconstitution but failed to aggravate graft-versus-host-disease while maintaining graft-versus-leukemia activity. This appears to be related to the only minimal expression of IL-7R in activated and memory alloreactive donorderived T cells from recipients of allogeneic bone marrow transplants (Alpdogan et al., 2001). IL-7 promotes the growth of lamina propria lymphocytes but inhibits their CD3–dependent proliferation (Watanabe et al., 1995). Like IL-2, IL-7 promotes the preferential expansion of (short-term, day 14) cultured tumor-infiltrating lymphocytes obtained from patients with colorectal cancer (Maeurer et al., 1997). Long-term in vitro culture of human iIEL harvested from patients with colorectal cancer with IL-7 results in preferential outgrowth of Vd1 T cells (Maeurer et al., 1995, 1996) which recognize colorectal cancer cells, renal cell cancer and pancreatic cancer cell lines (Maeurer et al., 1996). Such immune effector cells release significant amounts of IFNc. Human intestinal

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cells also express IL-7Ra, and stimulation of such cells with IL-7 leads to rapid tyrosine phosphorylation of proteins (Reinecker and Podolsky, 1995). The physiologic role of these IL-7-responsive epithelial cell lines is unclear, although IL-7 may represent a member of a family of epithelial growth factors that promote homing, maturation and maintenance of IL-7-responsive immune cells. Thus, IL-7 may be actively involved in creating an interactive environment of epithelial cells and lymphocytes. Murine cd T cells secrete keratinocyte growth factor (KGF), which promotes proliferation of epithelial cells (Boismenu and Havran, 1994). Conversely, human epidermal growth factor (EGF) (Reinecker and Podolsky, 1995) increases IL-7Ra mRNA expression in human colorectal cancer cell lines. Human keratinocyte growth factor stimulates IL-7 mRNA expression and IL-7 protein secretion by human intestinal cells (our unpublished observation). Therefore, EGF and/or KGF and IL-7 may represent cytokines involved in the homeostasis of epithelial and immune cells in vivo. The conditioning role of intestinal cells is further supported by the observation that bone marrow cells develop into phenotypically mature T cells when co-cultured with the intestine epithelial cell line MODE-K (Vidal et al., 1993; Maric et al., 1996). To what extent IL-7 is involved in mediating these effects is not known. The effects of IL-7 on IL-7Ra immune cells are well understood. Less is known about IL-7-mediated effects on non-hematopoietic cells. Recent data underlined the ‘trophic’ effects of IL-7 on gastrointestinal cells. Intestinal stem cells show increased radiosensitivity within intestinal crypts in IL-7Ra/ mice and IL-7 is able to confer protection of radiation-induced apoptosis in intestinal stem cells (Welniak et al., 2001). Several recent studies have addressed the roles of IL-7 and IL-7Ra mRNA expression in developing tissues. The observation that IL-7 stimulates maturation of embryonic hippocampal progenitor cells in culture indicates a role for IL-7 in the proliferation and differentiation of immature cells of non-hematopoietic origin (Mehler et al., 1993). IL-7 has also been observed to both enhance survival of hippocampal neurons in culture and increase numbers of astroglia and microglia (Araujo and Cotman, 1993). IL7 and IL-7R mRNA expression can also be observed in the developing brain, and treatment of culture of embryonic

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brain with exogenous IL-7 leads to increased neuronal survival and greater numbers of cells exhibiting neurite outgrowth. Changes in gap junction properties have been observed in hippocampal multipotent progenitor cells undergoing differentiation under the influence of IL-7 (Rozental et al., 1998). Such effects on neuronal differentiation may be the result of the restriction and differential modulation of glial and neuronal signaling compartments. IL-7 may also be involved in local immune reactions affecting the eye. The neuroectodermis derived retinal pigment epithelium (RPE) contributes to the blood–retina barrier that regulates infiltration of immune cells in retinal diseases. Activation of RPE cells leads to the expression of MHC class II antigens as well as adhesion molecules. IL-7 also induces monocyte chemotactic protein-1 and IL-8 in RPE cells (Elner et al., 1996). Further studies are needed, however, to address whether IL-7 can be detected in retina-associated diseases in vivo.

IL-7 and cancer IL-7 plays different roles in cancer-bearing hosts, depending both on the tumor and status of the immune system. IL-7 mRNA, IL-7 protein and IL-7Ra have all been identified in some hematologic malignancies, indicating that IL-7 functions as a growth factor in an autocrine fashion. Some tumor cells exhibit expression of IL-7Ra without IL-7 expression and these may be responsive to IL-7 provided by different cell types. IL-7 may be used as a treatment for cancer since IL-7 increases immune effector cell functions by T lymphocytes, NK cells and macrophages. IL-7 may be applied systemically, or it may be secreted by genetically engineered tumor cells in order to induce a strong and long-lived immune response. IL-7 may be one of several growth factors suitable for use in recovery from bone marrow transplantation both in the setting of treatment of hematological malignancy and of bone marrow rescue following high-dose chemotherapy treatment of solid tumors (e.g. breast cancer).

Expression of IL-7 and IL-7Ra in cancer Several solid tumors, including colorectal and renal cell cancers, have been found to be positive for IL-7

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mRNA and also to express IL-7 protein (Watanabe et al., 1995; Maeurer et al., 1997; Trinder et al., 1999). Cells from both tumor types express the IL-7Ra receptor as well as the common cc chain. Immunohistochemical staining of IL-7 has been observed in head and neck squamous cell cancer (Paleri et al., 2001) (see Plate 13.5) and IL-7 mRNA has been detected in tumor cells of nodular sclerosing and mixed cellularity type of Hodgkin’s disease (Bargou et al., 1993; Foss et al., 1995). The prominent immune cell infiltrate observed in most cases of Hodgkin’s disease may be attributed to local delivery of IL-7 in vitro. Increased IL-7 serum levels have been detected in patients with Hodgkin’s disease (Trumper et al., 1994; Gorschluter et al., 1995). Similarly, Sézary’s lymphoma cells express IL-7Ra and proliferate in response to IL-7. However, some of these lymphoma cells obtained from different patients (3/5) were also found to express IL-7 mRNA (Foss et al., 1994). It is believed that keratinocyte-secreted IL-7 serves as growth factor for cutaneous T-cell lymphomas. This hypothesis is substantiated by examination of IL-7 transgenic mice in which the IL-7 gene is expressed under the control of the mouse MHC class II (Ea) promotor (Mertsching et al., 1995). These mice develop a lymphoproliferative syndrome characterized by early polyclonal expansion of T lymphocytes followed by development of pro-pre-B and bipotential myeloid/ B-cell tumors, which can be observed in about 25% of C57Bl/6 and in up to 100% of Balb/c mice (Mertsching et al., 1995, 1996). If the IL-7 gene is controlled by of the Sra promotor, which is constitutively expressed in many tissues, development of cutaneous (cd TCR) lymphomas is observed (Uehira et al., 1993). A number of leukemia and lymphoma cells isolated from patients have been screened for their growth responses and/or dependence on IL-7: many but not all proliferate when exposed to IL-7 and the cell types include B- and T-cell malignancies (Eder et al., 1990; Touw et al., 1990; Makrynikola et al., 1991; Shand and Betlach, 1991; Skjonsberg et al., 1991; Lu et al., 1992; Yoshioka et al., 1992). Evidence of lymphoid maturation of the tumor cells in response to IL-7 incubation was not observed (Eder et al., 1990). In a separate study, pre-B cells transformed by a variety of oncogenes were tested for IL-7 production. None produced any IL-7 bioactivity. IL-7 overexpression achieved by removing portions of the 5 flanking

region was not associated with dramatic colony formation in agar and most clones were not tumorigenic in vivo (syngeneic mice) (Young et al., 1991). It seems therefore that production of IL-7 does not represent a final common step in the malignant transformation of lymphoid cells, but that in selected malignancies it may represent a target for therapeutic intervention. For instance, IL-7 may represent an ‘antiapoptotic’ factor for some hematopoietic malignancies: in murine T-cell lymphoma cells (CS-21) IL-7 induces expression of the Bcl2 protein and suppression of the CPP32-like protease (Lee et al., 1996). The observation that IL-7 up-regulates ICAM expression by melanoma cells, a phenotype correlated with metastatic behavior, indicates an additional role for IL-7 in malignant progression (for review see Moller et al., 1996). IL-7Ra expression in several types of cutaneous and nodal lymphomas has been studied in detail. IL-Ra is not expressed in cutaneous B-cell lymphomas, benign cutaneous lymphoid infiltrates or in reactive lymph nodes, but is expressed in over 50% of all histological types of cutaneous T-cell lymphomas (Bagot et al., 1996). IL-7 mRNA and protein are also readily detectable in chronic B-cell chronic lymphocytic leukemia (B-CLL). The coincidence of IL-7 mRNA down-regulation and apoptosis in B-CLL suggests that IL-7 gene expression may be required for B-CLL viability in vitro. Interestingly, IL-7 down-regulation and apoptosis could be prevented by co-culture of B-CLL cells with human umbilical cord endothelial hybrid cells (EA.hy926). Cell-cell contact appears essential as cell culture supernatant was unable to reconstitute the effect, suggesting that poorly defined integrins expressed on B-CLL cells may affect IL-7 gene expression and apoptosis (Long et al., 1995). Furthermore, IL-7 mRNA and protein elaborated by B-CLL cells may account for some of the clinical symptoms: some patients with CLL experience suppression of immune responses and also autoimmune symptoms (Frishman et al., 1993). Interestingly, cloning of the IL-7 gene product from B-CLL cells revealed that at least one different, alternatively spliced, IL-7 mRNA is expressed in tumor cells. The alternatively spliced form appears to be identical to an original IL-7 cDNA clone obtained by screening a (hepatocarcinoma) cDNA library for the human IL-7 gene (Goodwin et al., 1989). The alternative transcript

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lacks the entire exon 4 (132 bp) coding for 44 amino acid residues, as depicted in Plate 13.1 (Goodwin et al., 1989; Frishman et al., 1993). We have also observed that IL-7 mRNA expressing cells derived from renal or colorectal cancer cells contain the ‘canonical’ IL-7 full-length IL-7 mRNA and additionally differentially spliced IL-7 mRNA (our unpublished observations). The biology of these IL-7 mRNA species remains unclear. Thus, it appears that IL-7 exerts several differential effects on tumor cells. IL-7 may exert growthpromoting, but potentially also growth-arresting, activities. For instance, proliferation of some pre-B acute lymphoblastic leukemia (B-ALL) cells can be specifically inhibited by exogenous IL-7. This effect can be abrogated by blocking of the IL-7 receptor (Pandrau-Garcia et al., 1994). In contrast, other acute lymphoblastic leukemia cells appear to be IL-7 responsive (Greil et al., 1994). Furthermore, targeting of IL-7R-positive cells using a recombinant fusion toxin (DAB389-IL-7) has been suggested as a treatment for lymphomas (Sweeney et al., 1995). Thus, IL-7induced effects mediated by IL7Ra may also be dependent on the actual cell type, as proliferation of early pre-B cells can be augmented by IL-7.

IL-7 in cancer therapy Immunotherapy is in the process of becoming a feasible treatment option for some cancer patients. Theoretically, the approach assumes the existence of antigenic differences between malignant and normal cells, and that these differences can be manipulated in a manner beneficial to the host. In addition, it must be assumed that the tumor-bearing host is functionally immunodeficient in that the tumor somehow blocks or inactivates the patient’s own antitumor response. For instance, alterations in expression and function of signal transduction molecules associated with the TCR are responsible for inefficient immune responsiveness in T lymphocytes in several human malignancies, such as renal cell cancer, colorectal cancer, ovarian cancer and melanoma. Decreased CD3 expression and inefficient CD3mediated signaling in tumor infiltrating lymphocytes (TIL) and in PBL have been observed in tumor-bearing mice (Mizoguchi et al., 1992; Salvadori et al., 1994; Levey and Srivastava, 1995), and also recently in

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cancer-bearing patients (Finke et al., 1993; Nakagomi et al., 1993; Matsuda et al., 1995; Tartour et al., 1995; Zea et al., 1995; Lai et al., 1996; Rabinowich et al., 1996). One of several mechanisms of CD3 down-regulation and inefficient signaling involves reduced expression of the  chain of the TCR, presumably related to hydrogen peroxide secretion elaborated by tumor-derived macrophages (Kono et al., 1996). This defect can be reversed in vitro and in vivo using exogenous IL-2 or IL-2 transfected into tumor cells and used as a vaccine (Salvadori et al., 1994; Rabinowich et al., 1996). IL-7 is, however, also able to up-regulate the TCR (Ono et al., 1996) and can enhance protein expression of molecules associated with TCR expression and signaling functions (e.g. ZAP-70,  chain, p56lck and p59fyn, our unpublished data). Additionally, suppressive factors released by tumors may impair antitumor-directed immune responses, such as TGFb. Macrophagederived TGFb mRNA can be down-regulated by IL-7 (Dubinett et al., 1993). The same effect of IL-7 has been found to be true for TGFb down-regulation in a murine fibrosarcoma (Dubinett et al., 1995). In contrast, TGFb is able to reduce stromal IL-7 mRNA expression and protein secretion using a human in vitro lymphoid progenitor cell culture system (Tang et al., 1997). Biologic therapy approaches including immunotherapy seek to reverse this apparent state of anergy and to augment antitumor-directed immune responses. Clinical trials utilizing IL-2 as a cytokine-based immunotherapy have demonstrated that this approach is successful in treating some patients. The challenge for both clinicians and researchers is to increase the efficacy and decrease the non-specific effects of the therapy. IL-7 appears to have a number of ‘IL-2-like’ properties and preclinical testing is supportive of the use of IL-7 in clinical trials. The IL-7-mediated effects can be segregated into those due to non-specific, MHC non-restricted, lysis of tumor cell targets (e.g. due to lymphoid-activated killer (LAK) cells), MHC class Ior class II-specific recognition of tumor cells by ab T lymphocytes and tumor-restricted and presumably classical MHC non-restricted recognition by cd T-cell effectors. The LAK phenomenon was first reported in 1980 by Yron and associates (Yron et al., 1980) and describes the in vitro lysis of labeled fresh tumor targets by lymphoid cells that have been preincubated in IL-2 or other lymphokines. The effect is not MHC restricted

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and is relatively non-specific, in that a variety of different fresh tumors are lysed yet most normal cells are spared. IL-7 is able to generate LAK activity from thymocytes and peripheral blood mononuclear cells. Compared with IL-2, IL-7 is a relatively weak LAK inducer. IL-2 stimulates five-fold more LAK precursors than IL-7 (Alderson et al., 1990). Thymocytes from cultures grown in IL-2 are highly cytolytic whereas those grown in IL-7 exhibit minimal cytolytic activity, however, cultures grown in IL-7 and then switched to IL-2 become cytolytic. The addition of IL-4 does not induce cytolytic activity of the cells grown in IL-7 but rather down-regulates IL-2-induced proliferation and cytoytic activity (Widmer et al., 1990). IL-7 can generate human LAK activity in the absence of IL-2 and induces or up-regulates expression of CD25, CD54 and CD69. LAK generation is negatively influenced by TGFb and IL-4. Anti-IL-4 antibody and anti-IL-4 antisense enhances IL-7induced LAK activity (Stotter et al., 1991). IL-7 promotes secretion of TNF but not of IFNc. The nature of the LAK cell precursor for IL-7induced LAK is not totally clear. One study showed that LAK cell activity (comparable to that obtained using IL-2) could be generated from a population of NK cells (CD56) whereas no LAK activity was generated in PBMCs (Naume and Espevik, 1991). Another study using murine cells compared IL-7-induced LAK with IL-2 LAK. IL-7 LAK peaked on days 6–8. IL-7 was more effective at maintaining cytotoxic activity over longer periods of time than IL-2. IL-7 LAK were induced from secondary lymphoid tissue (spleen and nodes) but not from primary lymphoid tissue (thymus and bone marrow). LAK activity was abrogated by anti-CD8 or anti-Thy-1C and unaffected by antiCD4, anti-asialo GM1 or anti-NK1.1C suggesting that IL-7 LAK activity is probably mediated not by NK cells, but by T lymphocytes (Lynch and Miller, 1990). When compared with IL-2 and IL-12, IL-7 stimulates CD56 NK cells to secrete significantly lower amounts of soluble TNF receptor as well as lower levels of GM-CSF, but significantly higher GM-CSF levels (Naume et al., 1993). Assuming that IL-7-induced LAK activity resides within the T-cell population, it might then be possible to create a LAK immunotherapy treatment regimen that lacks some of the deleterious effects of IL-2 treatment which have been blamed on the NK cell popula-

tion. Circulating human T cells also proliferate when incubated in IL-7. Both CD4 and CD8 subsets respond to a similar degree; although, when T cells are separated on the basis of reactivity with an antibody (anti-CD45) that reacts with 220 kDa isoform (CD45RA) of the common leukocyte antigen, memory T cells (CD45RO) appear to respond more readily than naive T cells (CD45RA) (Welch et al., 1989). In addition, a variety of effects of IL-7 on monocytes has been reported. Activation of monocytes with IL-7 can result in the development of a tumor lytic phenotype using melanoma cells as targets (Alderson et al., 1991). Induction of mRNA for both IL-8 and macrophage inflammatory protein-1 b gene is induced in monocytes by IL-7 (Ziegler et al., 1991; Standiford et al., 1992), and monocytes incubated with IL-7 secrete large quantities of IL-6 as well as IL-1a, IL-1b and TNFa. This response can be abrogated by addition of IL-4. T cell-based immunotherapy has the advantage of increased specificity compared with LAK cell therapy. T-cell tumor lysis is MHC restricted and highly specific. Various cytokines including IL-2 and IL-4 are active in promoting the clonal expansion in vitro of T cells while maintaining their tumor lytic activity. IL-7 appears to have similar properties. IL-7 alone generates modest CTL activity which is augmented by IL-2, IL-6 or IL-4 (Bertagnolli and Herrmann, 1990; Hickman et al., 1990). Removal of CD8 cells results in decreased killing whereas removal of CD4 cells enhances the CTL response. IL-7 has been found to enhance cell proliferation and duration of growth more than IL-2. Addition of antiIL-4, anti-IL-2 or anti-IL-6 decreases the proliferation of CTL in culture (Bertagnolli and Herrmann, 1990; Jicha et al., 1992). CTLs harvested from draining nodes of tumor-bearing animals and incubated in IL-7 were four-fold more effective than CTL grown in media alone in adoptive transfer experiments (Lynch et al., 1991). CTLs incubated in IL-7 and adoptively transferred to mice bearing 3-day pulmonary metastases (MCA tumor) were effective in mediating tumor regression (Jicha et al., 1991). IL-7 stimulates proliferation of human TILs derived from renal cell carcinoma, but only if the TILs are first incubated in either IL-2 alone or in IL-2  IL-7. IL-7 stimulated proliferation of CD4 or CD8 TIL lines specific for renal cell carcinoma. IL-7 synergized with

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anti-CD3 in the induction of IFNc from short-term TIL cultures (Sica et al., 1993). Human T cells harvested from peripheral blood and incubated in IL-7 when restimulated with phorbol ester and ionomycin secrete IL-2, IL-4, IL-6 and IFNc. This effect was not seen as readily in cultures initiated with either IL-2 or IL-4. Both CD4 and CD8 subsets responded by cytokine secretion. Almost all the potential to secrete IL-4 and IL-6 in response to IL-7 pre-incubation resides within the memory subset as opposed to the naive population (Armitage et al., 1992a). The aforementioned observations suggest that IL-7, either alone or in conjunction with IL-2, acts to stimulate proliferation and tumor lytic activity in sensitized T cells and therefore may be clinically useful in the immunotherapy of malignancy. Some of the most promising data come from a study demonstrating that antitumor-specific T lymphocytes can be grown and expanded in vitro without restimulation for extended periods (up to 22 months) compared with T lymphocytes grown in IL-2 (Lynch and Miller, 1994). IL-7 alone (Maeurer et al., 1997), admixture of IL-7 to IL-2 and INFc also appears to preferentially expand and maintain tumor-specific and MHC class IIrestricted CD4 T lymphocytes (Cohen et al., 1993) from tumor-bearing patients. Of note, some of these tumor-reactive and MHC class II-restricted T lymphocytes preferentially secrete IFNc in response to autologous tumor cells (Maeurer et al., 1997). This observation substantiates an earlier report demonstrating IL-7-mediated effects in adoptive immunotherapy in human colorectal cancer xenografts in SCID mice. Exclusively the combination of IL-7 treatment and passive transfer of human autologous T cells resulted in enhanced survival of mice engrafted with the respective tumors; treatment with IL-7 alone showed no effect. The antitumor effects correlate with IFNc secretion by the passively transferred T cells and not by their cytolytic capacity (Murphy et al., 1993). The ability of IL-7 to generate antitumor-directed immune reactivity may also depend on the tumor type and the availability of T cells capable of recognizing tumor-associated peptides presented either in the context of MHC class I, or MHC class II molecules. For instance, application of IL-7 resulted in an up to 75% reduction in pulmonary metastases of the murine renal cell cancer line Renca (Komschlies et al., 1994). However, the pharmacoki-

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netics of IL-7 administered to humans have not yet been evaluated in detail. Some toxic side-effects have been observed in mice treated with IL-7 systemically (Komschlies et al., 1994). IL-7 may also be used to reconstitute the immune sytem in primary or secondary immunodeficiencies (e.g. induced by viral infections, by inherited abnormalities, such as Di George syndrome) or after BMT. For instance, the successful outcome of autologous BMT is limited by susceptibility to infections. Since the effective reconstitution of the immune system requires more than just the quantitative replacement of immune cells (usually achieved within 3–4 months after transplantation), the quality of the immune system is often impaired. Since IL-7 has not only growthpromoting, but also differentiation-promoting effects on both B- and T-cell lymphopoiesis, it may represent an attractive cytokine, potentially in combination with flt2/flt3, to reconstitute a competent immune system. Several studies have addressed this issue. For instance, IL-7 treatment of Balb/c mice after syngeneic BMT leads to increased thymic cellularity, increased RAG-1 expression, and to promotion of Vb8(D)J gene rearrangement of TCRs. The increased ‘quality’ of IL-7-treated mice is reflected in better mitogenic responses of thymic cells and in enhanced cytokine production provoked by influenza virus challenge (Abdul-Hai et al., 1996). Additionally, IL-7 accelerates PBL recovery of mice after cyclophosphamide, 5-fluorouracil treatment (Damia et al., 1992) or radiation (Faltynek et al., 1992). Using a metastatic breast cancer model in mice, IL-7 and BMT could significantly prolong survival, presumably due to enhanced immune cell reconstitution after splitdose chemotherapy using cyclophosphamide, cisplatin and nitrosourea (Talmadge et al., 1993). In another study IL-7 was shown to mobilize longterm reconstituting peripheral CD34 stem cells (Grzegorzewski et al., 1994). Such cells may be useful for stem cell transplantation, or for therapies using CD34 cells either for gene transduction or for maturation in vitro in order to generate potent antigen-presenting cells capable of initiating potent antitumor-directed cellular immune responses. Additionally, development of tumors in older individuals may reflect not only accumulative genetic alternations, but also a decreased capacity of the humoral and cellular immune system to identify and eradicate

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transformed cells efficiently. Several studies have demonstrated age-related alteration in T and B lymphocytes. In murine models, the ability of pro-B-cells to proliferate in response to stroma cells decreases with age (Stephan et al., 1996). This functional alteration is due to an impaired response of pro-B cells to IL-7, but not to other stromal-associated cytokines including stem cell factor or insulin-like growth (Stephan et al., 1997). The reduced IL-7 responsiveness appears not to be induced by inefficient IL-7R expression, but rather by as yet poorly defined intracellular signalling events mediated through the IL-7 receptor complex (Stephan et al., 1997). Thus, IL-7 may not only be implemented for primary or secondary immunodeficiency disorders: the functional impairment of the immune system in older individuals may in part be mediated by reduced IL-7responsive immune cells. Future studies may devise therapeutic strategies to overcome age-related immunodeficiencies which may play a role in decreased immune-surveillance. The effects of locally secreted IL-7 elaborated by genetically engineered tumor cells may overlap with some of the effects observed by systemic application. Transfection of cytokine genes into tumor cell lines has been developed as a theoretical strategy to increase the local regional response to the tumor in the hope that a heightened in situ response might translate to an enhanced systemic response, not only to the transfected tumor but also to the nontransfected or wild-type tumor. IL-7 transfection experiments have yielded some provocative results. Transfection of IL-7 into the murine tumor line (J5581) leads to tumor rejection in vivo. CD8 cells have been shown to be required for longterm tumor eradication although short-term regression has been noted in the absence of CD8 cells. While tumor transfected with IL-2, IL-4, TNF or IFNc regresses when placed in nude or SCID mice, IL-7transfected tumor requires the presence of CD4 cells for regression and no regression was observed in nude mice bearing tumor transfected with IL-7. In most of the murine studies, tumors were eventually rejected by the animals, while the in vitro growth was not affected by IL-7 (Aoki et al., 1992; Hock et al., 1991, 1993; McBride et al., 1992; Miller et al., 1993; Allione et al., 1994; Tepper and Mule, 1994). It appears that CD8 T cells play a major role in mediating tumor rejection

(Hock et al., 1991, 1993; Aoki et al., 1992; McBride et al., 1992; Miller et al., 1993) and that antigen-specific T cells are elicited upon immunization with IL-7secreting tumor cell lines (Aoki et al., 1992). However, other immune cells may also contribute to antitumor responses, since not only T lymphocytes, but also macrophages, eosinophils and basophils are present at the site of tumor rejection (Hock et al., 1991; McBride et al., 1992). In another study, the effects of locally secreted IL-7 and induction of tumor-specific cellular immune responses were examined (Cayeux et al., 1995). In B7-transfected mammary adenocarcinoma cells TS/A, T cells showed predominantly CD28 and CD25 marker expression and in IL-7 transduced tumor cells CD28 and CD25 marker expression, while in B7/IL7 tumor cells, the T-cell infiltrate typically showed CD28/CD25 expression. The doubletransfected tumor elicited enhanced immunity as compared with tumor cells expressing IL-7, or B7 alone, or non-transfected tumor cells admixed with Corynebacterium parvum (Cayeux et al., 1995). Human non-small-cell lung cancer cell lines infected with a retroviral construct containing the human IL-7 cDNA lead to alterations of cell surface expression of molecules (e.g. MHC class I, LFA-3) on co-cultured PBL favoring antitumor-directed immune responses (Sharma et al., 1996). In a rat model of glioma, IL-7transfected glioma cell clones, immunized into the hind leg, were found to inhibit growth of their intracerebral parental tumors, thereby enhancing recipient survival (Visse et al., 1999). Thus, despite the unfavorable location of intracerebral tumors, therapeutic immunization with IL-7-transfected tumor cells at a site distant to the brain, appears to be a viable therapy option. IL-7-transfected tumors cells may therefore represent a reasonable vaccine for eliciting strong antitumor-directed immunity in a variety of cancer types (Moller et al., 1996). Vaccination with IL-7 genemodified autologous melanoma cells was found to enhance antimelanoma cytolytic activity in patients participating in a phase I study (Moller et al., 1998). In three of six patients, antimelanoma cytolytic precursor cell frequencies increased by between 2.6- and 28fold, and two of these patients exhibited a minor clinical response. In a further clinical study, patients received autologous tumor cells which had been simultaneously transfected with IL-7 and GM-CSF using the MIDGE system (Minimalistic, Immunologically-

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Defined Gene Expression). All ten patients had progressive disease at trial onset. Cytotoxicity of patients’ PBLs increased significantly during treatment and five patients showed responses ranging from complete (1/10) response to stable disease (3/10). The remaining five patients progressed (Wittig et al., 2001).

IL-7 and the immune response to infection The role played by cytokines in regulating host immune responses to intra- and extracellular pathogens is becoming clearer. It is the TH1-type response, involving secretion of IFNc, IL-2 and IL-12, which represents the dominant reaction to obligate intracellular pathogens in mouse and man. Interestingly, a number of studies have recently suggested that IL-7 plays a central role in infections involving intracellular bacteria or parasites. IL-7 has been found to have both positive and negative effects depending on the time of IL-7 application and also on the infection model studied. IL-7 has been found to be beneficial in murine models of mycobacterial infection, or in infections with the parasite Toxoplasma gondii. Female A/J mice treated with IL-7 from the time of infection (2 mg daily for 2 weeks) with Toxoplasma gondii survived, while mice treated following infection died, as did untreated infected mice. Antibody-based depletion experiments revealed that both CD8 T cells and asialo-GM1 NK cells are required for protection against this intracellular parasite in vivo. It appears that these IL-7mediated effects are predominantly mediated by IFNc secretion, since in vivo depletion of IFNc abolished any IL-7 protective effects (Kasper et al., 1995). In another mouse model, combination of IL-7 with IL-1b augments anti-Listeria monocytogenes-directed immune responses. There is a predominance of peritoneal cd T lymphocytes which specifically react to heat-killed Listeria preparations in the presence of macrophages as accessory cells in an MHCindependent manner. The responsiveness of cd T cells to IL-7 was found to be enhanced in the presence of accessory cells. This effect could be replaced by exogenous IL-1 (Skeen and Ziegler, 1993). IL-7 also appears to be involved in the successful immune response to infection with mycobacteria. In Mycobacterium leprae infection the cellular immune

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response has been found to correlate both qualitatively and quantitatively with clinical manifestations. Increased IL-7 mRNA and IL-7-receptor mRNA expression correlates strongly with the tuberculoid form of the disease, in which the infection is limited. In contrast, no significant IL-7 mRNA expression is observed in the progressing lepromatous form of the disease (Sieling et al., 1995). Furthermore, IL-7 has been found to inhibit the intracellular growth of M. avium complex (MAC) in human macrophages in vitro (Tantawichien et al., 1996). MAC is a common opportunistic pathogen often found in patients with HIV infection. The major reservoir for MAC in the susceptible host appears to be the mononuclear phagocyte and consequently such infections are often resistant to standard treatment protocols. Additional treatment modalities may therefore be required in order to control MAC infections. Treatment of human macrophages with TNFa or GM-CSF, for example, leads to mycobacteriostatic or mycobactericidal activity (Denis, 1991). Furthermore, MAC infection of human macrophages leads to generation of TGFb which inhibits the capacity of infected cells to control bacterial growth (Bermudez, 1993). Despite this, treatment of human macrophages with IL-7 brings about a dose-dependent reduction in the number of intracellular bacteria. Addition of IL-7 to cultured macrophages prior to infection, results in diminished anti-MAC activity as compared with that obtained when IL-7 was added to cells post MAC infection (Tantawichien et al., 1996). We have obtained very similar results using virulent M. tuberculosis bacilli (Maeurer et al., 2000). IL-7 treatment of Balb/c mice preinfected with M. tuberculosis resulted in up to 100% increased survival when compared with untreated mice, or mice treated with IL-2 or IL-4. Such IL-7-mediated survival can be passively transferred to animals preinfected with mycobacteria, using spleen cells derived from IL-7treated and M. tuberculosis-infected animals. In contrast, transfer of cells from mice treated with IL-7 alone failed to increase survival as compared with control animals, indicating that priming with M. tuberculosis is required to elicit anti-microbial immune responses facilitated by IL-7 treatment (Maeurer et al., 2000). In other studies, IL-7-mediated effects were abolished by anti-human TNFa antibody. In contrast, IL-7 failed to decrease TGFb secretion by

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macrophages upon infection, an observation found to be true for IL-7-mediated down-regulation of IL-2 as well as LPS-induced TGFb mRNA expression in murine macrophages (Dubinett et al., 1993). Thus, IL-7 may exert some of its effects by inducing or potentiating proinflammatory cytokines, such as IL-1a, IL-1b, IL-6 and TNFa (Alderson et al., 1991). Additionally, some of the antibactericidal effects of macrophages involve nitric oxide and superoxide radicals, both of which are induced by IL-7 (Alderson et al., 1991; Gessner et al., 1993). Unfortunately, IL-7 can also negatively influence ‘clinical outcome’ in animals with intracellular infections. Previous studies have shown mediation by IL-7 of antimicrobial activity against the intracellular parasite Leishmania major in murine macrophages in vitro (Gessner et al., 1993). However, treatment of susceptible Balb/c mice with IL-7 at the onset of L. major infection leads to enhanced lesion development and accelerates death of treated animals correlating with up to 40-fold increased parasite burden in spleens and lymph nodes when compared with untreated animals. Analysis of cellular immune responses of such animals showed that lymphocytes from IL-7-treated mice produced comparable amounts of the TH2 cytokines IL-4 and IL-10, but significantly less IFNc in response to antigen (Gessner et al., 1995). In agreement with this observation, decreased IFNc production resulting from increased levels of IL-7, leads to aggravation of disease in Schistosoma mansoniinfected mice (Wolowczuk et al., 1997). These observations suggest that a number of other factors may be involved in the complex interactions of cytokines. IL-7 has, for instance, been shown to up-regulate anti-CD3, or anti-CD3/anti-CD28-induced IFNc and IL-4 mRNA expression in human T lymphocytes (Borger et al., 1996). An increase in total cell numbers in the B-cell compartment has been observed in IL-7-treated mice. In order to elucidate the nature of any potentially deleterious B-cell responses, mice with the XSCID immunodeficiency were evaluated. Typically, these mice lack B1 cells and have reduced numbers of B2 cells which are only partially functional. B1 cells (formerly referred to as Ly-1 B or CD5 B cells) represent a small subpopulation with a distinct phenotype, and developmental and functional properties. B1 cells express a unique array of cell surface molecules, in addition to expression of the CD5 marker; they are

preferentially generated from fetal or neonatal sources of progenitors, and the antibodies derived from B1 cells are predominantly of the IgM class, show minimal hypermutation and a high frequency of low-affinity, poly- or self-reactive specificities (for review see Stall and Wells, 1996). The absence of these cells leads to reduced susceptibility against infections with intracellular parasites (e.g. Leishmania species). Treatment of X-SCID mice with a single IL-7 dose concomitant with Leishmania infection resulted in a clinical course resembling that of susceptible Balb/c mice with up to 100-fold enhanced parasite load in treated animals. Again, examination of CD4 Leishmaniaspecific T lymphocytes revealed that IFNc secretion is reduced in IL-7-treated X-SCID mice compared with control animals, and that the population of B2 (B220, sIgM, MHC class II) cells appeared to be significantly enhanced. However, the mechanisms of the disease-aggravating effects of IL-7 remain unclear. One potential mechanism may be antigen presentation by B cells, an event which may lead to preferential activation and expansion of TH2 lymphocytes. As with parasitic infection, a similar dichotomy of IL-7 has emerged in infection with the human immunodeficiency virus (HIV). Previous studies indicated that exogenous rhIL-7 can augment the generation of antiviral CTL responses (Carini and Essex, 1994). Examination of HIV-infected individuals testing negative for anti-HIV-1-specific CTL reactivity revealed that CD8 and CD4 T cells lack cell surface expression of IL-7R, which may be attributed to production of insufficient numbers of IL-7R upon retroviral infection, or alternatively, to increased shedding of IL-7R (Carini and Essex, 1994; Carini et al., 1994). In the case of CD8 T cells, IL-7R expression is partially restored in patients undergoing effective antiretroviral therapy (MacPherson et al., 2001). Because HIV infection is associated with loss of cytotoxic CD8 T-cell activity, and also with reduced numbers in the CD4 cell compartment, several cytokines capable of modulating the immune system have been considered for treatment of HIV-positive individuals. IL-7 represents one of these candidates (e.g. IL-2, IL-12, or IL-15) as it not only enhances anti-HIV-directed CD8 T-cell responses, but also augments both CD4 T helper cell-dependent humoral immune responses and CD8 cytotoxic T-cell reactivity in mice immunized with the HIV envelope protein (Bui et al., 1994).

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Caution must be exercised, however, prior to incorporation of cytokines including IL-7 into clinical protocols, since addition of exogenous IL-7 induced virus replication and increased proviral DNA levels in PBMC cultures, and increased the levels of doubly spliced HIV-1 tat RNA (Smithgall et al., 1996). These effects are not inhibited by neutralizing IL-1b, IL-2, IL-6 or TNFa activity. Although CD8 T cells inhibited the increase in viral replication induced by IL-7 stimulation, they failed to prevent virus replication following CD3 ligation in the presence of IL-7, an event that can also be mimicked by adding IL-7 to anti-CD3 antibody-stimulated HIV PBMC cultures, resulting in enhanced HIV production (Moran et al., 1993). The results obtained from such studies have addressed the role of exogenous added IL-7 in HIV replication in vitro, but not the role of endogenous IL-7 on viral load or viremia. Serum levels of IL-7 are known to be elevated in HIV-1-infected subjects, and patients undergoing highly active antiretroviral therapy (HAART) continue to have elevated IL-7 serum levels despite successful treatment, thus bringing into question the impact of IL-7 immunotherapy on the process of immune reconstitution (Darcissac et al., 2001). This has, however, been studied. McCune and coworkers observed that not only were increased IL-7 serum levels accompanied by HIV-1-mediated T-cell depletion, but that they were also associated with increased viral load (Napolitano et al., 2001). The authors suggest that increased IL-7 production results from the existence of a compensatory feedback loop that leads to enhanced T-cell differentiation, and forms part of a homeostatic response to T-cell depletion. Raised IL-7 levels have also been observed in severe combined immunodeficiency syndrome, acute lymphocytic leukemia and various other lymphopenic conditions (Mackall, personal communication in Bui et al., 1994; Napolitano et al., 2001). The enhanced IL-7 levels associated with HIV-1 infection appear to be the result of increased synthesis by dendritic-like cells within peripheral lymph nodes. IL-7 is not just associated with enhanced viral load, it also accelerates HIV1 infection in vivo (mouse model, in Napolitano et al., 2001). Thus, although IL-7 has been touted as a possible treatment for HIV, its use may actually be detrimental, enhancing replication and therefore viral load and leading to accelerated disease progression. There are several other clinical conditions, in which elevated

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IL-7 levels have been determined. For instance, plasma and synovial fluid levels of IL-7 were significantly elevated in patients with systemic juvenile rheumatoid arthritis, but not in individuals with poly, or pauciarticular JRA, or in patients with other rheumatic diseases (De Benedetti et al., 1995). Activated T cells have been associated with increased osteoclast formation and bone resorption linked with inflammation. T cells stimulate osteoclast formation by producing osteoclastogenic cytokines. IL-7 stimulation of T cells leads to increased production of such osteoclastogenic factors from T cells and is therefore indirectly involved with osteoclast formation leading to bone loss (Weitzmann et al., 2000). In addition, IL-7 was found to be increased in patients with untreated Hodgkin’s lymphoma (Trumper et al., 1994) and in some patients with colorectal, or renal cell cancer (Trinder et al., 1999). However, the precise source, and the functional consequence of such elevated IL-7 serum levels has yet to be determined. IL-7 mRNA has been detected in a number of different infections, and exogenously added IL-7 either provided by the recombinant protein or by retroviral infections, has been shown to augment specific cellular immune responses. For instance, IL-7 mRNA has been detected in Helicobacter pylori-positive gastritis, but not in H. pylori-negative controls (Yamaoka et al., 1995). Studies with an IL-7R neutralizing antibody have indicated a key role for IL-7R in the development of H. felis-induced gastritis in mice (Ohana et al., 2001), and IL-7R has also been shown to be up-regulated in epithelial cells following their infection with enteropathogenic bacteria in culture (Yamada et al., 1997). Salmonella typhimurium, enteropathogenic Escherichia coli and enteroinvasive E. coli all induced IL-7R expression in the colonic epithelial cell line T84, suggesting that communication between the epithelium and mucosal lymphocytes may be involved in the modulation of infection-induced mucosal inflammation. Others have shown that IL-7 assists in induction of antiviral specific T-cell responses using synthetic peptides and IL-7 as an adjuvant (Kos and Mullbacher, 1992) and that IL-7 overcomes anergy in parasite-specific cellular immune responses (Sartono et al., 1995), and facilitates expansion of tetanus toxoid (Kim et al., 1994), or dengue-virus-specific cytotoxic CD4 T-cell clones (Berrios et al., 1996).

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T-cell homeostasis and ‘immune reconstitution’ Recent evidence has implicated IL-7 as being a key cytokine in at least two important clinical situations (Plate 13.6). First, IL-7 serves as a central cytokine in T-cell homeostasis and second, IL-7 appears to be enhanced, potentially as a feedback mechanism, in response to low lymphocyte counts (for review see Fry and Mackall, 2001). In general, lymphoid homeostasis is required to ensure immune responsiveness to a wide variety of antigens and to prevent immunodeficiency. The immune system has to maintain naive T cells as well as memory T cells which are able to react effectively to the target antigen upon reencounter. Naive and memory, as well as effector T-cell subsets are defined by different phenotypic markers, exhibit different homing characteristics (Jourdan et al., 2000), and may also show different requirements for cytokines in order to ensure survival in the absence of antigenic stimulation (Lantz et al., 2000). Moreover, (CD4) memory T cells are comprised of at least two populations, ‘central memory’ T cells, which express the chemokine receptor CCR7 and CD62 ligand (L) and home to secondary lymphoid organs, and ‘effector memory’ T cells, which are CCR7 negative and are capable of migrating into non-lymphoid tissues (Manjunath et al., 1999; Sallusto et al., 1999; Iezzi et al., 2001; Masopust et al., 2001; Reinhardt et al., 2001). IL-7 (as well as IL-15) is able to expand effector memory T cells effectively; central memory T cells do not respond well and naive T cells fail to respond to IL-7 (Geginat et al., 2001), which suggests that IL-7 may help in sustaining and expanding antigenexperienced T cells, a situation which is desirable in patients with chronic infectious disease or suffering from cancer. More interestingly, in T cell-depleted conditions, naive T cells undergo spontaneous proliferation in response to MHC–peptide complexes in a cytokine-dependent manner, a condition termed ‘homeostatic proliferation’. Exclusively IL-7, but not IL-4 or IL-15, is able to induce ‘homeostatic proliferation’ (Tan et al., 2001), which makes IL-7 a key candidate to facilitate T-cell expansion in T cell-depleted hosts. The nature of this expansion could formally be two-fold: first, IL-7 may preferentially expand distinct T-cell subsets, alter the T-cell repertoire and induce

T-cell activation. Second, IL-7 may not directly impact on the T-cell pool, but rather lead to ‘preservation’ of the immune repertoire. Indeed, the latter possibility appears to be the case: IL-7, but not IL-2, IL-4 or IL-6, expands and maintains naive (CD45RA) T cells and directly induces telomerase activity which may help in preserving the life-span of naive T lymphocytes (Soares et al., 1998; Webb et al., 1999; Geiselhart et al., 2001; Hassan and Reen, 2001; Rathmell et al., 2001; Vakkila et al., 2001; Vivien et al., 2001). Most of the data pertaining to T-cell homeostasis have been generated by analyzing the CD4 T-cell population. A similar central role emerged for IL-7 for CD8 T cells: IL-7 appears to be crucial for survival and maintenance of CD4 and CD8 T cells if the host is lymphopenic. CD8 (but not CD4) T-cell survival in normal hosts also depends on IL-7. In contrast, an immediate T-cell response to infection does not require IL-7, but the generation of a strong and longlasting memory T-cell response is again associated with the presence of IL-7 (Schluns et al., 2000). A physiological ‘immuno-deficiency’ is represented by non-efficient functional activity of the cellular immune system in later life. Thymic atrophy has been postulated to contribute to the contraction of the T-cell repertoire and to a decline in immune effectiveness. Recent studies have shown that IL-7 alone is able to reverse the age-associated effects in thymocyte development (Andrew and Aspinall, 2001), leading to improved TCR-b rearrangement at the stage of the triple negative thymocytes (see Figure 13.4). In addition, IL-7 impacts on the development of thymic dendritic cells (Varas et al., 1998) and may interact at different levels within the thymic architecture with thymocytes: MHC class II thymic epithelium and fibroblasts express fibronectin and heparan sulfate, extracellular matrix components which effectively bind IL-7 (Banwell et al., 2000). Recent studies linked age-associated thymic atrophy with a decline in IL-7 production (Andrew and Aspinall, 2002) and showed that a decline of thymic IL-7 is associated with the dose of (experimentally) applied radiation (Chung et al., 2001), which substantiates the notion that IL-7 may represent an interesting candidate to reverse an age-associated decline in immune responsiveness, particularly in the case of patients suffering from cancer at older ages which presumably requires a broad and effective immune repertoire. The second key

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REFERENCES

function mediated by IL-7 represents the capacity to expand and establish a functional immune system in lymphopenic hosts. As discussed above, Il-7 serum levels show an inverse relationship to the number of lymphocytes both in bone marrow recipients (Bolotin et al., 1999) as well as in patients with HIV infection (Llano et al., 2001; Napolitano et al., 2001). These data support the notion that IL-7 may be implemented as a therapeutic agent to restore immune competence. Indeed, IL-7 has been shown to facilitate engraftment and to restore protective immunity in athymic T celldepleted mice (Fry et al., 2001). Of note, IL-7 application after allogeneic bone marrow transplantation did improve immune reconstitution without the aggravating side-effects of graft-versus-host disease but with preservation of the beneficial effects of allogeneic transplantation. The graft-versus-leukemia effect remained intact, an observation which has been associated with low IL-7R expression on activated and memory alloreactive donor-derived T cells (Alpdogan et al., 2001). Thus, IL-7 represents a key mediator of immune competence and may hold great promise in clinical applications which require T-cell homeostatic expansion and maintenance of a strong and effective immune response.

SUMMARY IL-7 is an important lymphopoietin and plays a critical role in both B- and T-cell development. IL-7 promotes expansion of T lymphocytes exhibiting antigen-specific reactivity. IL-7 may be implemented to promote strong and effective immune responses against tumor cells, or directed against microbial or viral infections. It may also be useful in reconstituting an effective and functional immune system after bone marrow transplantation or helping to design novel strategies for immune reconstitution in patients with cancer or with HIV infection.

ACKNOWLEDGMENTS We thank Monika Wiedmann for expert secretarial assistance and editing. Dr Ingeborg Zehbe, Dept. of Med. Microbiology, contributed the picture of IL-7 in cervical cancer and Dr Torsten Reichert, the Dept. of

Oral and Maxillofacial Surgery, University of Mainz, provided the picture of IL-7 protein expression in head and neck cancer. This work was supported by grants DFG SFB 432/A9, SFB 490/C4 and the Stiftung für Innovation Rheinland-Pfalz (to M.M.).

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cells: a first clinical phase I/II trial. Hum. Gene Ther. 12, 267–278. Wolf, M.L., Weng, W.K., Stieglbauer, K.T. et al. (1993). Functional effect of IL-7-enhanced CD19 expression on human B cell precursors. J. Immunol. 151, 138–148. Wolowczuk, I., Delacre, M., Roye, O. et al. (1997). Interleukin-7 in the skin of Schistosoma mansoni-infected mice is associated with a decrease in interferon-gamma production and leads to an aggravation of the disease. Immunology 91, 35–44. Yamada, K., Shimaoka, M., Nagayama, K. et al. (1997). Bacterial invasion induces interleukin-7 receptor expression in colonic epithelial cell line, T84. Eur. J. Immunol. 27, 3456–3460. Yamada, N., Wakugawa, M., Kuwata, S. et al. (1996). Changes in eosinophil and leukocyte infiltration and expression of IL-6 and IL-7 messenger RNA in mite allergen patch test reactions in atopic dermatitis. J. Allergy Clin. Immunol. 98, S201–206. Yamaoka, Y., Kita, M., Kodama, T. et al. (1995). Expression of cytokine mRNA in gastric mucosa with Helicobacter pylori infection. Scand. J. Gastroenterol. 30, 1153–1159. Yasue, T., Nishizumi, H., Aizawa, S. et al. (1997). A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5. Proc. Natl Acad. Sci. USA 94, 10307–10312. Yasunaga, M., Wang, F., Kunisada, T. and Nishikawa, S. (1995). Cell cycle control of c-kitIL-7R B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium. J. Exp. Med. 182, 315–323. Ye, S.K., Maki, K., Kitamura, T. et al. (1999). Induction of germline transcription in the TCRgamma locus by Stat5: implications for accessibility control by the IL-7 receptor. Immunity 11, 213–223.

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Ye, S.K., Agata, Y., Lee, H.C. et al. (2001). The IL-7 receptor controls the accessibility of the TCRgamma locus by Stat5 and histone acetylation. Immunity 15, 813–823. Yoshida, H., Honda, K., Shinkura, R. et al. (1999). IL-7 receptor alpha CD3() cells in the embryonic intestine induces the organizing center of Peyer’s patches. Int. Immunol. 11, 643–655. Yoshioka, R., Shimizu, S., Tachibana, J. et al. (1992). Interleukin-7 (IL-7)-induced proliferation of CD8 T-chronic lymphocytic leukemia cells. J. Clin. Immunol. 12, 101–106. Young, J.C., Gishizky, M.L. and Witte, O.N. (1991). Hyperexpression of interleukin-7 is not necessary or sufficient for transformation of a pre-B lymphoid cell line. Mol. Cell Biol. 11, 854–863. Yron, I., Wood, T.A., Jr., Spiess, P.J. and Rosenberg, S.A. (1980). In vitro growth of murine T cells. V. The isolation and growth of lymphoid cells infiltrating syngeneic solid tumors. J. Immunol. 125, 238–245. Yu, H., Li, X., Marchetto, G.S. et al. (1996). Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation. J. Biol. Chem. 271, 29993–29998. Zea, A.H., Curti, B.D., Longo, D.L. et al. (1995). Alterations in T cell receptor and signal transduction molecules in melanoma patients. Clin. Cancer Res. 1, 1327–1335. Zeng, Y.X., Takahashi, H., Shibata, M. and Hirokawa, K. (1994). JAK3 Janus kinase is involved in interleukin 7 signal pathway. FEBS Lett 353, 289–293. Ziegler, S.F., Tough, T.W., Franklin, T.L. et al. (1991). Induction of macrophage inflammatory protein-1 beta gene expression in human monocytes by lipopolysaccharide and IL-7. J. Immunol. 147, 2234–2239.

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14 Interleukin-9 Jean-Christophe Renauld and Jacques Van Snick Ludwig Institute for Cancer Research, Brussels Branch and Experimental Medicine Unit, Catholic University of Louvain, Brussels, Belgium

We don’t know who we are until we see what we can do Martha Grimes

INTRODUCTION Originally described as a factor stimulating in vitro T cell and mast cell growth, IL-9 has seen its scope of action gradually expanding to include B cells, eosinophils, macrophages, hematopoietic and neuronal precursors as well as lung epithelial cells. More recently, significant progress has been made in the understanding of IL-9 biological activities in vivo. This work has led to the recognition of IL-9 as an important player in asthma and as a significant element in the defense against nematode infections. This chapter briefly retraces the discovery of murine and human IL-9 and focuses on our current knowledge of its cellular origins, cellular targets, signal transduction and biological functions. Mouse IL-9 was purified from helper T-cell supernatants based on its ability to support the growth of a murine T-cell line (Uyttenhove et al., 1988), and its structure was determined both by protein sequencing (Simpson et al., 1989) and cDNA cloning (Van Snick The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

et al., 1989). The purified protein, originally designated P40 on the basis of its apparent size in gel filtration, is characterized by a high level of glycosylation. Although recombinant IL-9 produced in E. coli recapitulates all in vitro IL-9 activities, glycosylation plays a critical role in the in vivo activity of the protein. Indeed, natural IL-9, purified from transgenic mouse serum, has a half-life of 40 min, which is reduced more than three-fold upon desialidation (unpublished data). Mouse IL-9 has also been described as a factor enhancing the proliferation of mast cell lines in response to IL-3 or IL-4. This activity was originally called mast cell growth-enhancing activity (MEA) (Hültner et al., 1990a). High levels of MEA were also found in the supernatant of a murine T-helper cell line derived by Schmitt and colleagues, who had observed that these cells produced a T-cell growth factor distinct from IL-2 and IL-4, which was called TCGF-III (Schmitt et al., 1989). Human IL-9 was cloned both by expression cloning Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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-10 1 10 20 30 h MLLAMVLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCS ** * * ** ** ** * ** * * ** ** *** ** m MLVTYILASVLLFSSVLGQRCSTTWGIRDTNYLIENLKDDPPSKCSCS 40 50 70 h ANVTSCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSV ******** * * ** ** * * * ** * * * *** * m GNVTSCLCLSVPTDDCTTPCYREGLLQLTNATQKSRLLPVFHRVKRIV 80 90 100 110 120 h EVLKNNKCPYFSCEQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI ***** ** **** ***** *** * ****** *** * m EVLKNITCPSFSCEKPCNQTMAGNTLSFLKSLLGTFQKTEMQRQKSRP

FIGURE 14.1 Alignment of human and mouse IL-9 protein sequences. Amino acids are indicated in the one-letter code. The 10 cysteine residues of the mature protein are boxed and arrows indicate the potential N-linked glycosylation sites. Amino acid number 1 refers to the N-terminus of the mature mouse protein. of a factor stimulating the growth of a human megakaryoblastic leukaemia (Yang et al., 1989) and by cross-hybridization with the mouse gene (Renauld et al., 1990a). A comparison of the mouse and human IL-9 sequences is shown in Figure 14.1. Both deduced protein sequences contain 144 residues with a typical signal peptide of 18 amino acids. The overall identity reached 69% at the nucleotide level and 55% at the protein level. Four potential N-linked glycosylation sites are present in both sequences. This glycosylation is responsible for the discrepancy observed between the predicted relative molecular mass (14 150) and that measured for native IL-9 (30–40,000). The sequence is also characterized by the presence of 10 cysteines which are perfectly matched in both mature proteins and a strong predominance of cationic residues, which explains the elevated pI measured with purified IL-9 (Uyttenhove et al., 1988).

IL-9-RESPONSIVE CELLS Mast cells Mast cells were among the first identified targets for IL-9. In 1989, Hültner and colleagues observed that a factor, present in spleen cell-conditioned medium, was able to synergyze with IL-3 for the proliferation of permanent bone marrow-derived mast cell lines (BMMC) such as L138.8A (Hültner et al., 1989). This

factor, provisionally designated MEA proved to be similar to IL-9 (Hültner et al., 1990a). Responses of BMMC to IL-9 are highly dependent on the time spent in vitro. When primary BMMC are derived from hematopoietic progenitors, IL-9 is not sufficient to sustain mast cell growth, but synergistically enhances the proliferation induced by IL-3 or the combination of IL-3 and IL-4. Similar results are obtained by combining IL-9 with stem cell factor (SCF) (Godfraind et al., 1998). Moreover, in the absence of other factors, IL-9 significantly increases the survival of primary BMMC. In addition to its activity on growth and survival, IL-9 appears to be a potent regulator of mast cell function and differentiation. IL-9 up-regulates the expression of the high-affinity IgE receptor in murine mast cell lines (Louahed et al., 1995; Rupp et al., 2000). IL-9 is also a major inducer of proteases, including granzyme B and mast cell-specific proteases of the mouse mast cell proteases (MMCP) family, particularly MMCP-1, -2 and -4 (Eklund et al., 1993; Louahed et al., 1995; Godfraind et al., 1998; Rupp et al., 2000). IL-9-induced expression of MMCP-1 may be due to endogenously expressed TGF-b1, because anti-TGFb antibodies block the effect of IL-9 on the percentage of MMCP-1 cells in primary BMMC cultures (Miller et al., 1999). Other cytokines induced by IL-9 in murine mast cell lines include IL-6, IL-13 and IL-22 (Hültner et al., 1990b; Dumoutier et al., 2000 and unpublished data from our laboratory). In vivo, IL-9 transgenic mice show a massive mast cell infiltration of the gastric and intestinal epithelium, as well as in the upper airway epithelium and kidneys, but not in other organs such as the skin (Godfraind et al., 1998). Lung-specific expression of an IL-9 transgene resulted in a similar mast cell accumulation in the airway epithelium (Temann et al., 1998). Mast cell responses to IL-9 do not need longterm exposure to the cytokine as administration for 2 consecutive days of 50 ng IL-9, purified from the serum of IL-9 transgenic animals, to normal mice is sufficient to activate mast cells, as judged by the increase of MMCP1 in the serum (unpublished data). This observation indicates that small amounts of IL-9 can activate mast cells in vivo even in the absence of any other stimulus, contrasting with in vitro responses, where IL-9 had little activity without additional cytokines such as IL-3 or SCF (Godfraind

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et al., 1998). However, injections of antibodies directed against c-kit, the SCF receptor, blocked mastocytosis in IL-9 transgenic mice. As a constitutive SCF expression was observed in both IL-9 transgenic and control mice, these observations indicate that neither SCF nor IL-9 are sufficient to induce mastocytosis but that the synergistic activity of these cytokines is responsible for the in vivo amplification of this cell population in IL-9 transgenic mice (Godfraind et al., 1998). By contrast, the effect of IL-9 inhibition on mast cell responses has not been extensively described. In a model of Schistosoma infection, a decrease in mast cells present in granulomas was reported in IL-9-deficient mice, suggesting that, at least under certain circumstances, IL-9 is required for optimal mast cell responses (Townsend et al., 2000).

B lymphocytes The involvement of IL-9 in mast cell activation and proliferation, as well as IL-9 production during parasite infections and TH2 activation, raised the hypothesis of a potential role for this factor in IgE-mediated responses. In a mouse model, IL-9 was shown to synergize with suboptimal doses of IL-4 for the IgE and IgG1 production by lipopolysaccharide (LPS) activated semi-purified B cells, but did not induce any IgE or IgG1 production in the absence of IL-4. By contrast, IL-9 did not affect the IL-4-induced CD23 expression by LPS-activated B cells, indicating that its activity did not consist of a simple up-regulation of IL-4 responsiveness by the B cells (Petit-Frère et al., 1993). Moreover, these experiments have not ruled out the possibility that the effect of IL-9 observed on murine B cells might be mediated by accessory cells. In the human, very similar observations have been reported using semi-purified peripheral B cells (Dugas et al., 1993). In this experimental system, IL-9 synergized with IL-4 for IgE and IgG but not for IgM production. Moreover, IL-9 also potentiated the IL-4induced IgE production by sorted CD20 cells upon co-stimulation by irradiated EL4 murine T cells, thereby suggesting a direct activity on B cells (Dugas et al., 1993). In addition, anti-IL-9 antibodies were reported to block the IgE and IgG4 induced by the combination of IL-4 and IL-7 (Jeannin et al., 1998). Although IL-9-deficient mice did not show obvious defects in antibody responses (Townsend et al., 2000),

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observations made in IL-9 transgenic mice illustrated the potential activity of this cytokine in humoral responses in vivo. Both basal titers of all Ig classes and antigen-specific antibody responses are indeed increased in the serum of transgenic animals. In addition, these mice are characterized by a dramatic increase in the number of peritoneal B1 cells, but not in conventional B cells (Vink et al., 1999), suggesting that IL-9 may act preferentially on a B-cell population predominantly involved in autoimmunity. However, IL-9 transgenic mice do not have increased levels of autoantibodies, and the expansion of peritoneal B cells in these mice is mainly due to B1b cells, a functionally ill-defined B cell subset, which differs from B1a cells by the absence of the CD5 surface marker (Vink et al., 1999). Recent data obtained in a murine model of lung fibrosis may shed some light on the activity of this population of IL-9-responsive B cells. Intratracheal administration of crystalline silica particles induces lung fibrosis (Davis, 1986). When this treatment was applied to IL-9 transgenic animals or to mice receiving IL-9 injections, histologic examination and measurement of lung hydroxyproline content showed a clear reduction in the severity of the fibrosis (Arras et al., 2001). This protective effect was accompanied by a B-lymphocyte infiltration detected in bronchoalveolar lavage (BAL) and in pulmonary parenchyma, and by a reduced IgG1/IgG2a ratio in BAL. Most importantly, IL-9 failed to protect against silica-induced fibrosis in B-cell-deficient animals, and the protective effect of IL-9 was restored after injection of peritoneal B1 lymphocytes (Arras et al., 2001). Current investigations focus on the putative mediators by which B1 lymphocytes might modulate lung fibrosis.

T lymphocytes Although T lymphocytes were the first identified targets for IL-9, the physiological role of IL-9 for T cells remains puzzling. Initial observations in a murine system suggested that the activity of IL-9 was restricted to some T-helper cell clones (Uyttenhove et al., 1988). Noticeably, freshly isolated T cells never responded to this cytokine, but responsiveness became apparent after prolonged in vitro culture. Human T lymphocytes also need preactivation to respond to IL-9, and significant proliferations could

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be induced by IL-9 when human PBMC were preincubated for 10 days with PHA, IL-2 and irradiated allogeneic feeder cells (Houssiau et al., 1993). Similar results have been reported for T cells activated with PHA for 3 or 7 days (Lehrnbecher et al., 1994). These results suggest that responses to IL-9 require previous T cell activation. Transfection of mouse IL-9-dependent T-cell lines with an IL-9 cDNA expression vector and injection of these cells into syngeneic animals resulted in widespread lymphoma development (Uyttenhove et al., 1991). Similar observations have been reported in a rat model, using an IL-2-dependent T-cell lymphoma that, after infection with murine polytropic retroviruses, became IL-2 independent by induction of an autocrine loop involving IL-9 and its receptor (Flubacher et al., 1994). These results indicated that IL-9 could act as a T-cell oncogene. This hypothesis was validated by the fact that 5–10% of FVB mice expressing an IL-9 transgene, spontaneously developed lymphoblastic lymphomas (Renauld et al., 1994). The growth-promoting activity of IL-9 for T-cell tumors was confirmed in vitro, using other models of thymic lymphomas generated in normal mice (Vink et al., 1993). Moreover, IL-9 was found to protect such tumor cells against dexamethasone-induced apoptosis, even for those cell lines where in vitro proliferation is completely cytokine independent (Renauld et al., 1995). In the human, a link between dysregulated IL-9 production and lymphoid malignancies has been initially suggested by the observation that lymph nodes from patients with Hodgkin’s disease or large cell anaplastic lymphomas constitutively produce IL-9 (Merz et al., 1991). Constitutive IL-9 expression was also detected in HTLV-1-transformed T cells (Kelleher et al., 1991) and in Hodgkin cell lines (Merz et al., 1991; Gruss et al., 1992; Trümper et al., 1993). Moreover, an in vitro autocrine loop involving IL-9 has been reported for one of these Hodgkin cell lines (Gruss et al., 1992). Such an autocrine loop may also play a role in HTLV-I leukemias, as illustrated by the cis/trans-activation of the IL-9 receptor gene by insertion of the HTLV-I long terminal repeat (LTR) in one leukemia cell line (Kubota et al., 1996). The role of IL-9 in thymic maturation was studied

using human thymic precursor organ cultures, taking advantage of the fact that human CD34CD38Lin fetal liver cells or CD34Lin cells from cord blood can be cultured in vitro in thymic lobes from nude mice. An anti-IL-9 receptor antibody was found to inhibit growth of T cell precursors and more specifically prevented transition from CD34 to CD34CD1CD4CD3 immature thymocytes (De Smedt et al., 2000). These results raise the possibility that IL-9 is involved in normal T-cell development, but contrast with the fact that IL-9-deficient mice were reported to have a normal thymus (Townsend et al., 2000). This suggests either that IL-9 might be critical for human but not mouse thymus development, or that cytokines are less redundant in an in vitro experimental setting using human precursors and mouse thymus environment than in a pure murine model. Alternatively, the role of IL-9 in thymic development might also vary between fetal and adult life. In this respect, it was shown that fetal murine thymocytes, but not adult thymocytes, proliferate in the presence of IL-9 in vitro (Suda et al., 1990a, 1990b). Although IL-9 is specifically produced by TH2 but not TH1 cells (Gessner et al., 1993), its role in the TH1/TH2 balance in vivo remains unclear. Stimulation of lymph node cells in vitro with plate-bound antiCD3 antibodies induced similar IL-4, IL-5 and IFNc production in control and IL-9-deficient mice, and seric concentrations of IgG subclasses or IgE were identical in both strains (Townsend et al., 2000). Also, in vivo T-cell-dependent anti-ovalbumin antibody and cytokine production were not altered by IL-9 gene disruption. In IL-9 transgenic mice, IgG1 and IgE concentrations are preferentially increased, as expected from increased TH2 responses, but IgG2a concentrations are also significantly increased, as in TH1-like responses (Vink et al., 1999). By contrast, in immunoglobulin levels in BAL from silicotic mice, IL-9 overexpression surprisingly led to a decreased IgG1/IgG2a ratio, suggesting favored TH1 responses (Arras et al., 2001). Finally, analysis of in vivo cytokine expression in transgenic mice shows that IL-13 expression is strongly up-regulated in lungs and gut (unpublished observations from our laboratory). Further studies are definitely needed better to understand the role of IL-9 on the tuning of T-helper cell responses.

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Hematopoietic progenitors Although human IL-9 was identified as a growth factor for megakaryoblastic leukemia Mo7E, its activities as a hematopoietic growth factor have remained fairly modest. In fact, IL-9 does not seem to be active on normal megakaryoblastic precursors but supports the clonogenic maturation of erythroid progenitors in the presence of erythropoietin (Donahue et al., 1990). This activity was confirmed by several groups and reproducibly observed with highly purified progenitors after sorting for CD34 cells and T-cell depletion (Birner et al., 1992; Lu et al., 1992; Sonoda et al., 1992; Schaafsma et al., 1993), particularly in synergy with SCF (Lemoli et al., 1994; Sonoda et al., 1994). An activity on early multipotent progenitors was also observed by a two-step liquid culture assay with CD34CD33DR cells (Lemoli et al., 1994) In this assay, the majority of the colonies observed with IL-9 or IL-9 and SCF corresponded to CFU-GM. Noticeably, Schaafsma and colleagues observed that IL-9 also promoted some granulocytic as well as monocytic colony (CFU-GM) growth from CD34 CD2 progenitors from some bone marrow donors (Schaafsma et al., 1993). Experiments comparing the effects of IL-9 on fetal and adult progenitors have shown that addition of IL-9 to cultures of fetal progenitors induces maturation of CFU-Mix and CFUGM while IL-9 is also more effective on fetal cells of the erythroid lineage (Holbrook et al., 1991). In addition, IL-9 was found to increase the in vitro proliferation of human myeloid leukemic cells in a clonogenic assay in methylcellulose, suggesting a preferential activity on transformed myeloid cells compared with their normal progenitors (Lemoli et al., 1996). These observations are in line with findings on T cells. Thus, murine thymic lymphomas (Vink et al., 1993), but not normal adult thymocytes (Suda et al., 1990a, 1990b), respond to IL-9.

Monocytes and macrophages Besides the above-mentioned effect of IL-9 on myeloid progenitors and myeloid tumors, IL-9 receptor expression had been detected on certain mouse macrophage cell lines (Druez et al., 1990) but functional responses of macrophages to IL-9 have not been reported. Recently, it was found that IL-9 inhib-

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ited the oxydative burst induced by LPS in freshly isolated human monocytes and bronchial alveolar macrophages. This activity was paired with inhibition of TNFa and IL-10 production, and with induction of TGFb, which in turn inhibited activation of ERK by LPS (Pilette et al., 2002). Further experiments will be needed to evaluate the physiological significance of these observations, but experimental septic shock models illustrate the antiinflammatory activity of IL-9. Mice infected with Pseudomonas aeruginosa show massive production of inflammatory cytokines leading to death. Administration of IL-9 was found to protect mice against a lethal dose of these bacteria (Grohmann et al., 2000). This protection was accompanied by dramatic reductions in seric concentrations of TNFa, IL-12 and IFNc, and in strong induction of IL-10. It will be important to extend these observations to other infection models, and to investigate further the mechanisms involved in this activity.

Eosinophils IL-9 overexpression in transgenic mice leads to modest but significant eosinophilia in blood, peritoneal cavity and BAL of untreated mice (Dong et al., 1999 and unpublished data from our laboratory), and massive eosinophil accumulation in the peritoneal cavity after thioglycolate treatment (Louahed et al., 2001). Similar eosinophilia was seen in the lungs of IL-9 transgenic mice after challenge with Aspergillus fumigatus (McLane et al., 1998). The mechanisms underlying the effect of IL-9 on eosinophils remain unclear. IL-9 does not induce any eosinophilia in IL-5deficient mice (unpublished data from our laboratory). This raises the hypothesis that IL-9 induces eosinophilia by up-regulating IL-5 expression, as indeed was reported in the peritoneal cavity after thioglycolate injection (Louahed et al., 2001). Alternatively, this observation might simply reflect the fact that IL-5 plays a non-redundant role in eosinophil differentiation or proliferation, independently from any effect of IL-9. A synergistic action of IL-9 and IL-5 might also underlie the activity of IL-9 on eosinophilia, as suggested by the effect of these cytokines on in vitro eosinophil differentiation from murine bone marrow progenitors (Louahed et al., 2001). A similar synergy

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has been reported on human eosinophils, and it has been suggested that IL-9 up-regulates IL-5Ra expression (Gounni et al., 2000). In this report, the IL-9 receptor was found to be expressed by human eosinophils based on RT-PCR and immunochemistry experiments, indicating that IL-9 might act directly on eosinophils. However, IL-9 stimulation of purified mouse or human eosinophils does not result in the activation of the signaling pathways triggered by IL-9 in other target cells, namely phosphorylation of STAT3 and -5, or activation of the MAP-kinase pathway, raising some questions about the actual mode of action of IL-9 on this cell type.

Epithelial cells As mentioned above, eosinophilia in IL-9 transgenic mice is restricted to selected sites including lungs and particularly BAL fluid (Dong et al., 1999). This suggests that lung epithelial cells play a role in eosinophil recruitment through secretion of IL-9induced chemokines. This hypothesis was tested using primary lung cultures stimulated in vitro with IL-9. This stimulation induced eotaxin secretion and triggered enhanced eosinophil chemotaxis. Moreover, increased levels of eotaxin and MCP-1 were found in lungs of IL-9 transgenic mice by Western blot. Direct action of IL-9 on murine lung epithelial cells was confirmed with LA-4 and Mad/C3 lung epithelial cell lines which secreted MCP-1 and MCP-3 after in vitro stimulation with IL-9 (Dong et al., 1999). Bronchial epithelial cells have also been suspected to perpetuate airway inflammation in asthmatic patients by secreting chemokines attracting CD4 T cells. Several reports have indicated that IL-9 could modulate this activity. Human bronchial primary or immortalized epithelial cell lines were found to secrete IL-16 and RANTES upon stimulation with IL-9 (Little et al., 2001; Yoshida et al., 2001). IL-9 action on lung epithelial cells also results in mucin secretion. Lung cells from IL-9 transgenic mice showed enhanced expression of MUC2 and MUC5AC, a finding confirmed with human primary lung cultures and with the muccoepidermoid NCI-H292 cell line (Louahed et al., 2000). The importance of IL-9 in mucin production was demonstrated by elegant studies reported by Longphre et al. (1999). Cultured airway epithelial

cells incubated with samples of asthmatic BAL fluid produced more mucin (MUC5AC) than when incubated with BAL fluid from non-asthmatics. When similar experiments were carried out with bronchial lavage from allergen-challenged dogs, increased mucin production was also observed and this mucininducing activity was inhibited by anti-IL-9R antibodies. Size exclusion chromatography of the dog samples showed that IL-9 represented as much as 50–60% of the mucin-inducing activity of the lung fluids (Longphre et al., 1999). The mechanisms underlying up-regulation of mucus production by IL-9 are not clear. A recently described chloride channel, gob-5 or mCLCA3, might play a role in this process. Gob-5 was identified by subtraction cloning as a gene up-regulated in the lungs of IL-9 transgenic mice. This gene is also induced in the lungs of antigen-exposed mice, and its human homolog, hCLCA1, is upregulated by IL-9 in vitro in primary lung cultures (Zhou et al., 2001). This protein is expressed by lung epithelial cells and may play a key role in murine asthma. Intratracheal administration of adenoviruses expressing antisense gob-5 mRNA efficiently suppressed the asthma phenotype in a mouse model, and overexpression of gob5 exacerbated the asthmatic process. In addition, transfection of the gob-5 cDNA in a human mucoepidermoid cell line induced mucus production and MUC5AC expression (Nakanishi et al., 2001).

THE IL-9 GENE The human IL-9 gene is a single-copy gene and was mapped on chromosome 5, in the 5q31→q35 region (Modi et al., 1991), in a region of the genome that contains other cytokine genes such as IL-4, IL-13, IL-5, IL-3 and GM-CSF. However, in the mouse, the IL-9 gene does not seem to be linked to the same gene cluster as it has been localized on mouse chromosome 13 (Mock et al., 1990) while the IL-3, IL-4, IL-5 and GM-CSF genes are located on chromosome 11. As shown in Figure 14.2, the human and murine IL-9 genes share a similar structure with five exons and four introns stretching over about 4 kilobases (Renauld et al., 1990b). The five exons are identical in size for both species and show homology levels ranging from 56% to 74%. In contrast, no significant

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FIGURE 14.2 Map of the human and murine IL-9 genes. Closed boxes represent the coding regions and open boxes correspond to the 5 untranslated sequence. Exons are numbered and their respective size is indicated; homology levels are 56, 67, 64, 73 and 74%, respectively. sequence homology was found in the introns (except for intron 2, which is also the smallest one), although their size is roughly conserved. However, 3 and 5 untranslated regions show a high level of identity, supporting a possible involvement of these sequences in the transcriptional or post-transcriptional regulation of IL-9 expression. In articular, numerous ATTTA motifs were found in the 3 untranslated region of both genes. These sequences, frequently noticed in cytokine mRNAs, are thought to be related to the short half-life of these messengers by modulating their stability. The transcription start has been mapped by S1 nuclease protection 22 to 24 nucleotides downstream of a classical TATA box sequence. The promoter of the IL-9 gene contains potential recognition sites for several tetradecanoyl phorbol acetate (TPA)-inducible transcription factors such as activating protein-1 (AP-1) and AP-2, which could provide a structural basis for the induction of IL-9 expression by phorbol esters. A consensus sequence for interferon regulatory factor-1 (IRF-1) was also identified in both promoters but its physiological relevance remains more elusive (Renauld et al., 1990b). Kelleher and coworkers identified other consensus sequences in the 5 untranslated region of the human gene (SP1, NFjB, octamer, AP-3, AP-5, glucocorticoid responsive element, cAMP response element) and suggested that the NF-jB site and cAMP response element could be involved in the constitutive expression of IL-9 by HTLV-1-transformed T cells (Kelleher et al., 1991). The importance of the NF-jB site in this model was further confirmed by Zhu et al. (1996) who showed that this region of the promoter was critical for gene expression and bound various proteins including NF-jB, c-jun and an unidentified 35 kDa protein.

TH2 cells are the main source of IL-9 (Schmitt et al., 1989; Gessner et al., 1993) and its in vitro production by activated spleen cells is inhibited, like that of other TH2 cytokines, by IL-12, IFNc or CpG oligonucleotides (Schmitt et al., 1994; Lauwerys et al., 1998). in vivo, potent TH2 stimuli such as anti-IgD-triggered polyclonal activation (Svetic et al., 1991) and helminth infections (Grencis et al., 1991; Else et al., 1992; Svetic et al., 1993b) also induce IL-9 expression. Conversely, during Nippostrongylus braziliensis infection, administration of IL-12, which completely suppresses TH2 activation, abolished IL-9 expression as well (Finkelman et al., 1994). Viral infection concomitant to antigenic stimulation also suppresses IL-9 message (Monteyne et al., 1997). During Brucella abortus infection, a typical TH1 model, IL-9 expression is repressed by both type I and type II interferons (Svetic et al., 1993a). Finally, IL-9 expression reflects TH2 responses in Leishmania major infection, where susceptible TH2 responder mice produce IL-9 while resistant TH1 strains do not (Gessner et al., 1993). The mechanisms involved in the regulation of IL-9 production by human T cells have been studied quite extensively in vitro. After stimulation of peripheral T cells with lectins or other mitogenic agents, IL-9 mRNA expression peaks at 28 h, and is completely abrogated by cycloheximide, an inhibitor of protein synthesis, pointing to the involvement of secondary signals in this process. A complex cascade of factors acting in synergy seems to be involved, with IL-2 being required for IL-4 production, both IL-2 and IL-4 being needed for IL-10 production, and eventually IL-4 and IL-10 for IL-9 biosynthesis (Houssiau et al., 1995). The importance of IL-2 for IL-9 expression was confirmed in the mouse as IL-9 production was significantly reduced in IL-2-deficient mice (Schmitt et al., 1994). However, other regulatory mechanisms including IL-1 and TGFb could be involved in IL-9 expression by some T-cell lines and tumors (Schmitt et al., 1991, 1994). Another characteristic of IL-9 expression is its association with HTLV-I, a retrovirus involved in adult T-cell leukemia, which often produce IL-9 constitutively (Yang et al., 1989; Kelleher et al., 1991; Matsushita et al., 1997). The tax transactivator of HTLV-I might be implicated in this process through the NF-jB

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consensus site in the IL-9 promoter (Zhu et al., 1996). Interestingly, in another system of T-cell transformation by murine polytropic retroviruses, viral infection also resulted in IL-9 expression (Flubacher et al., 1994). Besides T cells, mast cells represent another source of IL-9. These cells produce IL-9 in response to ionomycin or IgE-antigen complexes (Stassen et al., 2000). The levels of IL-9 produced by these cells are strongly enhanced in the presence of added IL-10, IL-1 or lipopolysaccharide (Hultner et al., 2000; Stassen et al., 2001).

IL-9 AND DISEASES IL-9 and parasite infections Infections by helminths induce an immune response characterized by strong IgE production and mucosal mastocytosis resulting from TH2 cytokine production (Finkelman et al., 1997). During infection with the intestinal nematode Trichinella spiralis, IL-9 production is induced in mesenteric lymph nodes, as well as other TH2 cytokines such as IL-3, IL-4 and IL-5 (Grencis et al., 1991). Interestingly, IL-9 transgenic mice were found to be particularly resistant to infection with the intestinal nematode T. spiralis. Depression of the mast cell response with anti-c-kit antibodies resulted in the inability of these mice to expel the parasite (Faulkner et al., 1997), suggesting that the resistance conferred by IL-9 was mast cell dependent. IL-9 overexpression also conferred increased resistance to another intestinal nematode, Trichuris muris, which was paired with increased IgE production and also showed massive intestinal mastocytosis (Faulkner et al., 1998). While these observations indicate that IL-9 enhances anti-helminth resistance, they do not prove that normal IL-9 production plays any significant role in this process. This question was addressed in antiIL-9 vaccinated mice. Vaccination of mice with mouse IL-9 coupled with ovalbumin triggers the production of anti-IL-9 autoantibodies. Such vaccinated mice were unable to expel T. muris (Richard et al., 1999). Moreover, the eosinophilia characteristic of such infections was also strongly inhibited. By contrast, mast cell activation, evaluated through MMCP-1 serum levels, was not diminished.

Schistosoma mansoni infection is another model where mast cells are components of the host defense system. In infected mice, the liver and intestine are the major organs affected. The tissue damage in the liver is primarily caused by granulomatous inflammation surrounding parasite eggs trapped in hepatic parenchyma, whereas the intestine is subject to inflammation elicited by parasite eggs being translocated through the intestinal wall. In this model, IL-9 expression was found to be correlated with increased mast cell progenitors during the chronic phase of infection (Khalil et al., 1996). However, IL-9 overexpression is not beneficial in this model as is indicated by the dramatic increase in mortality of IL-9 transgenic mice infected with S. mansoni (86% death in transgenics and 7% in the parental FVB strain). This increased mortality correlated with increased IL-4 and IL-5, and reduced IFNc and TNF production by lymph node cells stimulated in vitro with parasite antigen. Dying animals showed ileum enlargement with muscular hypertrophy, mastocytosis, eosinophilia, goblet cell hyperplasia and increased mucin expression (Fallon et al., 2000). On the other hand, analysis of IL-9-deficient mice shows that IL-9 plays an essential role in goblet cell hyperplasia and mastocytosis in the pulmonary granuloma induced by S. mansoni eggs. In the latter model, eosinophilia and granuloma formation were not affected by IL-9 disruption (Townsend et al., 2000).

IL-9 and asthma Studies on the inheritance of susceptibility to asthma pointed to a cluster of candidate genes on chromosome 5q31–q33 (Marsh et al., 1994; Postma et al., 1995; Doull et al., 1996). In the mouse, homologies for these genes are localized in four regions of the mouse genome, two on chromosome 18, one on chromosome 11 and one on chromosome 13. When DBA/2 and C57BL/6 mice, respectively susceptible and resistant to experimental asthma, were crossed, susceptibility to the disease correlated with inheritance of chromosome 13 from the asthma-prone strain. IL-9, not IL-4 or IL-13, was located in the linked region and its expression was higher in bronchial hyperresponsive animals (Nicolaides et al., 1997). Further support for the role of IL-9 in asthmatic reactions was provided by studies of transgenic mice expressing IL-9 either sys-

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temically or under the control of a lung-specific promoter (McLane et al., 1998; Temann et al., 1998). These mice showed massive airway inflammation with infiltration by mast cells, eosinophils and lymphocytes (Godfraind et al., 1998; Dong et al., 1999). Moreover, a striking epithelial cell hyperplasia was noted with mucus accumulation and subepithelial deposition of collagen (Temann et al., 1998; Louahed et al., 2000). As a result, these mice showed a strong increase in airway response to methacoline or 5-hydroxytrytamine (McLane et al., 1998; Temann et al., 1998). Direct evidence for selective increased production of IL-9 in asthmatic airways, as compared with chronic bronchitis and sarcoidosis, was obtained by in situ hybridization and immunocytochemistry (Shimbara et al., 2000). The cells expressing IL-9 mRNA were identified as mainly CD3 lymphocytes, eosinophils and elastase-positive neutrophils. In vitro, preschool children with allergen-specific skin prick test reactivity showed increased IL-9 expression in response to house dust mite antigen, as compared with non-allergic controls. IL-9 is the TH2 cytokine most frequently associated with positive skin test responses (Macaubas et al., 1999). Taken together, the results gathered during the past few years provide a significant body of evidence for the important role played by this cytokine in asthma (Renauld, 2001). In addition, administration of antiIL-9 antibodies in mouse models of allergic asthma were reported significantly to inhibit IgE production, pulmonary eosinophilia, goblet cell hyperplasia and airway hyperresponsiveness (Kung et al., 2001; Cheng et al., 2002), raising some hopes for a new tool to be developed in human asthma disease. However, these results are in apparent contradiction to recent data obtained with IL-9-deficient mice, which showed normal antigen-induced bronchial responsiveness (McMillan et al., 2002).

IL-9 and cerebral palsy Beyond TH2-associated pathologies such as parasite infections and asthma, IL-9 might play a less expected role in cerebral pathologies. Cerebral palsy is the most common chronic motor disability of childhood. The causes of this complex syndrome remain a point of scientific debate, and may extend beyond hypoxic/ ischemic mechanisms to include multiple prenatal

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factors such as cytokine production. A striking correlation has been reported between increased levels of perinatal circulating cytokines, including IL-9, and subsequent occurrence of cerebral palsy in full-term infants (Nelson et al., 1998). Using a murine model of excitotoxic brain lesions, it was shown that mouse pups pretreated with IL-9, or IL-9-transgenic pups developed larger lesions than control pups (Dommergues et al., 2000). The deleterious effect of IL-9 may be mediated by mast cells and histamine as (1) IL-9 treatment increased mast cell numbers and MMCP-1 expression in the neopalladium of these mice, (2) IL-9 had no significant effect on brain lesions in mast cell-deficient animals, and (3) cromoglycate or anti-histamine drugs significantly reduced the lesions in IL–9-treated mice (Patkai et al., 2001). Alternatively, IL-9 might also act directly on neurones. Such a direct effect on this cell type has been described using immortalized murine embryonic hippocampal progenitor cell lines, with little evidence of morphological maturation. In combination with bFGF and TGFa, IL-9 enhanced neurite outgrowth as well as other morphological modifications, and conferred electrical excitability to these cells (Mehler et al., 1993).

THE IL-9 RECEPTOR STRUCTURE AND SIGNALING Characterization of the IL-9 receptor A single class of high-affinity binding sites for IL-9 (Kd ~100 pM) have been detected on various IL-9responsive murine cells, and cross-linking experiments demonstrated the binding of IL-9 to a 64-kDa glycoprotein (Druez et al., 1990). The murine IL-9 receptor (IL-9R) cDNA was cloned by expression cloning. It codes for a 468-amino acid polypeptide with an extracellular domain, composed of 233 amino acids, including a WSEWS motif and typical conserved residues from the hematopoietin receptor superfamily. The human IL-9 receptor consists of a 522-amino acid protein with 53% identity with the mouse molecule. The extracellular region is particularly conserved with 67% identity, while the cytoplasmic domain is significantly larger in the human receptor (231 versus

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177 residues), and shows little homology (Renauld et al., 1992). As observed for many members of the hematopoietin receptor superfamily, IL-9R mRNAs have been identified that lack the sequences encoding the transmembrane and cytoplasmic domains, as a result of alternative splicing. However, the frequency of these mRNA seems quite low and it is not yet clear whether they really encode a soluble IL-9-binding protein. A more frequent alternative splicing of the human gene generates an intriguing heterogeneity in the 5 untranslated region of the mRNA and introduces some short open reading frames that might represent an additional level in the regulation of IL-9R translation, as suggested for many genes involved in cell growth (Kozak, 1991; Kermouni et al., 1995). In the mouse, the IL-9R gene is a single-copy gene located on chromosome 11 and composed of nine exons and eight introns, sharing many characteristics with other genes encoding cytokine receptors (unpublished data; Vermeesch et al., 1997). By contrast, the human genome contains at least four IL-9R pseudogenes with ~90% homology with the IL-9R gene, which is located in the subtelomeric region of chromosomes X and Y (Kermouni et al., 1995). IL9R was thus the first gene to be identified in the long arm pseudoautosomal region. Using a polymorphism in the coding region of this gene, Vermeesch and colleagues showed that IL9R is expressed both from X and Y chromosomes and escapes X inactivation. In addition, comparative mapping of IL9R in mice, great apes and humans made it possible to reconstitute the evolution of this pseudoautosomal region (Vermeesch et al., 1997). Further studies will have to determine whether this unusual localization in the vicinity of the telomere may play any role in the regulation of transcription, as reported in various organisms (Biessmann et al., 1992).

Signal transduction through the IL-9 receptor Signalling through IL-9R requires association with the c chain of the IL-2 receptor. Antibodies directed against this molecule completely inhibit the activity of IL-9 without affecting the Kd of IL-9 binding (Kimura et al., 1995), indicating that this chain, also called cc, is required for signal transduction but not for IL-9 binding.

So far, the main function of cc seems to consist of recruiting the tyrosine kinase JAK3, while the IL-9R is associated with JAK1. This association of JAK1 with the IL-9R was ascribed to a 98-residue juxtamembrane region of the receptor (Demoulin et al., 1996). This region contains a Pro–X–Pro sequence preceded by a cluster of hydrophobic residues, which partially fits the box 1 consensus sequence shared by many cytokine receptors (Murakami et al., 1991). Downstream from this Pro–X–Pro motif, a striking homology was observed with the b chain of the IL-2 receptor resulting in a 40% identity for the first 33 amino acids of the cytoplasmic domains from the human IL-9R and IL-2Rb. This homologous region is likely to be involved in JAK association, and might explain that IL-9, like other cytokines such as IL-2, induces JAK1 and JAK3 phoshorylation (Russell et al., 1994; Yin et al., 1994; Demoulin et al., 1996). However, it must be stressed that a similar homology was found with this region from the EPO receptor, which activates JAK2 and not JAK1. Thus, the relationship between the juxtamembrane primary sequence of these receptors and specific association with a JAK kinase remains unclear. Upon IL-9 binding, both JAK1 and JAK3 become phosphorylated and catalytically active. These kinases are likely to be responsible for IL-9R phosphorylation on one of its five tyrosine residues. This single phosphorylated residue acts as a docking site for STAT-1, STAT-3 and STAT-5, three transcription factors which, after phosphorylation by the JAK kinases associated with the receptor, form hetero- or homodimere and migrate to the nucleus. Interestingly, mutation of a single tyrosine of the IL-9R abolished both STAT activation and cell growth control by IL-9, including protection against apoptosis and positive as well as negative effects on proliferation (Demoulin et al., 1996). Mutations of residues located just after the critical tyrosine allowed determination of the respective role of the different STAT activated by IL-9 (Demoulin et al., 1999a). When the leucine residue in position 1 was mutated into an arginine, STAT-5 activation was specifically lost. By contrast, when the glutamine residue in position 3 was mutated into a leucine, STAT-1 and -3 activation was lost, whereas STAT-5 activation remained unaffected. Using these mutants, we have shown that STAT-3 and STAT-1 mediate differentiation, growth

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inhibition and anti-apoptotic activities of IL-9 (Demoulin et al., 1999a, 1999b, 2001; Orabona et al., 2001). STAT-5 is mainly involved in proliferative responses and inhibition of apoptosis (Demoulin et al., 1999a, 2000a). The role of other signal transduction pathways for IL-9 activities remains more elusive. Opposing observations have been reported concerning the involvement of the ras–MAP kinase pathway. On the one hand, IL-9 did not induce or enhance the phosphorylation of the serine–threonine kinases Raf-1 or Erk in the Mo7E leukemia cell line (Miyazawa et al., 1992). On the other hand, Raf-1 antisense oligonucleotides inhibited 70% of the response to IL-9 for the same cell line (Brennscheidt et al., 1994). More recent data have shown that IL-9 can indeed induce Erk phosphorylation, but that this effect of IL-9 is highly dependent on the cell lines. In Baf3 cells tranfected with the human IL-9R, IL-9 induces Erk phosphorylation, and inhibition of this pathway by MEK inhibitors or by dominant-negative Ras isoforms partially inhibit IL-9-induced proliferation (Demoulin et al., submitted for publication). IL-9 stimulation also involves the phosphorylation of an adaptor protein called IRS2 (Yin et al., 1995; Demoulin et al., 1996), a feature shared with IL-4 signal transduction, where this pathway was shown to be critical for growth regulation (Keegan et al., 1994). Phosphorylation of IRS2 is not dependent on the phosphorylation of the IL-9 receptor, contrasting with the IL-4 system in which IRS2 associates with the IL-4 receptor through a phosphotyrosine residue. For the IL-9 response, IRS2 and JAK1 activation require the same region of the IL-9 receptor (Demoulin et al., 2000b). These two molecules were also shown to associate in response to IL-9 (Yin et al., 1995). Taken together, these observations raise the possibility that, upon IL-9 activation, IRS2 becomes phosphorylated by interacting directly with the JAK1 tyrosine kinase. After phosphorylation, IRS2 binds the SH2 domain of various signaling proteins including the p85 subunit of the phosphatidylinositol-3 kinase (Demoulin et al., 2000b). The IRS2 pathway seems to be functionally involved in proliferative responses. Overexpression in a T-cell line of IRS1, a protein closely related to IRS2, enhanced the sensitivity to IL-9 (Yin et al., 1995). When the IL-9 receptor was transfected into 32D cells, which lack IRS expression, transfected cells survived

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in the presence of IL-9, and ectopic IRS1 expression allowed IL-9-induced proliferation (Demoulin et al., 2000b). IL-9 can also modulate signal transduction pathways by modulating the expression of proteins involved in these pathways. This is the case for the NF-jB pathway. IL-9 indeed induces expression of Bcl-3, a protein that associates with p50/p50 NF-jB dimers (Richard et al., 1999). Whether Bcl-3 upregulation results in NF-jB activation or inhibition remains controversial. Bcl-3 expression was shown to inhibit apoptosis in IL-9-dependent cell lines (Rebollo et al., 2000), in line with well-described anti-apoptotic activities of NF-jB. On the other hand, IL-9 inhibits NF-jB activation by TNF in thymic lymphomas through induction of Bcl-3 (Richard et al., 1999). These apparent contradicting observations might be due to selective up- or down-regulation of various genes depending on NF-jB binding sites or on interactions with other transcription factors in the respective promoters. Another example of indirect modulation of signaling cascades by IL-9, is the induction of M-Ras/RRas3 in murine T cell lines (Louahed et al., 1999). This protein shows an amino acid identity of 49% and 46% with p21 H-Ras and p23 K-Ras. Similar to other members of the Ras family, activated M-ras is able to regulate cell proliferation and MAP kinase activation. To date, IL-9 has not been shown to activate M-Ras and induce its expression in the same cells. However, other cytokines such as IL-3 are potent M-ras activators, pointing to a putative cross-talk between signaling cascades induced by IL-9 and IL-3 (Louahed et al., 1999).

Signal attenuation mechanisms Negative feedback for signal transduction is a critical process in the regulation of cytokine activities. Cytokine-induced activation of phosphatases is a classical negative feedback mechanism. IL-9 induces the phosphorylation of SHP2 in a hematopoietic immature cell line (Wang et al., 1999). However, IL-9 signaling seems less sensitive to phosphatasemediated down-regulation than EPO signaling, a characteristic that can lead to more sustained STAT-5 activation and induction of distinct sets of genes (Imbert et al., 1999).

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Another mechanism responsible for negative feedback regulation consists of the induction of genes that code for inhibitors of JAK kinases. A family of such inhibitors has been described including CIS (for cytokine-inducible SH2–containing protein), and SOCS-1 to -3 (for suppressor of cytokine signaling). Other SOCS-related proteins have been described but their function remains to be established. In T-cell lymphomas, IL-9 induced the rapid induction of CIS, SOCS-2 and SOCS-3, peaking after 2 h of stimulation. However, only SOCS-3 was able to inhibit IL-9 signaling, and this effect was only seen when SOCS-3 was overexpressed in IL-9-responsive cells, suggesting that this process is not efficient in down-regulating IL-9 activities (Lejeune et al., 2001). However, induction of these genes by IL-9 might lead to inhibition of signaling by more sensitive cytokine receptors, suggesting a role in cytokine cross-talk rather than feedback inhibition. A schematic representation of the currently described signal transduction pathways triggered by IL-9 is shown in Figure 14.3.

CONCLUSIONS Identified as a T cell and mast cell growth factor with a narrow specificity, IL-9 has proved to be a broadly active cytokine with various targets including B cells, macrophages, eosinophils, hematopoietic precursors and epithelial cells. Its aggravating role in asthma is probably due to its action on the different cells involved in this disease. It will therefore be with great interest that the effect of IL-9 antagonists will be analyzed in the years to come. Another potential use of IL-9 in vivo stems from its ability to dampen excessive production of inflammatory cytokines by monocytes and macrophages, as has been shown in septic shock following infection with Pseudomonas aeruginosa. In view of the induction of IL-10 and TGFb by IL-9, it will also be of interest to evaluate the ability of IL-9 to modulate auto-immune reactions, a field that as yet has not been carefully investigated, but which may create further interesting perspectives for the use and study of this molecule. Finally, several aspects of IL-9 biology remain to be studied in more detail. The activities of this cytokine on peritoneal B1 lymphocytes or on neurones, for instance, deserve further investigation, and may unravel unexepected links with various pathophysiological situations.

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FIGURE 14.3 Schematic representation of the signal transduction pathways triggered by IL-9.

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Hültner, L., Moeller, J., Schmitt, E. et al. (1989). Thiolsensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4. J. Immunol. 142, 3440–3346. Hültner, L., Druez, C., Moeller, J. et al. (1990a). Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin-9). Eur. J. Immunol. 20, 1413–1416. Hultner, L., Kolsch, S., Stassen, M. et al. (2000). In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9. J. Immunol. 164, 5556–5563. Imbert, V., Reichenbach, P. and Renauld, J.-C. (1999). Duration of STAT5 activation influences the response of interleukin-2 receptor alpha gene to different cytokines. Eur. Cytok. Netw. 10, 71–78. Jeannin, P., Delneste, Y., Lecoanet-Henchoz, S. et al. (1998). Interleukin-7 (IL-7) enhances class switching to IgE in the presence of T cells via IL-9 and sCD23. Blood 91, 1355–1361. Keegan, A.D., Nelms, K., White, M. et al. (1994). An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth. Cell 76, 811–820. Kelleher, K., Bean, K., Clark, S. et al. (1991). Human interleukin-9: genomic sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)-I-transformed human T cells. Blood 77, 1436–1441. Kermouni, A., Van Roost, E., Arden, K.C., et al. (1995). The IL-9 receptor gene: genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of the sex chromosomes and identification of IL-9R pseudogenes at 9qter, 10pter, 16pter and 18pter. Genomics 29, 371–382. Khalil, R.M., Luz, A., Mailhammer, R. et al. (1996). Schistosoma mansoni infection in mice augments the capacity for interleukin-3 (IL-3) and IL-9 production and concurrently enlarges progenitor pools for mast cells and granulocytes–macrophages. Infect. Immun. 64, 4960–4966. Kimura, Y., Takeshita, T., Kondo, M. et al. (1995). Sharing of the IL-2 receptor c chain with the functional IL-9 receptor complex. Int. Immunol. 7, 115–120. Kozak, M. (1991). An analysis of vertebrate mRNA sequences: intimations of translational control. J. Cell Biol. 115, 887–903. Kubota, S., Siomi, H., Hatanaka, M. and Pomerantz, R.J. (1996). Cis/trans-activation of the interleukin-9 receptor gene in an HTLV-I-transformed human lymphocytic cell. Oncogene 12, 1441–1447. Kung, T.T., Luo, B., Crawley, Y. et al. (2001). Effect of antimIL-9 antibody on the development of pulmonary inflammation and airway hyperresponsiveness in allergic mice. Am. J. Respir. Cell Mol. Biol. 25, 600–605. Lauwerys, B.R., Renauld, J.C. and Houssiau, F. (1998). Inhibition of in vitro immunoglobulin production by IL-12 in murine chronic graft-vs.-host disease: synergism with IL-18. Eur. J. Immunol. 28, 2017–2024. Lehrnbecher, T., Poot, M., Orscheschek, K. et al. (1994). Interleukin-7 as interleukin-9 drives phytohemagglutininactivated T cells through several cell cycles: no synergism

between interleukin-7, interleukin-9 and interleukin-4. Cytokine 6, 279–284. Lejeune, D., Demoulin, J.B. and Renauld, J.C. (2001). Interleukin-9 induces expression of three cytokine signal inhibitors: cytokine-inducible SH2-containing protein, suppressor of cytokine signalling (SOCS)-2 and SOCS-3 but only SOCS-3 overexpression suppresses IL-9 signalling. Biochem. J. 353, 109–116. Lemoli, R.M., Fortiuna, A., Fogli, M. et al. (1994). Stem cell factor (c-kit ligand) enhances the Interleukin-9dependent proliferation of human CD34 and CD34CD33DR- cells. Exp. Hematol. 22, 919–924. Lemoli, R.M., Fortuna, A., Tafuri, A. et al. (1996). Interleukin9 stimulates the proliferation of human myeloid leukemic cells. Blood 87, 3852–3859. Little, F.F., Cruikshank, W.W. and Center, D.M. (2001). IL-9 stimulates release of chemotactic factors from human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 25, 347–352. Longphre, M., Li, D., Gallup, M. et al. (1999). Allergeninduced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J. Clin. Invest. 104, 1375–1382. Louahed, J., Kermouni, A., Van Snick, J. and Renauld, J.-C. (1995). IL-9 induces expression of granzymes and high affinity IgE receptor in murine T helper clones. J. Immunol. 154, 5061–5070. Louahed, J., Grasso, L., De Smet, C. et al. (1999). Interleukin-9induced expression of M-Ras/R-Ras3 oncogene in T-helper clones. Blood 94, 1701–1710. Louahed, J., Toda, M., Jen, J. et al. (2000). Interleukin-9 upregulates mucus expression in the airways. Am. J. Respir. Cell Mol. Biol. 22, 649–656. Louahed, J., Zhou, Y., Maloy, W.L. et al. (2001). Interleukin 9 promotes influx and local maturation of eosinophils. Blood 97, 1035–1042. Lu, L., Leemhuis, T., Srour, E. and Yang, Y.-C. (1992). Human interleukin (IL)-9 specifically stimulates proliferation of CD34DRCD33 erythroid progenitors in normal human bone marrow in the absence of serum. Exp. Hematol. 20, 418–424. Macaubas, C., Sly, P.D., Burton, P. et al. (1999). Regulation of T helper cell responses to inhalant allergen during early childhood. (1999). Clin. Exp. Allergy 29, 1223–1231. Marsh, D.G., Neely, J.D., Breazeale, D.R. et al. (1994). Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum IgE concentrations. Science 264, 1152–1156. Matsushita, K., Arima, N., Ohtsubo, H. et al. (1997). Frequent expression of interleukin-9 mRNA and infrequent involvement of interleukin-9 in proliferation of primary adult T-cell leukemia cells and HTLV-I infected T-cell lines. Leuk. Res. 21, 211–216. McLane, M.P., Haczku, A.H., van de Rijn, M. et al. (1998). Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice. Am. J. Resp. Cell. Mol. 19, 713–720. McMillan, S.J., Bishop, B., Townsend, M.J. et al. (2002). The absence of interleukin 9 does not affect the development of allergen-induced pulmonary inflammation nor airway hyperreactivity. J. Exp. Med 195, 51–57. Mehler, M.F., Rozental, R., Dougherthy, M. et al. (1993). Cytokine regulation of neuronal differentiation of hippocampal progenitor cells. Nature 362, 62–65. Merz, H., Houssiau, F.A., Orscheschek, K. et al. (1991). IL-9

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in the presence of lipopolysaccharide: NF-kappaB is decisively involved in the expression of IL-9. J. Immunol. 166, 4391–4398. Suda, T., Murray, R., Fischer, M. et al. (1990a). Tumor necrosis factor-alpha and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2. J. Immunol. 144, 1783–1787. Suda, T., Murray, R., Guidos, C. and Zlotnik, A. (1990b). Growth-promoting activity of IL-1a, IL-6, and tumor necrosis factor-a in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3/CD3 double-negative thymocytes. J. Immunol. 144, 3039–3045. Svetic, A., Finkelman, F., Jian, Y et al. (1991). Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody. J. Immunol. 147, 2391–2397. Svetic, A., Jian, Y.C., Lu, P. et al. (1993a). Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-g in CD4 cells. Int. Immunol. 5, 877–883. Svetic, A., Madden, K.B., Di Zhou, X. et al. (1993b). A primary intestinal helminthic infection rapidly induces a gutassociated elevation of Th2–associated cytokines and IL-3. J. Immunol. 150, 3434–3441. Temann, U.A., Geba, G.P., Rankin, J.A. and Flavell, R.A. (1998). Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J. Exp. Med. 188, 1307–1320. Townsend, J.M., Fallon, G.P., Matthews, J.D. et al. (2000). IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity 13, 573–583. Trümper, L.H., Brady, G., Bagg, A. et al. (1993). Single-cell analysis of Hodgkin and Reed–Sternberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood 81, 3097–3115. Uyttenhove, C., Simpson, R. and Van Snick, J. (1988). Functional and structural characterization of P40, a mouse glycoprotein with T cell growth factor activity. Proc. Natl Acad. Sci. USA 85, 6934–6938. Uyttenhove, C., Druez, C., Renauld, J.-C. et al. (1991).

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15 Interleukin-11 James C. Keith, Jr Wyeth Research, Andover, MA, USA

Our doubts are traitors, And make us lose the good we oft might win, By fearing to attempt. Measure for Measure, Shakespeare

INTRODUCTION Interleukin-11 (IL-11) was originally identified as a soluble factor in conditioned medium following IL-1a stimulation of a primate bone marrow stromal cell line, PU-34, which supported long-term hematopoiesis in cell culture (Paul et al., 1990). Subsequent investigation revealed that IL-11 was a highly conserved, 178 amino acid, cationic protein that is a member of the IL-6 ligand family as it utilizes gp130 as the signaling component of its receptor. Originally described as a hematopoietic cytokine, IL-11 is, in fact, a multifunctional cytokine derived from many cell types distributed throughout the body. The earliest in vitro and in vivo studies demonstrated a hematopoietic activity for interleukin-11, largely manifest as thrombopoiesis in humans, and recombinant human IL-11 (rhIL-11), Neumega, has been developed and approved by the Food and Drug Administration for use in the prevention of severe

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

thrombocytopenia occurring after cancer chemotherapy. Current clinical interests are focused on the immunomodulatory effects of orally delivered rhIL-11 in the setting of inflammatory bowel disease. The mature IL-11 protein is thought to exist as a thermally stable, four-helix bundle, although it contains no cysteine residues. Human and murine IL-11 share 88% homology at the amino acid level, while human and nonhuman primate IL-11 share 94% homology. Residues 59 (methionine), 42 (lysine) and 99 (lysine), critical for function of the protein, are completely conserved in mouse, nonhuman primate and human IL-11. Studies of cells and tissues from other organ systems indicate that IL-11 has activity in protection and restoration of the gastrointestinal mucosa; major effects as an immuno-modulating agent; and activity in bone metabolism. Developmental investigations in mice indicate a widespread distribution of IL-11 expression in the embryo, but of great interest is the

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finding that IL-11 signaling is an absolute requirement for normal development of placentation and survival to birth.

GENE STRUCTURE IL-11 was originally identified as an activity in conditioned medium from an IL-1a-stimulated primate bone marrow stromal cell line, PU-34 (Paul et al., 1990). The human cDNA, GenBank database Accession No. M37006, was derived from a human fetal lung fibroblast line (MRC5) treated with phorbol 12-myristate13-acetate and IL-1a. The nonhuman primate IL-11 cDNA sequence is found at database Accession No. M37007, while the murine cDNA is found at UO3421 (Morris et al., 1996). The nucleotide sequence of the human IL-11 gene seen in Figure 15.1, consists of five exons and four introns that contain 7 kbp of genomic DNA (McKinley et al., 1992; Schendel and Turner, 1998). Although IL-11 is considered a member of the IL-6 ligand family, since it utilizes gp130 as the signaling component of its receptor, the IL-11 gene shares no homology with IL-6 at the nucleotide level. Several regions of the 5 flanking sequence of the gene do contain small nucleotide sequences that share approximately 70% homology to other cytokines, such as IL-2, IL-3, IL-6, IL-9 and GM-CSF (McKinley et al., 1992).

CHROMOSOME LOCATION The human and the nonhuman primate genes for IL-11 are found on the long arm of chromosome 19 at band 19q13.3- q13.4 (McKinley et al., 1992), and the murine IL-11 gene is found on the homologous chromosome 7 (Morris et al., 1996; Stubbs et al., 1996). For both the mouse and human genomes, the IL-11 gene is near the protein kinase C, gamma subunit, gene and the genes for several zinc finger proteins. The cDNA for human interleukin-11 codes for a 199 amino acid polypeptide. Northern analysis reveals the presence of two transcripts, a 2.5 kb mRNA and a minor 1.5 kb mRNA. Both transcripts encode the same IL-11 protein. The transcript size difference occurs because of alternative use of the two polyadenylation sites located within the 3 noncoding region of the gene, resulting in different lengths of the 3 noncoding region (Paul et al., 1990; McKinley et al., 1992).

CELLS AND TISSUES THAT EXPRESS THE GENE The IL-11 gene is expressed by a wide variety of cells and tissues throughout the body and its distribution has been reviewed previously (Du and Williams 1997; Dorner et al., 1997; Schendel and Turner 1998). The

FIGURE 15.1 The nucleotide sequence of the human IL-11 gene is displayed. Underlined are the 5 and 3 untranslated regions of the sequence (from Schendel and Turner, 1998). THE CYTOKINES AND CHEMOKINES

PROMOTERS AND SUPPRESSORS OF IL - 11 GENE TRANSCRIPTION

expression is relatively low as mRNA is only detected by RT-PCR in most tissue or cell types (Dorner et al., 1997).

PROMOTERS AND SUPPRESSORS OF IL-11 GENE TRANSCRIPTION IL-11 mRNA expression and protein production can be elicited by several agents, including IL-1a, tumor necrosis factor-alpha (TNF-a); transforming growth factor-b (TGF-b) parathyroid hormone, retinoic acid, histamine, phorbol myristate acetate (PMA), or infection with a variety of respiratory viruses, including respiratory synctial virus (reviewed in Du and Williams 1997; Dorner et al., 1997). Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-kappa B and is inhibited by sodium salicylate and aspirin (Bitko et al., 1997). Mutational analysis of the IL-11 promoter identified a region 720 nucleotides upstream of the mRNA start site in the transcriptional induction of IL-11 by RSV. In this region, two 10-nucleotide-long sequences were found that resembled the NF-kappa B consensus region. Mutation of either sequence decreased RSV-evoked induction of IL-11 promoter activity. NF-kappa B sites are also required for RSVmediated induction of transcription in the promoters of the IL-1 alpha, IL-6 and IL-8 genes, and sodium salicylate and aspirin can prevent transcriptional induction of all these cytokines by RSV. Binding sites for several transcription factors, including AP-1, SP-1, CTF/NF-1 and EF/C are found in the 5 region of the gene. Interferon-inducible and phorbol ester TPA-inducible elements are also located in this region of the gene. Two polyadenylation sites exist at the 3 end of the gene, and several Alu repetitive sequences are also found within this region (Paul et al., 1990). Several studies of mononuclear cells obtained from patients with rheumatoid arthritis have been used to further examine IL-11 production (reviewed in Keith, 2000). IL-1a and TNF-a synergistically induced cPLA2, COX-2 and PGE2, that stimulated cultured rheumatoid synovial fibroblasts production of IL-11 mRNA and protein (Mino et al., 1998). Indomethacin was able to block the IL-11 production, but exogenous

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PGE2 could obviate this inhibition. Pan-inhibition of phosphokinase isoforms also resulted in complete inhibition of IL-11 production. The indomethacin inhibition of IL-11 production was confirmed by Taki et al. (1998), who demonstrated that exogenous PGE2 completely prevented the inhibition by indomethacin, but the inhibition caused by dexamethazone was not affected by PGE2. IFN-c inhibited the production of IL-11 from IL-1a-stimulated cultured rheumatoid synovial fibroblasts (RSC), but not the production of IL-11 by fresh RSC. Immunoreactive IL-11 (0.08 to 24.65 ng ml1) has been detected in the cultures of synovial membrane tissue, obtained from RA patients who underwent synovectomy or joint replacement surgery (Hermann et al., 1998 ). Endogenous IL-11 and IL-10 appeared to regulate TNF-a since inhibition of IL-11 activity in synovial membrane mononuclear cell cultures with neutralizing antibody resulted in a two-fold increase in TNF-a production, and inhibition of IL-10 with neutralizing antibody resulted in a three-fold increase in TNF-a production. When simultaneously neutralized, an exaggerated increase in TNF-a production (23-fold) was seen (Figure 15.2) from Hermann et al. (1998).

Isotype control Anti-IL-11 mAb+ Anti-IL-10 mAb

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Basal production 0

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FIGURE 15.2 The effects of endogenous cytokine inhibition, by neutralizing antibodies to IL-10 and IL-11, on TNF-a production can be seen in synovial membrane mononuclear cell cultures from rheumatoid arthritis patients. Neutralization of both IL-10 and IL-11 results in a marked elevation of TNF-a (from Hermann et al., 1998).

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Finally, interferon-alpha (INF-alpha) down-regulates IL-1-induced IL-11 in human bone marrow stromal cultures (Aman et al., 1996). Expression of IL-11 mRNA, assessed by Northern blots, was reduced in the presence of INF-alpha, but the reduction was prevented by the addition of cycloheximide.

PROTEIN Interleukin-11 is a 19 000 dalton, highly conserved, 199 amino acid precusor protein. The first 21 amino acids are the typical leader sequence for secreted proteins and are removed when the mature 178 amino acid protein is released from the cell. The protein contains no cysteine residues, yet it is thermally stable. The mature human and murine proteins share 88% homology at the amino acid level, while the human and nonhuman primate share 94% homology. Amino acid residues 59 (methionine), 42 (lysine) and 99 (lysine) are critical for receptor binding of the protein, and these residues are completely conserved in the mouse, nonhuman primate and human (Czupryn et al., 1995a). The comparative 199 amino acid sequences of the murine, nonhuman primate and human gene products are seen in Figure 15.3 (from Morris et al., 1996). Recently, the IL-11 gene has been cloned in the rats (Li et al., 2001), and the protein transcribed has a high degree of homology to the murine (97%) and the human (87%) proteins. IL-11 is an unusually basic cytokine with a calculated isoelectric point of 11.7, due to the high content in proline (12%), leucine (23%), and positively charged amino acid residues (14%). Since there are no

FIGURE 15.3 The comparative amino acid sequences of murine, nonhuman primate and human rhIL-11 are seen (from Morris et al., 1996).

sites for asparagine-linked glycosylation the protein has little, if any, carbohydrate modification. The crystal structure of IL-11 has not been solved, but the protein is thought to exist as a four-helix bundle as predicted by computer modelling based on the primary amino acid structure, and by the use of alaninescanning mutagenesis of the mature protein Czupryn et al. (1995a, 1995b). Recombinant human IL-11 is produced in Escherchia coli by genetic engineering and is 177 amino acids long, differing from the naturally occurring protein only by the absence of the amino terminal proline residue.

CELLULAR SOURCES AND TISSUE EXPRESSION As previously described, many cell and tissue types are capable of producing IL-11, dependent on the local environment of the cell or tissue. Basal and inducible IL-11 mRNA expression can be detected in fibroblasts, epithelial cells, chondrocytes, synoviocytes, keratinocytes, endothelial cells, osteoblasts and certain tumor cells and cell lines (Du and Williams, 1997; Dorner et al., 1997; Schendel and Turner, 1998).

IN VITRO ACTIVITIES With the advent of rhIL-11, many in vitro studies have demonstrated that rhIL-11 acts on multiple cell types, including hematopoietic cells, hepatocytes, adipocytes, macrophages, intestinal epithelial cells, tumor cells, and both osteoblasts and osteoclasts. rhIL-11 in synergy with either IL-3, IL-4, IL-6 or steel factor (SF) supports murine hematopoietic cell development (reviewed in Goldman, 1995), and Leary et al. (1992) have also found that rhIL-11 acts synergistically with either IL-3 or SF in supporting human blast-cell colony formation. Increased maturation of late human megakaryocyte progenitors and of megakaryocytes from gpIIb-IIIa+ (CD41) bone marrow cells was produced by rhIL-11, and megakaryocytes were larger and had higher DNA ploidy (Bruno et al,. 1991; Teramura et al., 1992). rhIL-11 regulates human megakaryo-cytopoiesis at several maturation levels, from the earliest recognizable megakaryocyte progenitor to the level of the

THE CYTOKINES AND CHEMOKINES

IN VITRO ACTIVITIES

nondividing megakaryocyte. In rodents, erythroid and monocyte lineages may also be affected, but no mitogenic activity of primary murine thymocytes or of human peripheral T cells was caused by rhIL-11. rhIL-11 can induce production of the same hepatocyte acute-phase proteins as IL-6 (Baumann and Schendel, 1991), but rhIL-11 had no effect on albumin synthesis. The rhIL-11 effect appeared to be less potent than IL-6 in inducing acute-phase protein synthesis. In HepG2 cells (Fukada and Sassa, 1993), rhIL-11 also increased the level of microsomal heme oxygenase (HO). The production of HO may contribute to rhIL-11’s immunomodulatory effects since HO appears to protect tissues against oxidative stress (Willis et al., 1996). rhIL-11 is able to reduce proinflammatory mediator production by lipopoly-saccharide (LPS)-activated, murine macrophages ( Trepicchio et al., 1996). LPSinduced TNF-a, IL-6, IL-1b, IL-12 p40, and nitric oxide production were reduced in the conditioned medium, when assessed by protein or nitrate levels, respectively. Redlich et al. (1996) demonstrated that pretreatment of primary murine alveolar macrophages with 100 ng/ml rhIL-11 also caused a 65% reduction in LPS stimulated TNF-a production. However, rhIL-11 does not inhibit LPS-induced cell surface expression of ICAM-1, MHC class II molecules, B7.2 antigen, or the LPS-binding protein receptor CD14 (Trepicchio et al., 1996). Treatment with rhIL-11 also did not alter the levels of IL-10 or TGF-b in the conditioned medium as assessed by ELISA or bioassay. Interleukin-11 inhibits human macrophage interleukin-12 production (Leng and Elias, 1997). Inhibition of IL-12 protein production was associated with a proportionate decrease in IL-12 p35 and p40 mRNA accumulation. Nuclear run-on assays revealed comparable decreases in IL-12 p35 and p40 gene transcription. IL-11 did not similarly regulate monocyte/macrophage production of IL-8 or macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-6 did not similarly inhibit IL-12 elaboration. These studies demonstrate that IL-11 is a potent inhibitor of monocyte/macrophage IL-12 production and that this inhibitory effect appears to be transcriptionally mediated. Analysis of rhIL-11 effects on proinflammatory cytokines indicated that the level of LPS-induced

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NF-kappaB binding activity in the nucleus of rhIL-11treated, murine peritoneal macrophages was significantly reduced (Trepicchio et al., 1997). The block to NF-kappaB nuclear translocation was related to the ability of rhIL-11 to maintain or increase protein levels of the inhibitors of NF-kappaB, IkappaB-alpha, and IkappaB-beta following LPS treatment. Treatment of LPS-stimulated macrophages resulted in significant elevation of IkappaB-alpha and IkappaB-beta mRNA levels. These results demonstrated for the first time the regulation of IkappaB-beta by an antiinflammatory cytokine. Treatment of intestinal epithelial cells (IEC-6) cells in culture with rhIL-11 reduced the cellular growth rate (Peterson et al., 1996; Booth and Potten, 1995). This direct antiproliferative effect of rhIL-11 on the IEC-6 cell line was not mediated through induction of endogenous transforming growth factor-b1 production, and was associated with transient cell cycle arrest, mediated through inhibition of pRb phosphorylation. However, rhIL-11 did not affect the growth of the human colonic epithelial transformed cell lines SW620 and HT29 (Booth and Potten, 1995). Likewise, rhIL-11 had no effect on the proliferation of three human melanoma cell lines derived from primary lesions or four lines derived from metastatic tumors (Paglia et al., 1995). The response of rhIL-11-treated tumor cells exposed to cytotoxic therapies has also been evaluated (Teicher et al., 1996). rhIL-11 treatment did not alter the response of HT29 cells to radiation in culture or to 5-FU treatment in vivo or ex vivo. rhIL-11 treatment did not alter the ex vivo survival of EMT-6 mammary carcinoma cells following treatment with melphalan or cyclophosphamide. In in vivo tumor growth delay experiments, rhIL-11 treatment did not adversely affect treatment with melphalan, thiotepa, cyclophosphamide, or carboplatin. The data from these studies indicate that for tumor cells of nonhematopoietic origin, rhIL-11 does not stimulate proliferation or alter the tumor response to cytotoxic cancer therapies when assessed in vitro, in vivo, or ex vivo. rhIL-11 also acts on osteoclasts and osteoblasts, cell types commonly believed to be derived from the monocyte lineage. However, in vitro effects appear to be different than those seen with therapeutic administration of rhIL-11 in vivo. IL-1, IL-6, IL-11 and tumor

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necrosis factor (TNF) can stimulate osteoclast development and affect bone resorption (reviewed in Manolagas, 1995). IL-6 has been implicated in excessive osteoclastic bone resorption, typically associated with loss of either ovarian or testicular function. However, it remains unclear whether IL-6 is the sole pathogenetic factor or whether IL-1, TNF-alpha and IL-11 may also be involved. The in vitro results suggest that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblastmediated osteoid degradation rather than stimulating osteoclast migration and activity. Matrix metalloproteinases and products of arachidonic acid metabolism are also involved (Hill, P.A. et al., 1998). IL-11 is produced by human bone-derived endothelial cells (BDECs) in response to IL-1 and TNF-a and its role in the formation of osteolytic bone metastasis has been postulated based on in vitro studies (Zhang et al., 1998). Thus, endothelial cells in bone may be important in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions. Kim et al. (1997) have demonstrated that rhPTH (1-34) and rhIL-a dose-dependently stimulate IL-6 and IL-11 production from human bone marrow stromal cells (hBMSCs). Agonists for protein kinase A and protein kinase C also stimulated IL-6/IL-11 production, but stable forms of cAMP that are inhibitors of PKA significantly inhibited PTH-stimulated IL-6/ IL-11 production, but did not inhibit IL-1-stimulated IL-6/IL-11 production. Regulation of cytokine production by estrogen in hBMSCs was selective, as the IL-1-induced IL-6 production mediated by PKC pathway was inhibited, but PTH-induced IL-6 production and PTH/IL-1-induced IL-11 production were not inhibited by estrogen. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin induced production of IL-11 by osteoblasts, but IL-6, IL-4 and TGF beta did not (Romas et al., 1996). Osteoblasts, as well as osteoclasts, expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.

IL-11 EXPRESSION, REGULATION AND IN DISEASE STATES Information regarding the regulation of IL-11 expression in vivo in humans is limited. Under normal conditions, circulating serum levels of IL-11 are very low. This may be related, in part, to the unique and marked cationic nature of the protein that can predispose its binding with negatively charged surfaces throughout the body. For example, IL-11 has been detected in the ovarian follicular fluid of women treated with gonadotropins in the setting of in vitro fertilization (Branisteau et al., 1997). However, the protein was not detected in the patients’ sera. IL-11 plasma levels were significantly elevated in patients with severe thrombocytopenia secondary to myeloablative therapy or in patients with immune thrombocytopenia (Chang et al., 1996). Platelet counts and endogenous IL-11 levels following myeloablative therapy were inversely related. Circulating IL-6 levels were not elevated in these thrombocytopenic patients, decreasing the possibility that elevated IL-11 levels were due to a global induction of the gp130 cytokines. Low, but detectable, levels of IL-11 (20 pg ml1) have been reported in a significant portion of patients with disseminated intravascular coagulation with or without sepsis (Endo et al., 1996). IL-11 has been found in seminal plasma and is elevated in seminal plasma of infertile patients with urogenital infection (Matalliotakis et al., 1998). Recently, measurable serum levels of IL-11 have also been seen in RA patients (DeBenedetti et al., 1997; Hermann et al., 1998). Cells from the synovium of RA patients expressed mRNA for IL-6, IL-11, LIF and OSM at higher levels than did synovial cells from osteoarthritis (OA) patients, and spontaneously released greater quantities of these proteins in culture (Okamoto et al., 1997). Immunoreactive IL-11 has also been reported in aseptic loosening of total hip replacement implants (Xu et al., 1998). Chronic inflammatory response to abrasion particles from total hip replacement (THR) is believed to cause osteolysis and to contribute to prosthetic loosening. IL-11 was found in the interface and pseudocapsular tissues in the aseptic loosening of THR. This is not an unexpected finding since inflammatory mediatorinduction of IL-11 has been previously discussed.

THE CYTOKINES AND CHEMOKINES

IL - 11 RECEPTOR

Interleukin-11 production is increased in organ cultures of lesional skin of patients with active plaquetype psoriasis, as compared with nonlesional and normal skin (Ameglio et al., 1997). Cytokine levels, IL-11 and three other proinflammatory cytokines (IL-1 beta, IL-6 and IL-8), were determined in supernatants of skin organ cultures from lesional and nonlesional psoriatic skin and in supernatants of biopsies from normal volunteers using commercially available ELISA kits. The amounts of IL-11 and the other three modulators were increased in lesional areas, and the levels of IL-11 were also correlated with the disease severity index. Trepicchio et al. (1999) examined the expression of over 35 genes in psoriasis patients receiving subcutaneously administered rhIL-11 in an open label study. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Proinflammatory genes were elevated in psoriatic tissue compared with nonlesional skin. Amelioration of disease by rhIL-11 in seven of 12 patients, shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of disease-related genes, including K16, iNOS, IFN-c, IL-8, IL-12, TNF-a, IL-1b and CD8, and with increased expression of endogenous IL-11. This was the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. Several tumor types are known to produce cytokines and express cytokine receptors. Human renal cell carcinomas (RCC) produce IL-6, IL-10, IL-11 and TGFbeta 1 in primary cultures and modulate T lymphocyte blast transformation (Knoefel et al., 1997). Immunomodulation is a common feature of RCC, and breast cancer cells are also known to produce IL-6 and IL-11 (Sotiriou et al., 1999). However, Soda et al. (1999) , who studied the effects of rhIL-11 proliferation of human tumors taken directly from patients found no evidence of stimulation of multiple solid tumor types.

IL-11 RECEPTOR Although interleukin-11 utilizes the gp130 molecule as the signaling component of its receptor, specificity

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is conferred through the use of a unique IL-11 alpha chain receptor that must interact with the ligand and the gp130 complex. The cDNA for the murine IL-11 receptor alpha chain (mIL-11Ra) of the receptor complex for IL-11 was published in 1994 (Hilton et al., 1994), and the human IL-11 receptor alpha chain (hIL-11Ra) has been reported (Cherel et al., 1995, 1996; Nandurkar et al., 1996; Van Leuven et al., 1996). The mIL-11Ra consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular region exhibits structural features typical of hematopoietic receptors such as proline residues preceding each 100 amino acid subdomain, a motif of four conserved cysteines and one tryptophan residue, a series of polar and hydrophobic amino acid residues, and the WSXWS domain between the cysteine and transmembrane domain. IL-11 binding to the IL-11Ra alone occurs with low affinity (kDa ~10 nM) and does not signal. A high affinity receptor complex capable of transducing a signal (kDa ~400–800 pM) is produced with co-expression of IL-11Ra and gp130 (Nandurkar et al., 1996). Barton et al. (2000) have recently reported that IL-11 signals through the formation of a hexameric receptor complex, requiring two molecules of IL-11, and two each of the receptor subunits. The IL-11R a-chain does not have to be membrane bound to elicit a biological effect, because soluble forms of the a-chain receptor have been generated that can bind IL-11 and activate gp130 (Neddermann et al., 1996; Baumann et al., 1996; Curtis et al., 1997). The mIL-11Ra gene includes 14 exons with alternative use of the first two exons regulated in a developmental fashion (Nandurkar et al., 1997a). The gene contains two loci (1 and 2), while locus 2 is restricted to only some mouse strains. Two alternatively spliced exons (1a and 1b) encode the 5 untranslated region (5UTR) of the murine locus 1. A second mIL-11Ra locus has been identified (IL-11Ra2) adjacent to the IL-11Ra1 gene (Robb et al., 1997). It shares 99% sequence identity with IL-11R a1 in the coding exons, but contains differences in the 5 untranslated region (5UTR) (Bilinski et al., 1996; Robb et al., 1997). The mIL-11Ra1 gene is expressed at relatively low levels in several tissues including bone marrow, spleen, thymus, lung, bladder, heart, brain, kidney, muscle, salivary gland, small and large intestine, ovary, testis and uterus. Primary cell types such as macrophages,

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osteoblasts, and osteoclasts also express the murine IL-11Ra1 gene (Romas et al., 1996; Trepicchio et al., 1997) while expression of the IL-11Ra2 gene is restricted to the testis, lymph node and thymus (Robb et al., 1997). The human locus has 10 kilobasepairs (kb) and contains 14 exons. Two alternatively spliced first exons (1a and 1b), encoding the 5UTR, shared 76 and 73% nucleotide identity with murine exons 1a and 1b. Multiple transcription start sites were seen for human exon 1a. The promoter regions of both human exons 1a and 1b did not display a canonical TATA box. A predominant 1.8 kb transcript for the hIL-Pa was present in heart, brain, skeletal muscle, lymph nodes, thymus, appendix, pancreas and fetal liver. The hIL-11R alpha gene was localized to chromosome 9p13. The hIL-11R alpha gene was highly related to locus 1 of the murine gene, but there was no evidence of a second hIL-11R alpha locus. The hIL-11Ra chain shares 85% nucleotide identity and 84% amino acid identity with the murine gene

(Van Leuven et al., 1996; Nandurkar et al., 1997b). The gene is composed of 13 exons comprising nearly 10 kb of DNA that was completely sequenced. The intron–exon boundaries were determined based on the mouse Etl2 and interleukin-11 receptor cDNAs that were recently cloned. The protein sequence predicted by the human gene was over 83% identical with its murine counterpart, with very strict conservation of functionally important domains and signatures. Two isoforms of the hIL-11Ra protein have been identified which differ in the structure of their cytoplasmic domains. One isoform, a 422 amino acid protein, has a short cytoplasmic domain similar to the IL-6Ra and mIL-11Ra. The second isoform, a 388 amino acid protein, lacks the cytoplasmic domain and is similar to the human CNTF receptor (CNTFR). Figure 15.4 shows a comparison of the full length mIL-11Ra and hIL-11Ra proteins (Van Leuven et al., 1996). The mature human and murine IL-11 ligand proteins share 88% homology at the amino acid level,

FIGURE 15.4 The comparative amino acid sequences of murine and human rhIL-11Ra are displayed (from Van Leuven et al., 1996). THE CYTOKINES AND CHEMOKINES

IL - 11 RECEPTOR SIGNAL TRANSDUCTION

while the human and nonhuman primate share 94% homology. Amino acid residues 59 (methionine), 42 (lysine) and 99 (lysine) are critical for function (receptor binding and signaling) of the protein, and these residues are completely conserved in the mouse, nonhuman primate and human proteins (Czupryn et al., 1995a). Specific alkylation of a single methionine residue, Met59, produces a 25-fold reduction of in vitro biological activity of rhIL-11 on mouse plasmacytoma cells. Modification of the amino-terminal amino group and partial labeling of two lysines, Lys42 and Lys99, causes a three-fold decrease in activity. Removal of the last four carboxyl terminal residues reduces rhIL-11 activity 25-fold, whereas removal of eight or more amino acids results in an inactive molecule. Using the four-helix bundle model, the Met59, Lys42 and Lys99 are located on the surface of the molecule, it is postulated that Met59 and the carboxyterminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys42 and Lys99 may be a part of binding site II.

RECEPTOR EXPRESSION AND DISTRIBUTION The expression of murine IL-11 ligand and mIL-11Ra in adult mouse tissues, in embryos, and during development of embryonic stem (ES) cells into cystic embryoid bodies in vitro has been examined by RNAase protection assays (Davidson et al., 1997). The testis showed a high level of IL-11 gene expression, and a much lower level of expression was detected in the lung, stomach, small intestine and large intestine. Expression of the IL-11 ligand was not detected between day 10.5 and day 18.5 post coitum of embryonic development or in differentiating ES cells in vitro. However, the mIL-11Ra was expressed in all adult tissues examined, during embryonic development, and in totipotent and differentiating ES cells. Murine megakaryocytes (MKs) are direct targets of rhIL-11 since they expressed functional mIL-11Ra (Weich et al., 1997). Exposure of purified bone marrow derived MKs to rhIL-11, enhanced phosphorylation of both its signal transduction subunit, gp130, and evoked the transcription factor, STAT3, showing a direct activation of receptor signaling by the cytokine. Consistent with the lack of effect of rhIL-11 on human

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platelets in vivo, hIL-11Ra mRNA and protein were not detected in isolated human platelets. These data indicate that rhIL-11 acts directly on MKs and MK progenitors, but not on platelets. Since rhIL-11 has immunomodulating activities in several animal models and biologic activity in patients with inflammation (Dorner et al., 1997), the relative expression of mIL-11Ra has been determined in immune cells (Treppicchio and Dorner, 1998). RNase protection assays revealed the receptor component in murine blood marrow cells, peritoneal macrophages and spleen cells, all known to be responsive to rhIL-11 treatment. However, only very low levels of hIL-11Ra were detectable by RT-PCR in human neutrophils (Bozza et al., 1998), and rhIl-11 treatment had no effect on neutrophil function.

IL-11 RECEPTOR SIGNAL TRANSDUCTION The activities of murine (Barton et al., 1999) and human (Czupryn et al., 1995a, 1995b) IL-11 ligand mutants in receptor binding and cell proliferation assays have been used to characterize the critical residues involved in the binding of murine and human IL-11 to both the IL-11R and gp130. The location of these residues, as predicted from structural studies and a model of IL-11, suggest that murine and human IL-11 have three distinct receptor binding sites, structurally and functionally analogous receptor binding sites I, II and III of interleukin-6. These data support the concept that IL-11 signals through the formation of a hexameric receptor complex and suggests that site III is a common feature of cytokines that signal through gp130. The gp130 signaling mechanisms have been reviewed previously (Hirano et al., 1997). Both IL-6 and IL-11 in a complex with their specific alpha-receptors, interact with gp130 and, as a consequence, activate the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signaling pathway in their target cells. Dahmen et al. (1998) studied the interaction of IL-11 and IL-11R with human gp130 by use of a soluble hIL-11Ra, expressed in baculovirus-infected insect cells. Coprecipitation binding assays revealed that IL-11 and IL-6 compete for binding to gp130. Then, deletion and point

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mutations of gp130 were used to show that IL-11IL-11R and IL-6-IL-6R recognize overlapping binding motifs on gp130. The intracellular domain of IL-11R alpha has no significant effect on IL-11 ligand binding and signaling (Lebeau et al., 1997). Treatment of 3T3-L1 cells with IL-11, IL-6, LIF and oncostatin M was shown to induce overlapping, but distinct patterns of tyrosine phosphorylation and activation indistinguishable primary response genes (Yin et al., 1994). IL-11 and other IL-6 family cytokines activate mitogen-activated protein (MAK) kinases and the 85–92-kDa ribosomal S6 protein kinase (pp90rsk). A 130-kDa tyrosine-phosphorylated protein induced by IL-11 in 3T3-L1 cells was identified as JAK2 tyrosine kinase (Yin et al., 1994). In 3T3-L1 cells, rhIL-11 activates phospholipase D to produce the second messenger phosphatidic acid (PA). The increased PA then enhanced tyrosine phosphorylation of p44 and p47, members of MAP kinase family (Siddiqui and Yang, 1995). Tyrosine phosphorylation of Syp was also seen (Fuhrer et al., 1995). Syp was inducibly associated with both gp130 and Janus kinase 2 (JAK2). IL-11 promotes the formation of the active GTP-bound form of Ras, suggesting that IL-11 actions may be transduced in part through the Ras/MAPK signaling pathway (Wang et al., 1995). Spencer and Adunyah (1997) studied the possible

involvement of PKC in the IL-11 signaling pathway. IL-11 stimulated rapid PKC activation and markedly induces cytosolic to particulate (membrane) association of alpha and beta PKC isoforms, suggesting that PKC may be involved in the IL-11 signaling cascade. Dependent on the cell type and the condition of that cell, damaged or normal, the effect of IL-11 receptor activation may vary. The consequences of gp130 signalling and the subsequent activation of STAT3 are seen in Figure 15.5 (Hirano et al., 1997).

DOWNSTREAM GENE ACTIVATION Depending on cell type, a variety of early response genes are induced, including tis8, tis11, tis21 and junB (Du and Williams, 1997). Of course the acute phase protiens are activated as is the case with IL-6, but the potency of IL-11 appears weak, compared with IL-6 (Fukada and Sassa, 1993).

IL-11 RECEPTOR FUNCTION IL-11 activity is required for normal development of the placenta and implantation site since the second-

642(1) JAKs

JAKs

709(68) 749(108) Y759 Y767 774(133) STAT3 c-Myc

S

G2/M

Cell cycle progression

Bcl-2 Anti-apoptosis

Growth

P P

MAPK

STAT3

p19INK4D c-myc c-myb Growth arrest

Neurite outgrowth in PC12 cell Acute phase response ?

Differentiation in M1 cells

THE CYTOKINES AND CHEMOKINES

FIGURE 15.5 The signaling sequence for IL-11 receptor is shown (from Hirano, 1997).

IN VIVO PHARMACOLOGY OF rh IL - 11

ary decidual response and subsequent successful placental formation fails to occur in mice with a null mutation of the mIL-11Ra gene (Nandurkar et al., 1997a; Robb et al., 1998). These mice were healthy and had normal peripheral white blood cell, hematocrit and platelet levels (Nandurkar et al., 1997b). However, female mice were infertile because of defective decidualization (Robb et al., 1998). Another mouse with expression of a mutant, low activity, mIL-11Ra exhibited low fertility with very small litters, indicating that slight function of the receptor complex could allow very low production of offspring without complete infertility (Bilinski et al., 1998). Therefore, IL-11, as well as LIF, appears to play a critical role during reproduction. Murine studies of the IL-11 receptor alpha chain knockout mouse indicate that activity of IL-11 is an absolute requirement for successful reproductive function (Robb et al., 1998). Blastocysts were able to initially implant, but then decidualization did not occur and pregnancy failed. The secondary decidual zone did not develop, and the space normally occupied by mesometrial decidua was filled with trophoblast giant cells at 6.5 days. Most embryos were dead by 7.5 days, and no viable embryos were present after 10.5 days.

IN VIVO PHARMACOLOGY OF rhIL-11 Thrombopoietic pharmacology The in vivo pharmacology effects of rhIL-11 have been extensively reviewed (Goldman, 1995; Dorner et al., 1997; Schwertschlag et al., 1999; Keith, 2000). Since rhIl-11 was initially characterized as a hematopoietic cytokine, rhIL-11 has been evaluated in multiple murine models of chemotherapy- and/or irradiation-induced myelosuppression and in murine models of bone marrow transplantation. Although administration of rhIL-11 as a single agent to normal animals primarily stimulated megakaryocytopoiesis and thrombopoiesis, in myelosuppressed animals, rhIL-11 stimulated multilineage recovery of hematopoietic progenitor cells in many murine models. Multilineage stimulation of both bone marrow and splenic hematopoietic progenitors was seen in the peripheral circulation as significant improvements in

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the platelet nadir and in the red blood cell nadir, accompanied by a shorter period of thrombocytopenia and anemia. Therapy with rhIL-11, begun the day following chemotherapy in carboplatin-induced myelosuppression in cynomolgus monkeys significantly improved platelet nadirs and recovery in this model of myelosuppression (Schlerman et al., 1996). Platelets from rhIL-11-treated nonhuman primates were functionally and ultrastructurally normal, supporting the contention that the physiologic process of platelet production was stimulated by rhIL-11 treatment (Kaviani et al., 1996; Schlerman et al., 1996; Dorner et al., 1997). Slight differences in relative potency of rhIL-11 between species appear to be related to size/metabolic rate determined differences in clearance (reviewed in Keith, 2000). Allometric scaling pharmacokinetic estimates based on clearance indicate an approximate 10-fold difference in the relative potency between the mouse and humans. Linear pharmacokinetics were seen after both i.v. infusion and s.c. administration. Comparison of half-life and mean residence values after i.v. administration with those after s.c. administration indicated that rhIL-11 SC pharmacokinetics were absorption rate-limited. Further studies of the absorption and elimination of subcutaneously administered rhIL-11 in normal volunteers and in patients with renal failure have confirmed that the kidney is the site of elimination and metabolism since anephritic animal models and patients with renal failure have delayed elimination of rhIL-11 (Hutabarat et al., 1997).

Clinical results Seven days of rhIL-11 subcutaneous administration at a dose of 25 lg kg1 increased plasma levels of von Willebrand Factor (vWF) and fibrinogen 94% and 140%, respectively, in normal volunteers (Kaye et al., 1994). vWF and fibrinogen are important proteins in the hemostatic process, as vWF mediates the attachment of platelets to basement membranes after injury, contributes to normal platelet aggregation, and complexes with coagulation factor VIII to protect it from proteolysis. This activity could be especially beneficial in the thrombocytopenic patient. Recently, Denis et al. (2001) examined the mechanism of the

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effect of recombinant human (rh)IL-11 on vWf and factor VIII (FVIII) secretion in mice. In vitro, rhIL-11 did not increase vWf production by endothelial cells, and in vivo, plasma vWf was not elevated in mice after a single intravenous (i.v.) bolus injection of 250 or 1000 lg/kg rhIL-11. Continuous exposure to rhIL-11 by subcutaneous therapy increased plasma vWf and FVIII levels two-fold after 4 and 7 days. This occurs despite no increase in vWf or FVIII messenger RNA. Heterozygous vWf mice responded to rhIL-11 treatment by a significant increase in platelet counts and vWf and FVIII levels. In vWf-deficient mice, rhIL-11 caused significant increases in FVIII and reduced skin bleeding time. The results of rhIL-11 clinical trials in cancer patients have been reported (Gordon et al., 1996; Tepler et al., 1996; Vrendenburgh et al., 1998). In a phase I study in patients with breast cancer (Gordon et al., 1996), rhIL-11, at doses ranging from 10 to 100 μg kg1, was administered once daily by subcutaneous injection to women for 14 days prior to chemotherapy (cycle 0). Patients then received up to four 28-day cycles of cyclophosphamide and doxorubicin, followed by rhIL-11 therapy at their assigned dose on days 3–14. Treatment with rhIL-11 produced a dose-dependent increase in peripheral platelet counts in cycle 0. Patients receiving rhIL-11 at doses 25 μg kg1 experienced less thrombocytopenia in the first two cycles of chemotherapy. rhIL-11 had thrombopoietic activity at all doses tested and was well tolerated at doses of 10, 25 and 50 μg kg1. The effects of rhIL-11 were subsequently evaluated in a randomized placebo-controlled trial in cancer patients who had previously received platelet transfusions for severe chemotherapy-induced thrombocytopenia (Tepler et al., 1996). Ninety-three patients who had received platelet transfusions for nadir platelet counts 20 000 μl1 during the chemotherapy cycle immediately prior to entry into the study were randomized to receive placebo or rhIL-11 at doses of 25 or 50 μg kg1 subcutaneously once daily for 14 to 21 days beginning 1 day after chemotherapy. Therapy with rhIL-11 reduced the need for platelet transfusions. This study demonstrated that rhIL-11 at a dose of 50 μg kg1 reduced the chance of platelet transfusion in subsequent chemotherapy cycles for patients who had required platelet transfusions for thrombocytopenia in a previous cycle.

Having been approved by the FDA, rhIL-11 (Neumega) is indicated for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in patients with nonmyeloid malignancies who are at high risk of severe thrombocytopenia. Kurzrock et al. (2001) have recently reported their initial experience with low-dose rhIL-11 in patients with bone marrow failure states. In this small study, patients received rhIL-11 subcutaneously at 10 μg kg1 daily. Thirty-eight percent of the patients responded with a median increase in platelet count of 95  109 l1 above baseline. This pilot study suggests that low doses of rhIL-11 can increase platelet counts without toxicity in this patient population.

Effects of rhIL-11 on gastrointestinal mucositis During studies of rhIL-11’s ability to produce hematopoietic reconstitution following chemotherapy, increased survival of rhIL-11-treated mice that had been subjected to severe cytoablative therapy was reported (Du et al., 1994). Extensive damage to the small intestinal mucosa resulted in death of all vehicle-treated animals. However, treatment with rhIL-11 (250 μg kg1 day1) s.c., beginning on the day of irradiation, increased survival to 85% and was associated with a rapid recovery of small intestine. Further study of rhIL-11 treatment in this model demonstrated that rhIL-11 suppressed apoptosis in the small intestine following cytoablative therapy and increased villus length (Orazi et al., 1996). Studies of rhIL-11’s abilitiy to reduce chemotherapyinduced mucositis were continued by Sonis et al. (1995, 1997). rhIL-11 reduced oral mucositis in a 5-fluroruracil (5-FU) hamster model in a dosedependent manner (Figure 15.6) and increased animal survival at all doses tested. Oral mucosal tissue damage was reduced when rhIL-11 was administered subcutaneously at the time of chemotherapy, as well as when rhIL-11 administration was delayed until chemotherapy was complete. Combined with the data from Booth and Potten (1995) and Peterson et al. (1996), these results suggest that in models of mucositis using cell cycle specific cytoablative agents, the activity of rhIL-11 may be due in part to inhibition of cell cycle progression in intestinal epithelial cells.

THE CYTOKINES AND CHEMOKINES

IMMUNOMODULATORY EFFECTS OF rh IL - 11

100 90 80

Survival %

70 60 50 40

Group 1 - Placebo Group 2 - 3lg/day Group 3 - 10lg/day Group 4 - 30lg/day Group 5 - 100lg/day

30 20 10 0 6

7

8

9

10

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FIGURE 15.6 The mucositis scores for hamsters with 5-FU-induced oral mucositis are shown. All doses of rhIL-11 increased animal survival (from Sonis et al., 1995, with permission). Radiation-induced damage to the murine small intestinal crypt clonogenic stem cells has been reduced by rhIL-11 (Potten, 1995). Since the regenerative capacity of the intestinal crypt stem cells following injury determines the survival of crypts, these results suggest another mechanism by which rhIL-11 can maintain mucosal integrity. Treatment with rhIL11 was also shown to enhance survival in a murine model of radiation-induced pulmonary injury in which TNF-a is involved in lung damage (Redlich et al., 1996). The reduced injury in rhIL-11-treated animals correlated with a decrease in TNF-a mRNA in lung tissue. The radioprotection was apparently specific for normal thoracic structures since rhIL-11 did not affect the development or radiosensitivity of EMT6 tumor cell lung metastases. Hill, G.R. et al. (1998) investigated the pharmacologic properties of rhIL-11 in the setting of allogeneic bone marrow transplantation (BMT). Mice received rhIL-11 (500 mg kg1 s.c. divided by twice daily doses) or its diluent from day 2 to 7 or 14. GVHD was severe with mortality beginning on day 4. Treatment with rhIL-11 significantly decreased lethal GVHD (day 50 survival: 90 versus 20%, P 0.001) when compared with treatment with diluent. Survival in syngeneic BMT recipients was 88% at day 45. In 9–11-week-old mice receiving syngeneic BMT, rhIL-11 therapy in the same regimen resulted in 100% long-term survival at 90 days versus 60% survival in diluent-treated animals.

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In separate experiments, where mice received allogeneic BMT with lower whole body irradiation dose (1300 cGy) and more T cells (2  106), the same rhIL-11 therapy described previously increased survival from 35 to 70% at day 70. With rhIL-11 therapy, donor T cell cytokine responses to host antigen following BMT were polarized toward TH2 with a 10-fold increase in IL-4 occurring as 50% decreases in IFN-c and IL-2 were observed. Serum IFN-c levels were reduced, and decreased IL-12 production was present in mixed lymphocyte cultures. Histologic evaluation of the small bowel, large bowel and liver at day 45 revealed that the total GVHD score was reduced from 27  3.2 in allogeneic control animals to 16.1  1.5 in rhIL-11-treated mice (P 0.02). The GVHD score in syngeneic recipients was 13.3  2, different from allogeneic animals (P 0.05), but not different from rhIL-11-treated mice. These data indicate that therapy with rhIL-11 reduced mortality and morbidity following allogeneic BMT by polarizing T cells, protecting the small intestine and suppressing proinflammatory cytokine release. Teshima et al. (1999) extended these findings to investigate whether rhIL-11 could maintain a graftversus-leukemia (GVL) effect while decreasing GVHD. Lethally irradiated B6D2F1 mice were given either T cell-depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. They also received host-type P815 mastocytoma cells at the time of BMT. The animals received subcutaneous rhIL-11 or diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All controland IL-11-treated allogeneic animals rejected their leukemia, but rhIL-11 also reduced GVHD-related mortality. Examination of the mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8() T cells.

IMMUNOMODULATORY EFFECTS OF rhIL-11 Endogenous production of IL-11 appears to be evoked by several proinflammatory mediators. When

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this fact is combined with the inhibitory activities produced by exogenous rhIL-11 or the synergistic anti-inflammatory activity of endogenous IL-11 with IL-10 (Hermann et al., 1998), a strong rationale for immunomodulating effects of IL-11 can be constructed. Studies of rhIL-11, conducted to determine its biological activity in systemic inflammatory responses and during inflammation of the gastrointestinal tract, are reviewed in detail elsewhere (Keith, 2000).

Models of systemic inflammatory conditions Dose-dependent reductions in mortality were produced by rhIL-11 in a model of T cell-mediated toxic shock triggered by superantigen (Barton et al., 1996). At 500 μg kg1, mortality was 55% compared with 100% in BSA-treated animals. Therapy with rhIL-11 in a rabbit model of endotoxemia reduced histologic lesions of the gastrointestinal tract and prevented hypotension that occurs as a consequence of endotoxemia (Misra et al., 1996). In murine endotoxemia, rhIL-11 reduced the peak serum levels TNF-a, IFN-c and IL-1b serum levels, ranging from 80 to 95% reductions, compared with animals which received only LPS (Trepicchio et al., 1996). Treatment with rhIL-11 improved survival, reduced gastrointestinal bacterial translocation, and ameliorated bone marrow suppression in burned mice (Schindel et al., 1997). rhIL-11 improved survival in the neutropenic rat model of Pseudomonas sepsis (Opal et al., 1998). rhIL-11 administered once daily intravenously for 3 days, beginning at the onset of fever in cyclophosphamide-treated rats with Pseudomonas aeruginosa 12.4.4, significantly reduced the quantitative level of lung infection, pulmonary edema and injury to the gastrointestinal epithelium of the small and large intestine. Forty percent of rhIL-11, 60% of ciprofloxacin, 100% of rhIL-11-ciprofloxacin-treated animals survived, but none of the vehicle group survived. These results indicate that rhIL-11 supports the integrity of the gastrointestinal tract and decreases the systemic inflammatory response to experimental Gram-negative infection in immunocompromised animals. Similar beneficial effects were seen when rhIL-11 and rhG-CSF were administered subcutaneously to the neutropenic rat model of Pseudomonas

aeruginosa sepsis (Opal et al., 1999). rhG-CSF did not provide a survival advantage (0% survival) compared with the placebo group (8% survival). rhIL-11 was partially protective (40% survival) and the combination of rhG-CSF and rhIL-11 resulted in a survival rate of 80%. rhIL-11 alone or in combination with rhG-CSF resulted in preservation of gastrointestinal mucosal integrity, lower circulating endotoxin levels, and reduced quantitative levels of P. aeruginosa in quantitative organ cultures. Endotoxin-inducible cytokine production was suppressed by a single dose of rhIL-11 for greater than 24 h in human subjects (Reznikov et al., 1999). Volunteers and renal failure patients received rhIL-11 (50 μg kg1 subcutaneously) and blood was obtained before therapy and at multiple timepoints to 48 h following the single dose. LPS (10 ng ml1) was used to stimulate cytokine production for 24 h, and TNF-a, IL-8, and IL-1b levels were determined. Maximum supression on the order of 50% occurred at 1 h, and the levels remained lower for 24 h. These data indicate that rhIL-11 is able to suppress proinflammatory cytokine production in humans as in animals.

Models of intestinal ischemia and short bowel syndrome Ischemic bowel necrosis has been reduced in mice by treatment with rhIL-11 (Du et al., 1997). Pretreatment with rhIL-11 in mice with bowel ischemia (induced by occluding the superior mesenteric artery for 90 min) significantly decreased morbidity and mortality. rhIL-11-treated mice demonstrated rapid recovery of intestinal mucosa, a concurrent increase in mitotic activity, a suppression of apoptosis in intestinal crypt cells, and an increased peripheral platelet and leukocyte count. Vehicle-treated mice developed thrombocytopenia after ischemia, but no rhIL-11-treated mice developed thrombocytopenia. Administration of rhIL-11 produces intestinal mucosal trophic effects in rats with experimental short bowel syndrome (Liu et al., 1996). After 90% small bowel resection, weights of animals that received IL-11 were significantly greater at the beginning of post-operative day 4 in comparison with that of the BSA groups. The rats treated with IL-11 also had significantly greater villus height and crypt cell mitotic rates.

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The effects of interleukin-11 and epidermal growth factor on residual small intestine were examined after massive small bowel resection in rats (Fiore et al., 1998). Animals treated with rhIL-11 and rhIL-11-EGF had increased mucosal mass at days 4 and 8 compared with controls and EGF. rhIL-11 had a trophic effect on small intestinal enterocytes, causing cell proliferation and increased mucosal thickness. EGF has a more generalized effect on intestine causing proliferation of both enterocytes and myocytes. Modulation of mucosal atrophy and bowel shortening by rhIL-11 were explored after creation of ThiryVella fistulas (TVF) in rats (Dickinson et al., 2000). On day 14, the TVF were examined, and enterocyte apoptosis was measured using the TUNEL assay. Administration of rhIL-11 resulted in a significantly greater weight gain and less shortening of TVF. TVF from the rhIL-11-treated group showed evidence of hyperplasia and hypertrophy and increased crypt to villus ratio. Vehicle animals had substantial mucosal atrophy. Recombinant human IL-11 prevented mucosal atrophy and shortening of defunctionalized intestinal loops. Nadler et al. (2001) measured intestinal cytokine mRNA in 21 infants with histologically confirmed NEC, 18 with other inflammatory conditions, and in nine patients without intestinal inflammation. IL-8 and IL-11 mRNA levels were up-regulated in patients with acute NEC compared with the other groups, and the levels returned to baseline by the time of stoma closure. The increased IL-11 mRNA decreased the likelihood of pan-necrosis (odds ratio, 0.93; P = 0.002), as did increased IL-12. The researchers postulated that local up-regulation of IL-11 may be an adaptive response that could limit intestinal damage in NEC.

Models of inflammatory bowel disease The effects of rhIL-11 on acute colonic injury were assessed in acetic acid-induced colitis in rats (Keith et al., 1994). These animals with acetic acid usually develop diffuse colonic lesions, characterized by ulceration, hemorrhage, edema, depletion of goblet cells and infiltration of leukocytes. The gross and histologic lesions of rhIL-11-treated animals were significantly reduced compared with vehicle control animals. Administration of rhIL-11 (10 to 1000 μg kg1 s.c.) for only the first 5 days following acetic acid

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injury was as effective as administration for 14 days after injury. rhIL-11 was also protective or therapeutic in a hapten-induced model of inflammation, intracolonic administration of trinitrobenzene sulfonic acid (TNBS) colitis in rats (Qiu et al., 1996). Dosedependent reductions in colonic ulcer indexes of rhIL-11-treated rats were achieved. Myeloperoxidase activity was increased during the TNB-induced colitis and was reduced by rhIL-11 administration. Mucus production was enhanced with the rhIL-11 therapy. HLA-B27 transgenic Fisher 344 rats, expressing HLA-B27 and b2-microglobulin genes, have lesions of the gastrointestinal system similar to those of Crohn’s disease patients. Treatment with rhIL-11 reduced the clinical signs and histologic lesion scores of inflammatory bowel disease in these rats (Keith et al., 1994, Peterson et al., 1998). RT-PCR analysis of colonic RNA revealed that treatment with rhIL-11 down-regulated expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IFN-gamma. rhIL-11 reduced the level of myeloperoxidase activity in the cecum, and anti-CD3 antibody-stimulated spleen cell cultures from rhIL-11-treated rats produced less IFNgamma, TNF-alpha and IL-2 than those from vehicletreated rats. The feasibility of oral treatment of HLA-B27 transgenic rats with rhIL-11 was investigated by Greenwood-Van Meerveld et al. (2001). Contractility of jejunal and colonic longitudinal muscles was examined. Muscle strips were isolated from HLA-B27 transgenic rats with spontaneous inflammation following treatment with enteric-coated rhIL-11 multiparticulates (500 μg kg1), or placebo multiparticulates were given orally every 48 h for 2 weeks. The HLA-B27 rats receiving placebo had chronic diarrhea, and MPO activity was increased in the jejunum and colon. Intestinal inflammation was associated with a decreased ability of the muscles to generate active tension in response to electrical field stimulation, carbachol, or high KCl. In the jejunum of placebo-treated HLA-B27 rats, concentration–effect curves for carbachol were shifted to lower concentrations yielding a higher EC50. Oral treatment of HLA-B27 rats with rhIL-11 suppressed the symptoms of diarrhea (Figure 15.7), normalized MPO activity, and improved the colonic damage score. Neurally mediated responses were improved, and the maximal tension generated

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diarrhea

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FIGURE 15.7 Orally delivered rhIL-11 decreased clinical signs of inflammatory bowel disease in HLAB27 transgenic rats treated every 48 h for 2 weeks (from Greenwood Van Meerveld et al., 2001, with permission). by carbachol or KCl returned toward normal in the jejunum and colon. The response to carbachol in the jejunum of HLA-B27 rats was also normalized by rhIL-11 treatment. These data show that oral administration of enteric-coated rhIL-11 suppresses intestinal inflammation and reverses intestinal smooth muscle dysfunction in HLA-B27 transgenic rats, despite the fact that it is apparently acting locally and is not available systemically (Tseng et al., 2000).

SUMMARY Interleukin-11 (IL-11), initially described in 1990, as a bone marrow stroma-derived hematopoietic cytokine, is a highly conserved, 178 amino acid cationic protein. The human and the nonhuman primate genes for IL-11 have been localized to the long arm of chromosome 19 at band 19q13.3-q13.4. IL-11 is a member of the IL-6 ligand family as it utilizes gp130 as the signaling component of its receptor, and amino acid residues 59 (methionine), 42 (lysine) and 99 (lysine) are critical for function of the IL-11 protein, being completely conserved in the mouse, nonhuman primate and human proteins. Interleukin-11 has multiple activities that continue to be characterized. Initial, in vitro and in vivo studies demonstrated a

hematopoietic activity for interleukin-11, largely manifest as thrombopoiesis in humans, and rhIL-11 has been developed and approved by the United States Food and Drug Administration for use in the prevention of severe thrombocytopenia occurring after cancer chemotherapy. Studies of cells and tissues from other organ systems indicate that IL-11 is produced by a variety of other cell types and has activity in protection and restoration of the gastrointestinal mucosa, major effects as an immunomodulating agent, and activity in bone metabolism. Developmental investigations in mice indicate a widespread distribution of IL-11 expression in the embryo, but of great interest is the finding, from IL-11 receptor alpha chain knock-out mice, that IL-11 signaling is an absolute requirement for normal development of placentation and survival to birth.

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Reznikov, L.L., Puren, A.J., Fantuzzi, G. et al. (1999). Suppression of endotoxin-inducible cytokines in whole blood from human subjects following a single dose of recombinant human interleukin-11. J. Endotoxin Res. 5, 197–204. Romas, E., Udagawa, N., Zhou, H. et al. (1996). The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures. Exp. Med. 183, 2581–2591. Robb, L., Hilton, D.J., Brook-Carter, P.T. and Begley, C.G. (1997). Identification of a second murine interleukin-11 receptor alpha-chain gene (IL11Ra2) with a restricted pattern of expression. Genomics 40, 387–394. Robb, L., Li, R., Hartley, L. et al. (1998). Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation. Nat. Med. 4, 303–308. Schendel, P.F. and Turner, K.J. (1998). Interleukin-11. In: Mire-Sluis, A.R., Thorpe, R. (eds). Cytokines. SanDiego: Academic Press, pp. 169–182. Schindel, D., Maze, R., Liu, Q. et al. (1997). Interleukin-11 improves survival and reduces bacterial translocation and bone marrow suppression in burned mice. J. Pediatr. Surg. 32, 312–315. Schlerman, F.J., Bree, A.G., Kaviani, M.D. et al. (1996). Thrombopoietic activity of recombinant human interleukin 11 (rhIL-11) in normal and myelosuppressed nonhuman primates. Stem Cells 14, 517–532. Schwertschlag, U.S., Trepicchio, W.L., Dykstra, K.H. et al. (1999). Hematopoietic, immunomodulatory, and epithelial effects of interleukin-11. Leukemia 13, 1307–1315. Siddiqui, R.A. and Yang, Y.C. (1995). Interleukin-11 induces phosphatidic acid formation and activates MAP kinase in mouse 3T3-L1 cells. Cell. Signal. 7, 247–259. Soda, H., Raymond, E., Sharma, S. et al. (1999). Recombinant human interleukin-11 is unlikely to stimulate the growth of the most common solid tumors. Anticancer Drugs 10, 97–101. Sonis, S.T., Muska, A., O’Brien, J.O. et al. (1995). Alteration in the frequency, severity and duration of chemotherapyinduced mucositis in hamsters by interleukin-11. Oral Oncology, Eur. J. Cancer 31B, 261–266. Sonis, S.T., Van Vugt, A.G., McDonald, J. et al. (1997). Mitigating effects of interleukin 11 on consecutive courses of 5-fluorouracil-induced ulcerative mucositis in hamsters. Cytokine 9, 605–612. Sotiriou, C., Lacroix, M., Lagneaux, L. et al. (1999). The aspirin metabolite salicylate inhibits breast cancer cells growth and their synthesis of the osteolytic cytokines interleukins-6 and -11. Anticancer Res. 19, 2997–3006. Spencer, G.C. and Adunyah, S.E. (1997). Interleukin-11 induces rapid PKC activation and cytosolic to particulate translocation of alpha and beta PKC isoforms in human erythroleukemia K562 cells. Biochem. Biophys. Res. Commun. 232, 61–64. Stubbs, L., Carver, E.A., Shannon, M.E. et al. (1996). Detailed comparative map of human chromosome 19q and related regions of the mouse genome. Genomics 35, 499–508. Takai, H., Kanematsu, M., Yano, K. et al. (1998). Transforming growth factor-beta stimulates the production of osteoprotegerin/osteoclastogenesis inhibitory factor by bone marrow stromal cells. Biol. Chem. 273, 27091–27096.

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Taki, H., Sugiyama, E., Mino, T. et al. (1998). Differential inhibitory effects of indomethacin, dexamethasone, and interferon-gamma (IFN-gamma) on IL-11 production by rheumatoid synovial cells. Clin. Exp. Immunol. 112, 133–138. Teicher, B., Chen, Y.N., Ara, G. et al. (1996). Interaction of interleukin-11 with cytotoxic therapies in vitro against stem cells and in vivo against EMT-6 murine mammary carcinoma. Int. J. Cancer 67, 864–870. Tepler, I., Elias, L., Smith, J.W. 2nd. et al. (1996). A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. Blood 87, 3607–3614. Teramura, M., Kobayashi, S., Hoshino, S. et al. (1992). Interleukin-11 enhances human megakaryocytopoiesis in vitro. Blood 79, 327–331. Teshima, T., Hill, G.R., Pan, L. et al. (1999). IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation. J. Clin. Invest. 104, 317–325 Trepicchio, W.L. and Dorner, A.J. (1998). Interleukin-11 A gp130 cytokine. Ann. NY Acad. Sci. 856, 12–21. Trepicchio, W.L., Bozza, M., Pedneault, G. and Dorner, A.J. (1996). Recombinant human interleukin-11 attenuates the inflammatory response through downregulation of proinflammatory cytokine release and nitric oxide production. J. Immunol. 157, 3627–3634. Trepicchio,W.L.,Wang, L.L., Bozza, M. and Dorner, A.J. (1997). IL-11 regulates macrophage effector function through the inhibition of nuclear factor-kappaB. J. Immunol. 159, 5661–5670. Trepicchio, W.L., Ozawa, M., Walters, I.B. et al. (1999). Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions. J. Clin. Invest. 104, 1527–1537. Tseng, C.M., Albert, L., Peterson, R.L. et al. (2000). In vivo absorption properties of orally administered recombinant human interleukin-11. Pharm. Res. 17, 482–485. Van Leuven, F., Stas, L., Hillicker, C. et al. (1996). Molecular cloning and characterization of the human interleukin11 receptor a-chain gene, IL11RA, located on chromosome 9p13. Genomics 31, 65–70. Wang, X.Y., Fuhrer, D.K., Marshall, M.S. and Yang, Y.C. (1995). Interleukin-11 induces complex formation of Grb2, Fyn, and JAK2 in 3T3L1 cells. J. Biol. Chem. 270, 27999–28002. Weich, N.S., Wang, A., Fitzgerald, M. et al. (1997). Recombinant human interleukin-11 directly promotes megakaryocytopoiesis in vitro. Blood 90, 3893–3902. Willis, D., Moore, A.R., Frederick, R. and Willoughby, D.A. (1996). Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat. Med. 2, 87–90. Xu, J.W., Li, T.F., Partsch, G. et al. (1998). Interleukin-11 (IL-11) in aseptic loosening of total hip replacement (THR). Scand. J. Rheumatol. 27, 363–367. Yin, T., Yasukawa, K., Taga, T. et al. (1994). Identification of a 130-kilodalton tyrosine-phosphorylated protein induced by interleukin-11 as JAK2 tyrosine kinase, which associates with gp130 signal transducer. Exp. Hematol. 22, 467–472. Zhang, Y., Fujita, N., Oh-hara, T. et al. (1998). Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis. Oncogene 16, 693–703.

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16 Interleukin-12 Family [IL-12, 23, 12RA and 27] Pawel Kalinski, Walter J. Storkus, Angus W. Thomson and Michael T. Lotze University of Pittsburgh, PA, USA

When you know a thing, to hold that you know it; and when you do not know a thing, to allow that you do not know it – this is knowledge Confucius

INTRODUCTION The pleiotropic immunomodulatory effects of IL-12, complex patterns of regulation of the production of each of its subunits p35 and p40 in individual cell types and the recent emergence of novel family members provide ample areas for investigation. Some of the old mysteries related to IL-12 biology have recently been clarified, following the identification of IL-23 (Oppmann et al., 2000), a p19–p40 heterodimer with IL-12-like functions (Table 16.1). In contrast to IL-12 and IL-23, the two members of IL-12 family which support TH1-type responses and cell-mediated immunity, the roles of the IL-12 receptor-antagonistic p40 homodimer (p40–p40; Mattner et al., 1993; Germann et al., 1995; Gillessen et al., 1995; Ling et al., 1995) and IL-27 (EBI3–p28; Devergne et al., 1996, 1997) are less well-defined and may involve reciprocal,

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

TH2 promoting activities. In the face of the discovery of novel IL-12 family members that, similar to the p40–p40 homodimer, share one of the original IL-12 subunits, the traditional name of p40 homodimer as IL-12p40 is potentially misleading. Therefore, we will refer to the p40–p40 molecule as the IL-12 receptor antagonist (IL-12RA).

IL-12 FAMILY: BIOCHEMISTRY, GENETICS AND THE FAMILY HISTORY IL-12 (p40–p35) IL-12 was simultaneously described as a CTL maturation factor (CLMF) (Gately et al., 1986; Wong et al., 1988; Stern et al., 1990), a T cell-stimulating factor

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TABLE 16.1 IL-12 family members Subunit composition

Name(s)

Receptor interactions

Major activities

References

p40–p35

IL-12; IL-12p70; TSF; CLMF; NKSF;

IL-12b1/b2

NK, CTL, TH1, enhancement of IFNc, TNFa, cytotoxicity

Gately et al., 1986, 1991 Germann et al., 1987, 1988; Wong et al., 1988; Kobayashi et al., 1989; Stern et al., 1990

p40–p19

IL-23

IL-12b1; additional receptor chain

IL-12-like

Oppmann et al., 2000

p40–p40

p40 homodimer, IL-12 receptor antagonist (IL-12RA)

IL-12b1/b2, competitive inhibitor of IL-12R

Antagonizes IL-12 receptor interactions

Mattner et al., 1993; Germann et al., 1995; Gillessen et al., 1995; Ling et al., 1995

EBI-3–p28

IL-27

WSX-1/TCCR

Promotes clonal expansion of naive CD4 T cells; synergizes with IL-12 in IFNc induction, may be involved in TH2-type inflammation (?)

Devergne et al., 1996, 1997; Pflanz et al., 2002

(TSF) (Germann et al., 1987, 1988), and a natural killer cell stimulatory factor (NKSF) (Kobayashi et al., 1989), based on its ability to promote CTL and NK cytolytic and effector activity. Cloning of the genes encoding NKSF and CLMF revealed that they are identical, and the unifying term IL-12 was applied (Gubler et al., 1991). The active factor proved to be a 70-kDa heterodimeric protein, composed of a heavy chain (p40) and a covalently associated light chain (p35). The p40–p35 heterodimer is currently known as interleukin-12 or IL-12p70. Both IL-12 genes are localized on different chromosomes, p35 on chromosome 5 (5q31–q33) and p40 on chromosome 3 (3p12–13.2) (Sieburth et al., 1992; Ma et al., 1996, 1997). The expression of each of the IL-12 subunits is regulated independently. Most cell types have the capacity to express p35, either constitutively or after stimulation. In contrast, the IL-12 p40 gene is expressed in a far more restricted manner, primarily by antigen-presenting cells (APCs). Since the biologically active IL-12 can only be produced in cell types co-expressing both the p35 and p40 chains of IL-12 (Gubler et al., 1991), the ability of a given cell type to produce p40 dictates their ability to produce and secrete bioactive IL-12p70. However, in contrast to several other cell types including T cells, that produce p35 spontaneously, APCs, including monocytes,

macrophages and dendritic cells (DCs), depend on additional stimuli to produce p35, thereby preventing any uncontrolled IL-12 production. This results in a strict pattern of regulation of IL-12p70: it is the p40 subunit that determines the ability of a given cell type to produce IL-12, but the absolute amount of the cytokine produced by APCs is determined by the amount of p35 produced (Snijders et al., 1996; Goriely et al., 2001). This complex pattern of regulation is consistent with the crucial role of IL-12 in regulating nearly every aspect of the immune response. This includes modulation of APC function, the efficacy of antigen processing and/or presentation and the activation, expansion and differentiation of T cells and/or B cells, and regulation of both effector and regulatory functions of these cell types. The predominant role of IL-12 is its positive impact on cell-mediated immunity, both at the level of its induction as well as effector mechanisms.

IL-23 (p40–p19) The deficit in TH1-related functions in p35 knockout animals is less pronounced than that in animals lacking the IL-12p40 subunit (Mattner et al., 1996; Piccotti et al., 1998; Carr et al., 1999; Cooper et al., 2002). This suggested the possible existence of an alternative

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p40-binding partner(s) able to bind the IL-12 receptor. Identification of p19 as the a subunit of the recently discovered IL-23 (Oppmann et al., 2000) helped to resolve the discrepancy. The gene encoding human p19 is located on chromosome 12q13 and encodes a 189-amino acid long, four-helical cytokine with five cysteine residues and no N-glycosylation sites (Oppmann et al., 2000). Its mouse analog shows 70% amino acid sequence homology and encodes a 197-amino acid long protein. The calculated molecular weight of mature human and mouse p19 protein is 18.7 kDa and 19.8 kDa, respectively. Structurally, p19 is most closely related to IL-12p35, IL-6 and G-CSF.

IL-12RA (p40–p40) Cells capable of producing biologically active IL-12 typically express far more IL-12 p40 chain than IL-12 p35 chain (Germann et al., 1995; Snijders et al., 1996, 1998; Goriely et al., 2001). In addition to binding p19, excess p40 can be also secreted as p40 homodimers. The IL-12 p40 homodimer binds to the IL-12R, but fails to mediate a signal, thereby serving as a functional antagonist to IL-12 p70 heterodimers (Mattner et al., 1993; Germann et al., 1995; Gillessen et al., 1995; Ling et al., 1995) IL-12 p40 homodimers inhibit IL-12induced murine Con A-blast proliferation, splenocyte secretion of IFNc, and NK activation (Gillessen et al., 1995; Ling et al. 1995; Gately et al., 1996). mIL-12 p40 homodimer shows a distinct IL-12 antagonistic activity following in vivo administration (Piccotti et al., 1996; Mattner et al., 1997a; Cooper et al., 2002). A similar, TH2-driving role has been also postulated for the endogenously produced p40 (Camoglio et al., 2002). Although human p40 homodimer also shows an IL-12 antagonistic function in vitro, its inhibitory function is less pronounced and its physiological role is less clear (Germann et al., 1995). Part of the problem with analyzing the role of human p40–p40 is its very low stability. In aqueous environments it rapidly dissociates into p40 monomers with substantially less binding of the IL-12R.

IL-27 (EBI-3/p28) An EBV-induced protein identified by Mark Birkenbach and colleagues, Epstein–Barr virusinduced gene 3 (EBI-3), is a novel hematopoietin

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receptor family member with homology to IL-12p40 and ciliary neurotrophic factor (CNTF) p35. EBI-3 maps to chromosome 19 (19p13.2–3), near genes encoding the erythropoietin receptor and the cytokine receptor-associated kinase, Tyk2 (Devergne et al., 1996). This gene, which was designated EBV-induced gene 3 (EBI-3), encodes a 34-kDa glycoprotein lacking a membrane-anchoring motif and can be secreted to a limited extent after formation of a p35-linked heterodimer (Devergne et al., 1996). Recently, the authentic binding partner, p28, a member of the longchain four-helix bundle cytokines, has been identified (Pflanz et al, 2002). The gene encoding human p28 is located on chromosome 16p11 and encodes a 243–234-amino acid long (human and mouse, respectively) cytokine with 13 negatively charged glutamic acid residues and no N-glycosylation sites although several O-glycosylation sites are predicted (Pflanz et al., 2002). Its mouse analog shows 73% amino acid sequence homology and likewise contains 14 negatively charged residues interrupted by one lysine with one potential N-glycosylation site at N85. The calculated molecular weight of mature human and mouse p28 protein is 24.5 kDa and 23.6 kDa, respectively. Structurally, p28 is most closely related to IL-11, CNTF, and the closely related NNT-1/CLC binding to the CNTF receptor.

CELLULAR SOURCES AND REGULATION OF THE PRODUCTION OF IL-12 FAMILY MEMBERS IL-12 Biologically active IL-12 (IL-12p70) can only be produced in cell types coexpressing both the p35 and p40 chains of IL-12 (Trinchieri and Scott, 1994; Gately et al., 1998) (see Table 16.2). Therefore, the ability of a given cell type to produce p40 dictates their ability to produce and secrete bioactive IL-12p70. This eliminates several cell types including T cells, constitutively expressing p35, as a cellular source of IL-12, restricting IL-12 production to APCs. However, in contrast to several other cell types such as T cells which produce p35 spontaneously, production of p35 in APCs, including monocytes, macrophages and DCs,

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TABLE 16.2 Production of IL-12 family members in different cell types Cell Type

Production of IL-12 family members

References

Blood monocytes

Inducible production of low amounts of p35, p40, secretion of (low amounts of) p70, inducible expression of EBI-3, p28

D’Andrea et al., 1992; Hayes et al., 1995; van Der Pouw Kraan et al., 1995; Hashimoto et al. 2000

Macrophages

Inducible expression of p40 and p35 as well as p70 secretion. Inducible expression of p28

Macatonia et al., 1993; Pflanz et al, 2002

Langerhans cells

Secretion of p40, initial demonstration of p35 expression and p70, was not confirmed by later reports. Negative in our experience

Kang et al., 1996; Teunissen et al., 1998

Spleen DCs

p40, p35, p70. Low in freshly isolated DCs high in cultured DCs

Macatonia et al., 1993, 1995; Murphy et al., 1994

Blood-isolated DCs

Expression of p40 and p35. Inducible secretion of p70; enhancement upon culture in the presence of IFNc

Ghanekar et al., 1996; Ito et al., 2001

Monocyte-derived DCs and BM-derived DCs

Inducible p40, p35, p70, EBI-3, p28; very high levels. Optimal production in relatively immature DC, reduced upon maturation. Most DC preparations spontaneously express low levels of p40, possibly due to cell culture-related stimulation

Cella et al., 1996; Koch et al., 1996; Hilkens et al., 1997; Hashimoto et al., 2000; Pflanz et al., 2002

Neutrophils

Mostly IL-12p40, low amounts of p70

Cassatella et al., 1995; Romani et al., 1997

Mesoglia

Low-level production of p40, p35 and p70

Aloisi et al., 1997

Keratinocytes

p40, p35 expression, p40 secretion, lack of p70 expression

Muller et al., 1994; Asada et al., 1997; Kondo and Jimbow, 1998

Endothelial cells

Inducible p40, p35, p70

Lienenluke et al., 2000

Trophoblast

EBI-3 and p28/IL-27 (spontaneous)

Devergne et al., 1996; Pflanz et al., 2002

requires strictly defined stimulatory requirements. Therefore, it is the p40 subunit that determines the ability of a given cell type to produce IL-12, but the absolute amount of this cytokine produced by APCs is determined by the amount of p35 produced (Snijders et al., 1997). The IL-12-inducing agents can be roughly divided into two categories. A group of exogenous IL12 inducers consists of a large array of different pathogen-related products signaling via such DC molecules as CD14, TLR-2, TLR-9, CD11b/CD18 (Manetti et al., 1995; Brightbill et al., 1999; ThomaUszynski et al., 2000; Tsuji et al., 2000; Seki et al., 2001; Campos et al., 2001; Kadowaki et al., 2001; Krug et al., 2001; Hume et al., 2001; Adachi et al., 2001; Vogel et al., 2001; Perera et al., 2001). IL-12 is induced by the very process of phagocytosis of bacteria (Corinti et al.,

1999; d’Ostiani et al., 2000). Pathogen-related agents capable of inducing IL-12 production include bacterial LPS, whole bacteria (SAC, BCG), nucleic acids: CpG motifs, double-stranded RNA or poly-IC, microparticulate ingestion, LPS, and numerous additional pathogens or derived products from species such as Staphylococcus, Mycobacterium, Neisseria, Yersinia, Candida, Toxoplasma, Listeria or Plasmodium (Hsieh et al., 1993; Tripp et al., 1994; Hayes et al., 1995; Cooper et al., 1995; Manetti et al., 1995; van Der Pouw Kraan et al., 1995; Scheicher et al., 1995; Snijders et al., 1996; Sparwasser et al., 1998). The second type of the IL-12-inducing stimulus is the interaction of APCs with Th cells. CD40–CD40L (CD154) interaction appears to be a dominant pathway of ‘endogenous’ IL-12 induction during the

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interaction of CD40L-expressing T helper cells with monocytes, macrophages or DCs. CD40 human and murine macrophages interacting with activated CD40L T cells or clones are induced to produce and secrete IL-12 (Armant et al., 1996; Kennedy et al., 1996). While the CD40 signaling pathway seems to be most important in the induction of IL-12 and the subsequent TH1 response, the effective induction of IL-12 also involves additional surface-bound and soluble factors. TRANCE-RANK–TRANCER-RANKL interactions, crosslinking of MHC class II by TCR or CD4 all induce IL-12 (Koch et al., 1996). Two soluble T helper cell factors, IFNc and IL-4, play an even more important role. They do not induce IL-12 by themselves but provide powerful co-stimulating signals allowing efficient production of this factor. IFNc enhances IL-12 production induced by bacterial products as well as following CD40 engagement (Hayes et al., 1995; Hilkens et al., 1997; Snijders et al., 1998). The presence of IFNc during DC maturation results in a stable modulation of maturing cells and the induction of type-1polarized DCs with strongly enhanced capacity to produce IL-12 during subsequent CD40L stimulation (Vieira et al., 2000; Mailliard et al., 2002). IL-4 shows a more complicated pattern of IL-12 regulation. While IL-4 appears to be a similarly potent enhancer of CD40L-induced IL-4 production, it is a general suppressor of cytokine production (including IL-12) to such bacterial products as LPS (Kalinski et al., 2000; Hochrein et al., 2000). In addition to the abovementioned T-cell products, recently also IL-1b a common inflammatory mediator, was reported to costimulate the CD40L-induced IL-12p70 production (Wesa and Galy, 2001; Luft et al., 2002). Therefore, the effective induction of bioactive IL-12 generally requires at least two different signals. Effective combinations include: (1) CD40 ligation and a costimulatory cytokine; (2) a bacterial product and IFNc; and (3) CD40L and a bacterial product (Snijders et al., 1998; Schultz et al., 2000). Although some bacterial products and very high levels of CD40L can induce some IL-12 by themselves, each of the above combinations results in at least a 100-fold enhancement of IL-12 production. Strict regulation of IL-12 production is consistent with its central role in the induction of potentially auto-destructive TH1 responses. The production of IL-12 is also negatively regulated by powerful inhibitors. These include IL-10 (D’Andrea

387

et al., 1995), glucocorticoids, PGE2 (van Der Pouw Kraan et al., 1995), histamine (van Der Pouw Kraan et al., 1996, 1998), and other cAMP-elevating agents (Caron et al., 2001; la Sala et al., 2001), TGFb (D’Andrea et al., 1995), IFNa (Cousens et al., 1997), and such neuropeptides as calcitonin gene-related peptide (CGRP) (Liu et al., 2000a), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclaseactivating polypeptide (PACAP) (Xin and Sriram, 1998; Delgado et al., 1999). Many of these factors have been shown to suppress IL-12 production in a stable fashion (Moser et al., 1995; De Smedt et al., 1997; Kalinski et al., 1997; Vieira et al., 1998; Vanderheyde et al., 1999). IL-12 production is strongly enhanced in IL-10deficient animals, but suppressed in animals deficient in, or unresponsive to IFNc (Heinzel et al., 1996; Wu et al., 1999; Salkowski et al., 1999; Maldonado-Lopez et al., 2001) (Table 16.2).

IL-23 In order to be secreted in a biologically active form, p19 (also called IL-23a) needs to be coexpressed with IL-12p40 (Oppmann et al., 2000). Similar to the IL-12 p35–p40 heterodimer, p19 forms a disulfidelinked heterodimer with IL-12p40, as shown by coimmunoprecipitation with antibodies against either subunit and silver staining of two-dimensional SDS gels (Oppmann et al., 2000). High levels of p19 mRNA are expressed by polarized TH1 cells and stimulated macrophages (Oppmann et al., 2000). Human and mouse monocyte-derived DCs generated in the presence of GM-CSF and IL-4 secrete IL-23 (p19–p40 complex) after activation with TNFa, LPS and anti-CD40 antibody (Oppmann et al., 2000).

IL-12RA In most APC types, p40 is produced at up to 100-fold excess, when compared with p35 (Gillessen et al., 1995; Ling et al., 1995; Germann et al., 1995; Gately et al., 1996; Snijders et al., 1996, 1998). Most IL-12inducing stimuli also induce p40 homodimer. In several cases p40 is induced as a sole factor. Interestingly, the production of p40 homodimer correlates with enhanced production of other TH2-driving factors, including IL-10 and PGE2 (Rieser et al., 1997; Kalinski et al., 2001). Overproduction of this factor is noted in

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several tumor models and in chronic inflammatory states (Gately et al., 1996; Piccotti et al., 1996b; Kato et al., 1996; Chen et al., 1997; Rothe et al., 1997; Heinzel et al., 1997; Schmidt et al., 1998; Yoshimoto et al., 1998a; Nitta et al., 2001). It is identified as an early event in the course of the induction of UVinduced immunosuppression (Schmitt and Ullrich, 2000), associated with PGE2 induction in keratinocytes (Shreedhar et al., 1998). In humans, overproduction of p40 was associated with reduced NK cell function in women with endometriosis (Mazzeo et al., 1998).

IL-27 Studies performed in EBI-3-transfected cells showed that, similar to other members of the family, it is not secreted in a monomeric form. Its association with p28, and potentially at a much lower level with p35, enhances the secretion rate of both these subunits (Pflanz et al, 2002, Devergne et al., 1996, 1997). Expression of EBI-3 has been detected in spleen and tonsils, as well as in the human trophoblast (Devergne et al., 1996). It is localized to interfollicular zones of tonsil tissue, expressed by cells associated with sinusoids in perifollicular areas of spleen tissue, and, at very high levels, by placental syncytiotrophoblasts. EBI-3 can be induced in vitro by pokeweed mitogen stimulation of peripheral blood mononuclear cells. Recently, Hashimoto and colleagues have performed a detailed analysis of gene expression in mature and activated DCs and contrasted them with LPSstimulated monocytes (Hashimoto et al., 2000). The most prominent genes expressed in these activated DCs was the p35 homolog, EBI-3 as well as the IFNainducible protein p27. LPS-stimulated monocytes did not express these molecules. Human p28 has been primarily detected in placenta, monocytes and DCs derived from monocytes. Murine p28 has been detected in bone marrow and tumor macrophages, particularly activated ones, as well as at low levels in the normal spleen and thymus, consistent with the presence of macrophages there. Although p28 peaks at 3–6 hours following activation and then rapidly diminishes, with EBI-3 mRNA is sustained for 12–72 hours (Pflanz et al., 2002). The notion that EBI-3 homodimers might antagonize this proposed heterodimeric receptor is based on similar notions related to p40 homodimers.

RECEPTORS, SIGNALING AND REGULATION OF IL-12 RESPONSIVENESS IL-12 receptor PHA-stimulated T-cell blasts express 1000–10 000 IL-12 binding sites per cell with three different affinities consistent with low-, intermediate- and highaffinity IL-12 receptors displaying Kds in the range of 2–6 nM, 50–200 pM and 5–20 pM, respectively (Chizzonite et al., 1992). IL-12 receptor is a multichain complex with an approximate Mr of 135–210 kDa. Expression cloning studies (Chua et al., 1994; Presky et al., 1996a; Wu et al. 1996) demonstrated that the IL-12 receptor is composed of a dimeric complex where the b1 and b2 chains each confer low– intermediate binding affinity of the complex for IL-12 while their combination allows for high-affinity binding (Chua et al., 1995). The IL-12Rb2, but not the IL-12Rb1 chain contains tyrosine residues in its cytoplasmic domain and determines the ability of the receptor to mediate IL-12 signaling (Chua et al., 1995; Presky et al., 1996a, 1996b, 1998). Co-transfection of the IL-12Rb1 and IL-12Rb2 chains into Ba/F3 cells yields high-affinity IL-12 receptor sites, allowing IL-12 to promote cell proliferation with an ED50  1 pM of IL-12p70. In mice, the IL-12Rb1 chain plays the dominant role in binding the IL-12 ligand. In human cells expressing the high-affinity IL-12R, both the b1 and b2 chains participate in IL-12 binding (Presky et al., 1996a, 1996b, 1998). The b1 chain binds the IL-12p40 chain while the IL-12Rb2 chain associates with the IL-12p35 chain (Gillessen et al., 1995; Presky et al., 1996a). This explains the capacity of the IL-12p40 homodimer to antagonize effectively the binding of the IL-12 heterodimer, largely due to its capacity to bind to the IL-12b1-binding site of the multimeric IL-12 receptor complex. Of note, antibodies directed against the IL-12Rb chains efficiently inhibit IL-12-induced IFNc production and activated T-cell proliferation, but have no effect on the ability of other T-cell growth factors (i.e. IL-2, -4, or -7) to promote T-cell expansion (Wu et al., 1996).

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Cell types and tissues expressing IL-12 receptor The presence of IL-12R is detected on activated T cells and NK cells (Desai et al., 1992). DCs express a single class of high-affinity IL-12R (Grohmann et al., 1998). In addition, the IL-12Rb1 subunit, which is necessary but not sufficient for expression of functional IL-12R (Wu et al. 1996), is detected on human B-cell lines and activated human peripheral blood or tonsillar B lymphoblasts (Benjamin et al., 1996; Wu et al., 1996). Mouse-activated B cells efficiently bind mIL-12 (Vogel et al., 1996, Wu et al., 1996). Both the IL-12Rb1 and b2 chains are inducible on naive (CD45RA) and memory CD4 T-cell clones within 48 hours of activation with antigen or antiCD3 treatment (Rogge et al., 1997). IL-7 augments the expression level of the IL-12Rb1 chain (Mehrotra et al., 1995). In the same study, TH2 cells could be induced to express low levels of IL-12Rb2 chains transiently and to secrete low levels of IFNc. The transience of this response may be due to IL-4 production by TH2 clones that promotes extinction of IL-12R expression and thereby IL-12 responsiveness (Rogge et al., 1997). The expression of the high affinity IL-12R by activated T cells is reciprocally regulated by IL-4 and IFNc (Szabo et al., 1997). IL-4 inhibits IL-12Rb2 expression and, concomitantly, IL-12-mediated signaling. IFNc promoted the sustained expression of the IL-12Rb2 on developing TH2 cells, but did not promote their IFNc production upon IL-12 stimulation (Szabo et al., 1997). Murine TH2 cells become resistant to repolarization at about day 9 of their differentiation (Murphy et al., 1996). In humans, only a limited percentage of the most extremely polarized TH2 clones lacks a functional IL-12 receptor, whereas most human TH2 clones can regain IL-12 responsiveness (Hilkens et al., 1996a; Smits et al., 2001). Clearly, the factors that promote the expression or maintenance of IL-12Rb2 do promote TH1 responses (Szabo et al., 1997; O’Garra and Arai, 2000).

Signaling pathways IL-12 signaling involves JAK kinases STAT1, 3, and 4 (Trinchieri, 1998), NFjB and c-Jun (Zhang et al., 1999). Crosslinking of IL-12 receptor results in the

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activation of the receptor-associated tyrosine kinases JAK2 and TYK2 (Bacon et al., 1995a), as well as activation of MAP kinase and the src family lck tyrosine kinase (Pignata et al., 1994, 1995). TYK2 interacts with the b1 subunit of the IL-12 receptor and JAK2 with the b2 subunit (Zou et al., 1997). JAK kinases phosphorylate the IL-12 receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for transcription factors termed signal transducers and activators of transcription (STAT). IL-12 binding to its receptor results in activation of STAT1, 3 and 4. STAT1 can dimerize with either STAT3 or STAT4 and STAT4 can dimerize with STAT3 (Jacobson et al., 1995; Yu et al., 1996). Tyrosine phosphorylation of STAT proteins induces their dimerization and subsequent translocation to the nucleus where they bind to related DNA sequences and regulate transcription (Darnell et al., 1994). STAT4, but neither STAT1 nor STAT3, is activated directly through the IL-12 receptor (Naeger et al., 1999). In humans, both IL-12 and IFNa induce tyrosine phosphorylation and DNA binding of STAT4 (Bacon et al., 1995b; Cho et al., 1996) whereas only IL-12 apparently mediates this activity in the mouse. IL-12R activates STAT4 in a direct fashion while IFNa binds indirectly via STAT-2 (Farrar et al., 2000). The crucial role of STAT-4 signaling in IL-12 responsiveness is confirmed by observations that the STAT4 knockout mice are defective in IL-12 responses and reveal profound defects in TH1 immunity (Thierfelder et al., 1996; Kaplan et al., 1996). TH2 immune responses (and IL-4, IL-5 and IL-10 production) predominate (Kaplan et al., 1996). While TH1-associated immune responses do occur, they are generally far weaker than those observed in heterozygous littermates or in wild-type control animals in response to IL-12 or to pathogenic organisms including L. monocytogenes (Kaplan et al., 1996). IL-12 activation of NK cells does not occur in STAT4 knockout mice. IL-12-induced IFNc production is diminished and long-term antigen-specific cellular immunity is impaired in these knockout animals.

Immune defects related to the absence of functional IL-12 receptor Splenocytes of IL-12Rb1 knockout (IL-12Rb1/) mice are deficient in all IL-12-induced biologic activities

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including proliferation, IFNc secretion, TH1 development and NK cell lytic activity, while their TH2 cell responses are moderately enhanced (Wu et al., 1997b). The observed deficit in TH1 and NK cell function is reminiscent of STAT4 knockout mice (Thierfelder et al., 1996; Kaplan et al., 1996). Several identified individuals with a mutated IL-12Rb1 gene display diminished IFNc production, and recurrent mycobacterial and Salmonella infections (de Jong et al., 1998; Altare et al., 1998). Lack of the IL-12Rb chain expression results in immunodeficiency in both humans and mice, demonstrating the essential role of IL-12 in resistance to infections with intracellular bacteria. Although IL-12 is involved in antiviral responses, lack of IL-12 signaling still allows efficient responses to many viruses (Schijns et al., 1998; Altare et al., 1998; de Jong et al., 1998), indicating the existence of effective compensatory mechanisms.

Regulation of IL-12 receptor expression IL-12 biologic effects are regulated not only by modulation of its production, but also by modulation of responsiveness. The potent TH2-driving activity of IL-4 is at least in part mediated by the ability of this factor to down-regulate IL-12Rb2 chain expression and signaling (Szabo et al., 1997; Wu et al., 1997a; Himmelrich et al., 1998). Similarly, inhibition of b2 expression and IL-12 signaling is a key feature of TGFb and IL-10 biologic effects (Wu et al., 1997a; Gorham et al., 1998; Heath et al., 2000). Prostaglandin E2 (PGE2) and dexamethasone inhibit IL-12Rb1 expression and mRNA encoding IL-12Rb2 (Wu et al., 1998). Another cAMP-elevating factor, cholera toxin inhibits the expression of both b1 and b2 chains (Braun et al., 1999). In contrast, factors such as IFNc maintain the ability to signal through IL-12Rb2 both in vitro (Szabo et al., 1997) and in vivo (Himmelrich et al., 1998), at least in the mouse. Reciprocal action of IFNc and IL-4 upon IL-12 signaling is probably best defined during T helper cell differentiation. In this case, differential regulation of IL-12 receptor expression by these factors is involved in the development of TH1 and TH2 cells and their stability (Szabo et al., 1995, 1996; Gollob et al., 1997; Murphy et al., 1999). In addition to IFNc also such cytokines as IL-2, IL-7 and IL-15

induce IL-12Rb1 expression (Wu et al., 1997a). The natural killer T (NKT)-cell ligand a-galactosylceramide (a-GalCer) induces mRNA for both IL-12Rb1 and IL-12Rb2 (Kitamura et al., 1999). Up-regulation of the receptor correlates with the ability of the cells to proliferate in response to IL-12 (Chizzonite et al., 1992; Desai et al., 1992).

Receptor interactions of other members of IL-12 family IL-23 Binding studies in cell lines transfected with either IL-12Rb1 or b2 chain or both chains revealed that a p19–p40 fusion protein binds to IL-12Rb1 but not to IL-12Rb2. Since p19p40 does not induce proliferation in cell lines co-transfected with both IL-12Rb chains and biologic effects in responsive cells can be blocked by IL-12Rb1 antibodies, it was concluded that IL-12Rb1 but not IL-12Rb2 is a component of the functional IL-23R complex (Oppmann et al., 2000). IL-23 binds to IL-12Rb1 (but not IL-12Rb2) along with another recently identified IL2Rb homolog (R Kastelein, personal communication). Similar to IL-12, the p19–p40 fusion protein induces STAT4 signaling in PHA-activated T cells (Oppmann et al., 2000).

IL-12RA The p40 homodimer binds to the IL-12R, but fails to mediate a signal, thereby serving as a functional antagonist of the IL-12 receptor (Mattner et al., 1993, 1997a; Ling et al., 1995; Germann et al., 1995).

IL-27 The orphan receptor WSX-1/TCCR was identified as one of the chains of the functional IL-27 receptor following a search with transfectants (Pflanz et al., 2002). Interestingly, the initiation of TH1 responses but not their continuation has been shown to require this receptor to be expressed on the same chromosome as the IL-12Rb1 chain (chromosome 19) (Yoshida et al., 2001). This receptor is expressed in adult lymphoid tissues such as peripheral blood lymphocytes, thymus

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and spleen with the highest expression on CD4 T cells and NK cells. Down-regulation seems to occur with memory-type differentiation from naive T helper cells (Chen et al., 2000). The notion that another chain is responsible for signaling through this receptor is suggested by WSX-1 transfectant studies which proliferate in response to IL-3 but not to IL-27.

BIOLOGIC ACTIVITIES OF IL-12 FAMILY MEMBERS IL-12 The early descriptive names of IL-12, cytotoxic lymphocyte maturation factor (CLMF) (Gately et al., 1986; Wong et al., 1988), T cell-stimulating factor (TSF) (Germann et al., 1987, 1988; Stern et al., 1990), and natural killer cell stimulatory factor (NKSF) (Kobayashi et al., 1989), focused on its ability to support the effector mechanisms of cell-mediated (type-1) immunity. Later studies added a much wider spectrum of activities of the IL-12 heterodimer, as a key factor in the induction of TH1-type responses of CD4 T cells, as well as a factor promoting the functions of B cells, APCs, vascular and stromal cells.

Central role of IL-12 in determining the TH1/TH2 response pattern IL-12 is the primary and by far the best studied TH1 inducing factor (O’Garra and Arai, 2000). IL-12 production is demonstrably responsible for TH1-cell development in numerous models in the mouse (Hsieh et al., 1993; Macatonia et al., 1993, 1995; Murphy et al., 1994; Openshaw et al., 1995) and in humans (Hilkens et al., 1996b; Heufler et al., 1996, 1997; Ohshima and Delespesse, 1997). Lack of IL-12 producing capacity makes it impossible for the animals to develop protective TH1 responses to such pathogens as Leishmania, Listeria and Mycobacteria, resulting in inefficient TH2-dominated responses and in succumbing to infection (Mattner et al., 1996, 1997b; Stobie et al., 2000; Wakeham et al., 1998). Similarly, IL-12-deficient animals demonstrate impaired TH1 reponses to individual antigens and adjuvants

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(Magram et al., 1996; Galbiati et al., 2000; Liu et al., 2000b). As mentioned above, a similar susceptibility to intracellular infections results from the absence of STAT4, the key signaling element dictating IL-12 responsiveness (Jacobson et al., 1995). Individuals lacking the functional IL-12 receptor demonstrate a similar deficit in TH1 responses to intracellular pathogens in humans (Altare et al., 1998; de Jong et al., 1998). Importantly, IL-12 is a critical mediator not only for development of pathogen-eliminating TH1 responses, but is also essential in sustaining TH1 responses (Yap et al., 2000). However, TH1 immunity to some pathogens can also effectively develop in the absence of the p40-containing cytokines, IL-12 and IL-23. Indeed, IL-27 may play this critical role (Jankovic et al., 2002). Although the response to some viruses is relatively intact in the absence of IL-12 (Schijns et al., 1998), IL-12 was demonstrated to be important for virus eradication in several other models (Orange and Biron, 1996a, 1996b; Monteiro e t a l ., 1998; Williams et al., 1998; Arulanandam et al., 1999; Delleher et al., 1999a; Marshall et al., 1999; Werle et al., 1999; Malmgaard et al., 2000). Numerous viruses target IL-12 production as a means of escape from immunity (Chougnet et al., 1996; Karp et al., 1996; FugierVivier et al., 1997; Taoufik et al., 1997; Levy et al., 1998; Karp, 1999; Kelleher et al., 1999b; Inagaki-Ohara et al., 2000; Servet-Delprat et al., 2000). Two cytokines from outside the IL-12 family, IFNa (only in humans, not in mouse) and IL-18 play a similar role in the optimal development of TH1 responses. However, several lines of evidence suggest that IL-12 plays the dominant role. The p40 knockout phenotype and the IL-12Rb2 phenotype demonstrate the strongest TH1 deficiency, suggesting a key role for both IL-12 and IL-23. IL-18 promotes TH1 responses largely in synergy with IL-12, enhancing several effector mechanisms with other cytokines such as IL-2 and IL-4 as well (Robinson et al., 1997; Yoshimoto et al., 1998b; Walker et al., 1999). The ability of IFNa to activate STAT4 and exert an IL-12-like effect is present in humans but not in mouse (Rogge et al., 1998) suggesting a phylogenetically later development of this pathway. Although antiviral responses can occur in the absence of IL-12 (Schijns et al., 1998; Altare et al., 1998; de Jong et al., 1998; Oxenius et al., 1999; Feng

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et al., 1999), this is probably due to effective compensatory mechanisms.

Role of IL-12 in the induction of TR1-type regulatory cytokines/cells The effect of IL-12 upon T helper-cell responses are apparently context dependent. While exogenous administration of IL-12 suppresses UV-induced enhancement of IL-10 production (Schmitt et al., 2000), IL-12 enhances the production of IL-10 by B cells (Skok et al., 1999). High levels of IL-10 are also produced by T helper cells following continuous culture in IL-12 and restimulation in the presence of IL-4 (Assenmacher et al., 1998). The significance of these seemingly paradoxical effects of IL-12 is not yet clear. They could represent negative feedback mechanism, reducing the likelihood of IL-12-mediated damage during chronic inflammation. In support of such a possibility, repetitive administration of recombinant IL-12 leads to progressively reduced IFNc response, enhanced IL-10 response, and reduced acute IL-12 toxicity, both in a mouse model (Coughlin et al., 1997; Leonard et al., 1997) and in cancer patients receiving experimental therapies with IL-12 infusions (Rakhit et al., 1999).

IL-12 in the effector phase of cellular immunity IL-12 was originally identified as a factor that synergized with IL-2 in the presence of hydrocortisone to generate lymphokine-activated killing (LAK) activity from human PBMC (Gately et al., 1986). IL-12 added alone provided only small levels of LAK induction when compared with IL-2 alone or combinations of IL-2  IL-12 (Gately et al., 1991; Zeh et al., 1993). Optimal IL-12 bioactivity was attained in the 10–50 pM range (Gately et al., 1991). Based on antibody neutralization studies, LAK induction by IL-12 could be effectively inhibited by up to 70% with either anti-TNFa or anti-IFNc, and was prevented by the combination of these antibodies (Gately et al., 1991). IL-12 in conjunction with B7–1 and IL-6 stimulation induce the generation of antigen-specific murine antitumor cytolytic T cells in vitro (Gajewski et al., 1995). IL-12 can also resurrect NK/LAK activity in the PBMC of HIV-infected individuals (Chehimi et al., 1992). Overall, the available data appear to support the

ability of IL-12 to enhance cytolytic effector function, rather than the number or frequency of effector cells generated in in vitro cultures (Bhardwaj et al., 1996). IL-12 enhances the proliferation of NK or T cells, but in most cases has been found to be inferior at comparable molar doses to other known T-cell growth factors including IL-2 and IL-7 (Naume et al., 1992). The bioactivity of IL-12 can be assessed in proliferation assays of PHA- or anti-CD3-stimulated T cells (CD4 and/or CD8) in the dynamic range of 1–100 pM IL-12 (Chizzonite et al., 1992, Desai et al., 1992, Zeh et al., 1993). Gately et al. (1986) first demonstrated that IL-12 enhanced the lytic potential of cytolytic effector cells in primary cultures of high-density peripheral blood lymphocytes stimulated with irradiated allogeneic HT144 melanoma cells. CTL effector cells principally targeting allospecificities developed at IL-12 doses 10-fold lower that those observed for IL-2. In a study of Bhardwaj et al. (1996), IL-12 enhanced antigen-specific CTL reactivity without a notable increase in the frequency of viral-specific pCTL. Thus, maturation rather than proliferation is the major impact of IL-12 on CTL generation, promoting the cytolytic apparatus of the CTL (i.e. up-regulation of perforin, serine proteases, and cytolytic granules (Chouaib et al., 1994; Mehrotra et al., 1993). IL-12 increases the anti-apoptotic transcription factor bcl-2, preventing the apoptotic death of cultured responder cells. This may be the case not only for T and NK cells but also APCs such as DCs (Williams et al., 1998; Pirtskhalaishvili et al., 2000). IL-12 protects human IL-2-dependent TH1 T-cell clones from apoptosis resulting from TCR ligation or IL-2 deprivation (Radrizzani et al., 1995). Similar activity of IL-12 was demonstrated in case the of CD4 (but not CD8) T cells derived from HIV patients and treated with anti-TCR or anti-Fas (CD95) antibodies (Estaquier et al., 1995) .

Role of IL-12 in B-cell responses The initial observations that IL-12 induced suppression of IgE synthesis by IL-4-stimulated B cells (Kiniwa et al., 1992; King et al., 1995) suggested a negative regulatory role of IL-12 on B-cell responses, at least of their TH2-dependent component. However, IL-12 was later demonstrated to enhance B-cell survival, to stimulate CD5 (B1a) expression on B cells

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(Jones, 1996), and was shown to be an important factor in the ability of DCs to promote the differentiation of naive (but not memory) B cells (Dubois et al., 1998). IL-12 promotes B-cell production of IL-10, initiating a B cell-dependent shift from TH1 to TH2 responses (Skok et al., 1999) which may further help B-cell survival and function. These observations may help to explain the role of the TH2 cell-induced IL-12 production (Kalinski et al., 2000; Hochrein et al., 2000). In accordance with this possibility of direct support for B cell Ig production, in the absence of IFNc, IL-12 enhances rather than diminishes the production of IgE (Wynn et al., 1995).

Impact on APC function In addition to acting as a source of IL-12, DCs and macrophages are also recipients of IL-12 signals. DCs display a unique STAT-independent pattern of IL-12 responsiveness mediated by the b1 chain of the IL-12R (Grohmann et al., 1998, 2001). IL-12 promotes nuclear localization of NFjB and may prime murine DCs for IL-12 production. Autocrine IL-12 production promotes activation of mouse DCs following CD40 triggering (Bianchi et al., 1999). Exogenous IL-12 synergizes with Flt-3 ligand in the maturation of Langerhans cells (Esche et al., 1999). Responsiveness to IL-12 is also observed in macrophages since IL-12p40 acts as a macrophage chemoattractant (Ha et al., 1999).

Role in hematopoiesis IL-12 is a direct activator of early hematopoietic progenitor cells (Jacobsen et al., 1993). It can also indirectly suppress hematopoietic colony formation by inducing IFNc and TNFa production in contaminating cells (Bellone and Trinchieri, 1994). IL-12 synergizes with other hematopoietic growth factors, including SCF, Flt-3 ligand, IL-3, IL-4, G-CSF, M-CSF and erythropoietin in promoting proliferation and differentiation of murine bone marrow progenitors (Jacobsen et al., 1993, 1995; Ploemacher et al., 1993a,b; Dybedal et al., 1995). Similar results were reported in human culture systems (Bellone and Trinchieri, 1994; Bertolini et al., 1995; FardounJoalland et al., 1995; Hirao et al., 1995). Mice receiving IL-12 injections over prolonged periods show IFNc-

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dependent suppression of bone marrow hematopoiesis, anemia and neutropenia, with an associated extramedullary hematopoiesis in the spleen (Jackson et al., 1995; Tare et al., 1995). In the absence of IFNc signaling, IL-12 only promotes (and does not suppress) hematopoiesis in bone marrow and spleen (Eng et al., 1995). IL-12-treated animals show enhanced myeloid dendropoiesis within lymphoid and non-lymphoid organs (Esche et al., 2000).

Angiogenesis IL-12 acts as an indirect inhibitor of angiogenesis in mice (Voest et al., 1995). This effect is mediated by IFNc induction of MIG and IP-10 (Sgadari et al., 1996) and may be mediated by NK-cell cytotoxicity of endothelial cells (Yao et al., 1999). IL-18 shows a synergistic effect with IL-12 in this respect (Coughlin et al., 1998).

Biologic functions of IL-23 IL-23 exerts an IL-12-like, IFNc-promoting effect, but in contrast to IL-12, it strongly promotes the proliferation of activated mouse memory T cells (CD4 CD45RBlow) but not of naive T cells (CD4CD45RBhigh) (Oppmann et al., 2000). With respect to human T cells, IL-23 has a more pronounced effect on activated CD45RO memory T cells than on activated CD45RA naive T cells. The effects could be antagonized by antiIL-12p40/70 but not by anti-IL-12p35 antibodies. Further evidence for the biologic importance of IL-23 comes from experiments with IL-23(p19) transgenic mice (Wiekowski et al., 2001). Ubiquitous expression of the transgene resulted in systemic inflammation in multiple organs, runting, infertility and premature death within 3 months. The inflammatory infiltrates consisted of lymphocytes, macrophages and to a lesser extent, neutrophils. Hypochromic, microcytic anemia, increased neutrophil counts in the peripheral blood and extramedullary hemopoiesis characterized IL-23 transgenic mice. In accordance with the histologic evidence of multiorgan inflammation, IL-23 transgenic animals had elevated serum levels of IL-1, TNFa, and IFNc and increased production of acute phase proteins in the liver. The levels of insulin-like growth factor 1, a molecule that promotes growth and influences

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fertility, were strongly decreased in transgenic animals. Importantly, tissue-specific expression of IL-23 in the liver resulted in a normal phenotype, whereas transfer of IL-23 transgenic bone marrow into lethally irradiated animals was sufficient to induce the phenotype described after ubiquitous expression of the transgene. These experiments suggest that IL-23 expression in hematopoietic cells is a prerequisite for its bioactivity (Wiekowski et al., 2001).

Biologic functions of IL-12RA (p40 homodimer) The IL-12 p40 homodimer binds to the IL-12R, but fails to mediate a signal, thereby serving as a functional antagonist to IL-12 p70 heterodimers (Mattner et al., 1993; Germann et al., 1995; Ling et al., 1995). IL-12 p40 homodimers inhibit IL-12-induced murine Con A-blast proliferation, splenocyte secretion of IFNc and NK activation (Gillessen et al., 1995). mIL-12 p40 homodimers block mIL-12 from binding the high-affinity IL-12 receptor, acting as a competitive IL-12 antagonist. This is also described with the porcine p40 homodimer (Foss et al., 1999). Although the human p40 homodimer also mediates an IL-12 antagonistic function in vitro, its inhibitory function is less pronounced and its physiological role is less clear (Ling et al., 1995). Part of the problem with analyzing the role of human p40–p40 is its very low stability. In aqueous solutions it rapidly dissociates into p40 monomers with lower ability to bind IL-12R (Gately et al., 1998). Both in mouse and in human, the p40 homodimer and also to a lesser extent the product of its dissociation, free p40 monomer, can suppress the responsiveness to IL-12 by competitively inhibiting IL-12 receptor binding (Gillessen et al., 1995; Germann et al., 1995; Gately et al., 1996; Presky et al., 1996).This activity is particularly pronounced in the mouse where the p40 homodimer exerts an antagonistic activity with an IC50 of 1–10 ng/ml. In humans at least 10-fold higher concentrations of p40 are required. In vivo, mouse p40 homodimer antagonizes IL-12 activity in animals with lethal LPS-induced shock (Mattner et al., 1997a) and suppresses the TH1dominated inflammatory responses in several models of chronic inflammation, autoimmunity, transplantation and cancer (Kato et al., 1996; Chen et al., 1997;

Heinzel et al., 1997; Rothe et al., 1997; Schmidt et al., 1998; Yoshimoto et al., 1998a). High levels of IL-12p40 in peritoneal fluid from patients with endometriosis may play an IL-12 antagonistic role, locally inhibiting the activity of NK cells (Mazzeo et al., 1998). Increased IL-12p40 production (monomer and heterodimer) by bronchial epithelium is observed in such TH2-driven conditions as bronchial asthma. The levels of p40 monomer and homodimer (as opposed to p70) detected in the circulation correlates positively with age (Rea et al., 2000). Interestingly, the production of p40 homodimer correlates with enhanced production of other TH2-driving factors, including IL-10 and PGE2, following UV irradiation of the skin (Schmitt and Ullrich, 2000). Production of p40 is strongly enhanced by PGE2 (Rieser et al., 1997; Kalinski et al., 2001), a mediator of UV-induced TH2 bias (Schmitt and Ullrich, 2000).

Biologic functions of IL-27 (EBI-3–p28) The function of IL-27 is not yet fully clarified. The recent publication from DNAX shows that EBI-3 pairs with p28 and interacts with the orphan novel receptor WSX-1/TCCR (Pflanz et al., 2002). The data suggest that IL-27 plays a major role in promoting the induction of TH1 immunity by enhancing proliferation, and possibly survival, of activated naive CD4 T cells, playing its most critical role early in T-cell biology at the level of the lymph node and proximal to that of IL-12 and IL-23. In two independent studies, high levels of expression of EBI-3 have been reported in virtually all patients with active ulcerative colitis (UC) but only in a small percentage of patients with Crohns disease (Christ et al., 1998; Omata et al., 2001). Oxazolone-induced TH2-mediated inflammatory bowel disease model in the mouse (RS Blumberg, personal communication) is associated with high levels of EBI-3. Interestingly, EBI-3 is produced at high levels in the human placenta, an environment associated with a selective suppression of TH1-type immunity and a TH2 bias. One possibility is that IL-12 antagonism is mediated by competitively inhibiting the IL-27 receptor (similar to the p40 homodimer). In line with this last option may be the observation that EBI-3 knockout mice are protected from TH2associated colitis but not TH1 disease (RS Blumberg, personal communication), suggesting an active

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role of EBI-3 in sustaining or inducing TH2-type autoimmunity.

IL-12 IN PATHOGENESIS AND THERAPY: EXPERIENCES FROM ANIMAL MODELS IL-12 therapy in cancer IL-12 is an important mediator of effective antitumor responses in numerous murine models. Systemic IL-12 administration markedly prolongs animal survival, inhibits tumor growth and metastasis formation in murine B16 melanoma, M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma (Brunda et al., 1993). This effect is mostly independent of NK cells but involves CD4 and/or CD8 T cells and IFNc as the downstream mediator of IL-12 (Brunda et al., 1993; Nastala et al., 1994). Gajewski et al. (1996) noted that endogenous IL-12 is responsible for a consistent 30% spontaneous regression rate in the P815 mastocytoma model in DBA/2 mice, which could be eliminated by treatment with an anti-IL-12 mAb. Daily i.p. administration of rIL-12 is therapeutic in a variety of transplanted syngeneic tumors. In both metastatic and subcutaneous delivery models, IL-12 has proven to be therapeutically effective, even if treatment is delayed for up to 3 weeks after tumor injection (Nastala et al.,1994; Brunda et al., 1995). Indeed, early administration of IL-12 is sometimes less effective, suggesting that T cells need to be selected before IL-12 can mediate its biologic effects. Systemic IL-12 administration is required for successful vaccination with a mutant p53 in Balb/c mice bearing Meth A sarcoma (Noguchi et al., 1995). The antitumor effect of IL-12 administration depends on the in situ production of IFNc, TNFa, and is mediated both by CD4 and CD8 T cells. Prolonged treatment of 3-methylocholantrene-injected mice with IL-12 resulted in delayed tumor appearance and reduced tumor incidence, associated with enhanced in vivo production of IFNc, TNFa, IL-10 and serum nitrates (Noguchi et al., 1996). The efficacy of IL-12 has also been evaluated using targeted delivery of IL-12 to the tumor microenvironment by gene therapy. Perilesional injection of NIH3T3 fibroblasts transfected with both the mIL-

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12p35 and mIL-12p40 chains reduced tumor area by 80% on day 32 in B6 mice bearing MCA 105 sarcomas, although it was not fully curative (Pappo et al., 1995). Systemic administration of IL-2 in conjunction with perilesional injection of 3T3-IL-12-transfected fibroblasts resulted in complete tumor regression in all treated animals. Using a polycistronic retroviral vector encoding the IL-12 p35 and IL-12 p40 subunits, as well as the neomycin phosphotransferase gene (neor ), Tahara et al. (1995) engineered tumor cells (MCA 207 fibrosarcoma, MCA 102 sarcoma) to stably secrete nanogram quantities of IL-12 in vitro. These IL-12 producing tumors, but not control-transfected tumors, failed to grow when injected intradermally into B6 mice. Non-adaptive and systemic tumorspecific immunity was induced, protecting animals against contralateral challenge with control tumors, with protection dependent upon the production of IFNc. In poorly immunogenic models (MCA 207 fibrosarcoma in B6 mice and TS/A mammary adenocarcinoma in Balb/c mice), IL-12 displays a synergistic antitumor effect with B7.1 gene delivery (Zitvogel et al., 1996). Co-inoculation of mice with tumors infected with retroviruses encoding IL-12 and B7.1 resulted in disease-free animals with long-term tumor-specific immunity in 80% of cases while solitary IL-12 or B7.1 transfectants progressed in 80% of animals. Therapeutic efficacy was dependent on the dose of IL-12 delivered by transfected tumor cells and was ablated by in situ administration of CTLA4–Ig or neutralizing antibodies against mIFNc or mTNFa.

IL-12 therapy in infectious diseases As a single agent, in combination with chemotherapeutic factors or as a vaccine adjuvant, IL-12 has demonstrated therapeutic potential in various murine models of infection (Gately and Mulqueen, 1996). IL-12 promotes a protective IFNc-mediated TH1 response, as well as the antibody-mediated protection, in Leishmania major-infected mice, reducing the parasite burden and IL-4 production in the regional lymph nodes (Heinzel et al., 1993; Sypek et al., 1993; Su and Stevenson, 2002). IL-12 therapy of susceptible A/J mice infected with blood-stage Plasmodium chabaudi AS decreased parasitemia at 0.1 lg levels and enhanced survival (Stevenson et al., 1995). IL-12 treatment enhances resistance of mice to

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Mycobacterium tuberculosis infection and promotes the induction of IFNc-producing CD4 T cells (Cooper et al., 1995). In addition, IL-12 as an adjuvant during vaccination promotes responses to microbial antigens which are not sufficiently immunogenic when administered alone (Afonso et al., 1994). Subsequent studies demonstrated the therapeutic efficacy of IL-12 in a variety of murine models of infectious diseases involving protozoans, mycobacteria and fungi (Gately and Mulqueen, 1996). IL-12 also mediates resistance to viral infections including lymphocytic choriomeningitis (Orange et al., 1994, 1995a), HIV (Gazinelli et al., 1994), cytomegalovirus infection (Orange et al., 1995b), vesicular stomatitis (Bi et al., 1995), encephalomyocarditis (Ozmen et al., 1995) and influenza (Monteiro et al., 1998).

liver allograft rejection reduce IL-12 concentration in serum and in grafts (Redaelli et al., 2002). Tacrolimus and intraportal donor-specific bone marrow infusion down-regulate intragraft IL-12 and IFNc gene transcripts in long-surviving rat intestinal allografts (Nakao et al., 2001). In allogeneic cornea transplantion in rats, an early (days 3–9) elevation in IL-12p40 transcripts (not different from that observed with syngeneic grafts) has been observed (King et al., 2000). APCs isolated from cervical lymph nodes of mice that accepted their corneal allografts produced significantly lower levels of IL-12 (Liu et al., 2001).

IL-12 in transplantation

Acute graft-versus-host disease (GVHD) is a major complication of bone marrow (BM) transplantation (BMT) and is characterized by hematopoietic dysfunction, immunosuppression and histopathological changes in the skin, intestinal mucosa and liver. The role of IL-12 in acute GVHD has been controversial. A single injection of as little as 50 U of murine IL-12 on the day of BMT inhibits GVHD while preserving graftversus-leukemia (GVL) effects of allogeneic CD8 T cells in lethally irradiated mice given fully MHCmismatched BM and spleen cells (Sykes et al., 1999). These protective effects are mediated by donorderived IFNc (Yang et al., 1998), the early secretion of which is induced by IL-12 administration. Moreover, the ability of IL-12 to protect against GVHD appears to be dependent on expression of Fas by donor T cells and on their Fas-dependent apoptosis (Dey et al., 1998). The timing of IL-12 administration and the nature of the conditioning regimen are critical for the induction of protection or toxicity in heavily irradiated allogeneic recipients. Thus, IL-12 given 1 hour before BMT was maximally protective, whereas delaying IL-12 administration until 36 hours after BMT obviated its protective effect. Conversely, neutralization of endogenous IL-12 ameliorates disease in a non-irradiated parent into F1 model of acute GVHD (Williamson et al., 1997). Others have used IL-12deficient mice to examine the contribution of donor and recipient sources of IL-12 in a fully MHCmismatched mouse model of acute lethal GVHD.

Experimental organ and tissue transplantation Treatment of wild-type murine cardiac allograft recipients with IL-12 results in high concentrations of serum IFNc and a 10-fold increase in IFNc production by recipient splenocytes after restimulation in vitro. However, this striking TH1 response failed to accelerate graft rejection (Piccotti et al., 1999). IL-12p70 treatment of fully allogeneic rat neonatal heart graft recipients significantly delayed graft rejection, an effect ascribed to induction of nitric oxide in donor hearts and draining lymph nodes (Verma et al., 2001). IL-12 antagonism on the other hand, exacerbates murine cardiac allograft rejection (Piccotti et al., 1996), but promotes liver allograft acceptance in recipients of transplants from Flt3L-treated donors (Li et al., 2001) and enhances allogeneic myoblast survival (Kato et al., 1996). High IL-12p40 mRNA expression has been reported in heart allografts of tolerant rats conditioned by donor-specific blood transfusion (DST) (Cuturi et al., 1997). By contrast, reduced IL-12 and IFNc production was observed with prolonged rat hepatic allograft survival after DST (Yamaguchi et al., 2000). Protection of rat lung transplants from ischemia reperfusion injury by prostaglandin E1 is associated with lower levels of IL-12 in lung tissue (De Perrot et al., 2001), whereas vitamin D3 analogues that inhibit rat renal or

Bone marrow transplantation/graft-versus-host disease

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THERAPEUTIC EFFICACY OF HUMAN IL - 12 IN CLINICAL TRIALS

Welniak et al. (2001) reported that IL-12 from both donor and recipient sources contributed to the exacerbation of acute GVHD although it was not required for its induction. In humans, IL-12 may play an important role in the development of acute GVHD after allogeneic stem cell grafting or donor leukocyte transfusion (Yabe et al., 1999). Thus, peripheral macrophage IL-12 production after LPS stimulation increased in patients who developed acute GVHD. In HLA haplotype-mismatched hematopoietic transplant recipients administered G-CSF, long-lasting type-2 immune reactivity and TH2-inducing DCs (not producing IL-12) were observed. Elimination of postgrafting G-CSF resulted in the appearance of IL-12producing DCs and accelerated immune recovery (Volpi et al., 2001). Cord blood transplantation has been associated with a low incidence of GVHD. Although cord blood contains similar numbers of monocytes that produce IL-12, when compared with adult PBMC cord blood CD3 and NK cells do not express the IL-12R (Han and Hodge, 1999). Syngeneic (S) GVHD dependent in part on IL-12, IFNc and TNFa develops following syngeneic BM transplantation and treatment with cyclosporine. Neutralization of IL-12, for example, abrogates development of the disease in mice (Flanagan et al., 2001), suggesting that activated APCs (macrophages and DCs) participate in the development of SGVHD.

IL-12 in human organ transplantion IL-12p40 mRNA was expressed in all human kidney allograft biopsies showing acute cellular rejection, as well as in all biopsies with evidence of focal interstitial necrosis. No IL-12 transcripts were detected in instances where normal graft histology was observed (De Mattos et al., 1997). IL-12 levels are elevated in human ischemic lung (De Perrot et al., 2002). Impaired APC function, including deficient IL-12 synthesis upon CD40 ligation contributes to T-cell hyporesponsiveness in stable human lung transplant recipients (Knoop et al., 2000).

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THERAPEUTIC EFFICACY OF HUMAN IL-12 IN CLINICAL TRIALS The therapeutic efficacy of IL-12 has been evaluated in multiple clinical trials in cancer (melanoma, renal cell carcinoma, cutaneous T-cell lymphoma, Kaposi’s sarcoma, AIDS, viral hepatitis, and asthma.

Clinical trials in cancer A phase I trial with escalating doses of rhIL-12 (3–1000 ng kg1 per day; i.v.) in 40 patients with advanced malignancy revealed one partial response (renal cell cancer) and one transient complete response (melanoma) (Atkins et al., 1997). A single test dose was followed 14 days later by cycles of five consecutive daily injections administered every 3 weeks. The 500 ng kg1 was determined to be the maximum tolerated dose (MTD), although one study death due to Clostridium perfringens sepsis was noted in this group. IL-12 therapy reversed the cancer-mediated defects in NK- and T-cell functions (Robertson et al., 1999). Based on the above results, a phase II trial of intravenous rhIL-12 in 17 patients with advanced renal cell carcinoma was initiated (Leonard et al., 1997). Patients were scheduled to receive 500 ng kg1 daily for 5 consecutive days every 3 weeks. Treatment resulted in severe toxicities with some patients unable to tolerate more than two successive doses. The study was halted following the death of two patients. This was unexpected given the relatively good tolerance observed in the earlier cohort of patients receiving IL-12 at twice higher doses and was attributable ultimately to IL-12 tachyphylaxis related to predosing with a single injection of IL-12 in patients in the phase I dose-escalation protocol. Subsequent experiments in mice revealed protection from acute rmIL-12 toxicity by pretreatment with rmIL-12 (Coughlin et al., 1997; Leonard et al., 1997). This finding has been confirmed in a phase I clinical trial (Rakhit et al., 1999). The diminished toxicity appeared to be associated with an attenuated IFNc response following subsequent IL-12 administration. Unfortunately, the clinical response requires sustained IFNc induction (Gollob et al., 2000a). A phase I trial of subcutaneous rhIL-12 in 50

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patients with advanced renal cell carcinoma resulted in one complete response (Motzer et al., 1998). A fixed-dose schedule of one injection per week revealed an MTD of 1 lg kg1, while 1.5 lg kg1 proved to be the MTD in a dose-escalation study. The low response with rhIL-12 single-agent therapy against metastatic RCC was confirmed in other trials (Motzer et al., 2001). The treatment at this low, non-effective dose was relatively well tolerated. A phase I study in patients with metastatic melanoma (0.5 lg kg1 rhIL-12 s.c. once weekly) resulted in regressions in three out of 10 patients (Bajetta et al., 1998). Treatment was well tolerated and had pronounced effects on immune parameters. A phase I vaccination of six patients with metastatic melanoma using autologous, IL-12 gene-modified tumor cells resulted in one minor clinical response. Two patients manifested increased melanomaspecific CTL activity in the peripheral blood. Vaccinations were well tolerated in both primary (Sun et al., 1998) and subsequent trials (Moller et al., 2000). Administration of rhIL-12 into melanoma patients resulted in a peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8 T cells (Mortarini et al., 2000). Vaccination of metastatic melanoma patients using rhIL-12 and peptide-pulsed PBMC induced different levels of clinical response in a proportion of patients, which positively correlated with the induction of specific immunity (Gajewski et al., 2001). Another option tested was the peritumoral injection of IL-12-producing autologous fibroblasts (Kang et al., 2001). Thirteen of 29 patients enjoyed a clinical response with two of 29 having an objective clinical response in non-injected lesions. At doses 7000 ng/ day a Schwartzman reaction was noted in several patients associated with severe local pain, swelling and tumor necrosis. This was defined as dose-limiting toxicity. A phase II study of 28 patients with recurrent or refractory epithelial ovarian cancer (250 ng kg1 rhIL-12 i.v.) demonstrated a low response rate (one partial responder, 13 cases of stable disease). However, grade 4 myelotoxicity occurred in 21% of the patients and two patients experienced capillary leak syndrome (Hurteau et al., 2001). A phase I trial in nine patients with cutaneous T-cell lymphoma (CTCL) (300 ng kg1 rhIL-12 s.c. twice weekly for up to

24 weeks) resulted in an overall response rate of 56% (Rook et al., 1999). Regression of cutaneous lesions was associated with tumor infiltrating cytotoxic T-cell responses. Adverse events were minor and limited, including low-grade fever and headache.

Clinical trials in viral diseases A phase I/II dose escalation trial (0.03, 0.10, 0.25 and 0.5 lg kg1 subcutaneous rhIL-12 once per week for 10 consecutive weeks) in 60 chronic hepatitis C patients demonstrated antiviral activity of IL-12 that was comparable to that of other current treatments (Zeuzem et al., 1999). Side-effects included flu-like symptoms, transient decreases in leukocyte counts and transient increases of aminotransferases and bilirubin. One patient experienced dyspnea and another abdominal pain, events considered unrelated to IL-12 administration. A phase I/II trial in 46 patients with chronic hepatitis B (0.03, 0.25 and 0.5 lg kg1 rhIL-12 s.c. once a week for 12 consecutive weeks) revealed dose-dependent responses and few adverse events (Carreno et al., 2000). A phase I dose-escalation trial of single doses of rhIL-12 (30–300 ng kg1 range) in 47 stable HIV patients with 100–500 CD4 T cells/L was well tolerated and revealed dose-related increases in absolute CD8 T- and NK-cell numbers, while having no apparent effects on plasma HIV RNA or absolute CD4 T-cell counts (Jacobson et al., 2000). Administration of 1000 ng kg1 rhIL-12 resulted in severe adverse events in this group of patients.

Clinical trials in atopic diseases A trial in asthmatic patients (increasing weekly doses of subcutaneous rhIL-12: 0.1, 0.25 and 0.5 lg kg1), demonstrated a reduction of blood and sputum eosinophil counts with no significant effects on airway hyperresponsiveness or the intensity of late asthmatic reaction (Bryan et al., 2000).

FUTURE DIRECTIONS Although systemic administration of rhIL-12 demonstrated the therapeutic potential of this cytokine in cancer and chronic infections, it was associated with

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significant toxicity reflecting pleiotropic immunomodulatory effects of this factor. Exploiting the therapeutic potential of this cytokine is likely to be facilitated by a targeted delivery of IL-12 as well as its combination with other factors modulating the responsiveness to IL-12 including cytokines and cells, including DCs.

ACKNOWLEDGEMENTS This work was supported by grants from NIH (CA82016) and from The Pittsburgh Foundation (to P.K.).

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17 Interleukin-13 Gabriele Grünig1, Jan E. de Vries2 and Rene de Waal Malefyt3 1

Luke’s Roosevelt Hosp Center, New York, NY, USA 2

Novartis Research Institute, Vienna, Austria 3

DNAX Research Inc, Palo Alto, CA, USA

Evolution reduces redundancy as no two cytokines are functionally identical

INTRODUCTION

IL-13 PROTEIN AND GENE

In 1989 Brown et al. isolated an unknown cDNA by differential hybridization of cDNA libraries from T helper 1 (TH1) and TH2 cells and designated it p600. However, it was not until 1992 that the human homolog of this murine sequence was cloned (McKenzie et al., 1993b; Minty et al., 1993a). Expression of this human cDNA led to the discovery that p600 encoded for a cytokine that had structural features and functional characteristics in common with interleukin-4. Following the demonstration of its immune-modulating effects on B cells and monocytes, the name interleukin-13 was proposed and accepted (Zurawski and de Vries, 1994). In recent years it has become clear that IL-13 plays a unique and essential role in the effector phase of allergic and asthmatic responses.

Human IL-13 is a secreted protein with a molecular mass of 12 kDa, which folds into four a-helical bundles. It contains four potential N-glycosylation sites and four cysteine residues, which pair to form two confirmed intramolecular disulfide bonds between amino acid residues 28–56 and 44–70 (McKenzie et al., 1993b; Minty et al., 1993a; Tsarbopoulos et al., 2000). IL-13 mRNA is 1280 nucleotides long and contains four copies of the (A/T)ATTTA(A/T) repeat which is present in most cytokine mRNAs and implicated in mRNA instability (GenBank accession No. L06801). Alternative splicing of IL-13 mRNA results in two functionally similar isoforms of mature hIL-13 consisting of 131 or 132 amino acids, the latter having an additional glutamine residue at position 98 (McKenzie et al., 1993b). The hydrophobic signal sequence of

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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IL-13 encompasses 20 amino acids. The mature IL-13 protein is ~25% homologous to IL-4, and all residues that contribute to the hydrophobic structural core of IL-4 are conserved, or have conservative replacements in IL-13 (Zurawski et al., 1993; Bamborough et al., 1994). Indeed, structural models of IL-13 based on the NMR and X-ray structure of IL-4, as well as the elucidation of a high-resolution solution structure of IL-13 itself, have demonstrated that the overall threedimensional structures of IL-4 and IL-13 are identical and predicted that helices A and C interact with the IL-4Ra and helix D with IL-13Ra1 (Eisenmesser et al., 2001; Moy et al., 2001; Zuegg et al., 2001). Interestingly, a lysine substitution of the glutamine residue at position 13 resulted in a variant, IL-13E13K, which bound the receptor with high affinity but acted functionally as an IL-13 antagonist (Oshima and Puri, 2001a). An IL-4 molecule with a tyrosine to alanine substitution at position 124 (IL-4Y124D) was previously identified and displayed similar antagonistic characteristics to both IL-4 and IL-13 activities (Aversa et al., 1993). Both human and mouse IL-13 are biologically active on human and murine cells. The gene encoding human IL-13 is located on chromosome 5q31, in the same 3000-kb cluster of the genes encoding IL-3, IL-4, IL-5, IL-9 and GM-CSF (Morgan et al., 1992; Smirnov et al., 1995). The IL-13 gene is only 12 kb upstream from the gene encoding IL-4 and lies in the same orientation, indicating that a gene duplication event took place during evolution. Mouse and human IL-13 genes are composed of four exons separated by three relatively short introns, which makes the total gene size approximately 3 kb (GenBank accession No. L13029) (McKenzie et al., 1993a). Two kilobases upstream of the IL-13 gene is a CpG island, but the overall homology between IL-13 and IL-4 5 flanking sequences is relatively low and allows for differences in the regulation of IL-4 and IL-13 gene expression patterns (Kishikawa et al., 2001). An IL-4P site which binds the NF-ATp transcription factor, has been identified in the IL-13 promoter, and is active in regulating expression of IL-13 in T cells (Dolganov et al., 1996). Furthermore, a regulatory region is found between the IL-4 and IL-13 genes. This regulatory region controls the coordinate expression of genes in the chromosome 5q31 gene cluster (Kelly and Locksley, 2000; Lacy et al., 2000; Loots et al., 2000). Differentiation of naive T cells into

TH2 cells producing IL-4 and IL-13 is functionally associated with chromatin remodeling of the flanking sequences of IL-4 and IL-13 genes, as well as of the intergenic region, and is dependent on antigen receptor stimulation, STAT-6 activation and GATA-3 function (Agarwal and Rao, 1998; Miyatake et al., 2000; Takemoto et al., 2000). A number of polymorphisms have been identified in both coding and flanking sequences of the IL-13 gene and some of these have been associated with enhanced production of IL-13 and susceptibility to allergic asthma (van der Pouw Kraan et al., 1999; Graves et al., 2000; Pantelidis et al., 2000; Howard et al., 2001; Leung et al., 2001).

IL-13R COMPLEXES IL-13 and IL-4 share many, but not all, biological properties which is partially due to the sharing and differential expression of IL-4 and IL-13 receptor components on various cell types (Zurawski et al., 1993). IL-13 receptor complexes are expressed on B cells, monocytes–macrophages, basophils, mast cells, endothelial cells, certain tumor cells and nonhematopoietic cells in many organs, including the lungs, but not on T cells (Zurawski et al., 1993; de Waal Malefyt et al., 1995; Poudrier et al., 2000). Receptor complexes in hematopoietic cells are usually present at 200–3000 sites per cell and bind IL-13 with high affinity (Kd 30 pM). The high-affinity IL-13 receptor complex comprises the 140-kDa IL-4Ra-chain and an IL-13 binding protein. The IL-4Ra chain alone binds IL-4 with a relatively high affinity (Kd ~ 1010 M), but does not bind IL-13. Two different cDNAs encoding IL-13 binding proteins have been cloned: IL-13Ra1 and IL-13Ra2. IL-13Ra1 was first identified in the mouse (Hilton et al., 1996) and subsequently three groups reported the cloning of the human homolog (GenBank accession No. Y09328, U62858) (Aman et al., 1996; Gauchat et al., 1997; Miloux et al., 1997). hIL-13Ra1 consists of a 427-amino acid protein that specifically binds IL-13 with low affinity (Kd approximately 4 nM) but not IL-4. Human IL-13Ra1 is 76% homologous to the mouse IL-13Ra1 and is encoded by a 4 kb mRNA. IL-13Ra1 expression is up-regulated on human B cells following activation through immunoglobulin and CD40 receptors (Graber et al., 1998; Ogata et al., 1998; Ford et al., 1999), but is down-

THE CYTOKINES AND CHEMOKINES

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regulated on human monocyte-derived macrophages (Hart et al., 1999). IL-13Ra1 and IL-4Ra are also coexpressed on keratinocytes, ciliated respiratory epithelial cells, heart muscle cells, faveola cells, hepatocytes and gastric, sebaceous and sweat glands (Akaiwa et al., 2001). The mouse IL-13Ra1 gene consists of 11 exons and alternative mRNA splicings can generate variants that encode a soluble receptor (Osawa et al., 2000). The promoter of human IL-13Ra1 gene does not contain TATA or CCAAT boxes (Ise et al., 1999). IL-13Ra2 is a 380-amino acid protein which binds IL-13 with high affinity (Kd ~50 pM) in the absence of the IL-4Ra chain (GenBank accession No. X95302) (Caput et al., 1996). Human IL-13Ra1 and IL-13Ra2 are 27% homologous, and human and mouse IL13Ra2 proteins are 59% identical. The IL-13Ra1 and IL-13Ra2 chains are expressed as ~65–70 kDa glycosylated molecules. Both IL-13Ra1 and IL-13Ra2 genes are located on the X chromosome (Donaldson et al., 1998) and show homology to IL-5Ra and prolactinR. They contain two consensus patterns characteristic for the hematopoietic cytokine receptor family. One is the WSXWS motif in the extracellular domain and the other is a consensus binding motif for a signal transducer and activator of transcription (STAT) protein present in their short cytoplasmic tails. However, binding of IL-13 to IL-13Ra2 does not lead to activation of signal transduction events including STAT-6 activation, and IL-13Ra2 may therefore function as a decoy receptor for IL-13 (Donaldson et al., 1998; Feng et al., 1998; Kawakami et al., 2001b). The IL-4Ra–IL-13Ra1 complex also functions as a second receptor for IL-4. In contrast, the IL-4R complex, consisting of the IL-4Ra chain and the common c-chain (cc), a shared component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, is specific for IL-4. These models of IL-4–IL-13 receptor complexes are supported by competitive binding studies which show that IL-4 can always completely compete for IL-13 binding on hematopoietic cells, but that IL-13 can only variably compete for IL-4 binding (Zurawski et al., 1993; Feng et al., 1995; Obiri et al., 1995). In addition, the IL-4 mutant protein in which the tyrosine residue at position 124 was replaced by aspartic acid could antagonize the effects of both IL-4 and IL-13 (Aversa et al., 1993). Furthermore, monoclonal antibodies against the 140-kDa IL-4Ra chain were able to block both IL-4 and IL-13 responses, and

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finally, some cell types that respond to IL-4 do not respond to IL-13 (Renard et al., 1994; Tony et al., 1994; Lefort et al., 1995; Zurawski et al., 1995). Additional insights that cc did not participate in IL-13 receptor complexes were obtained from data with T and B cells from patients with X-linked severe combined immunodeficiency (SCID). These patients have mutations in the cc gene and their T cells are unable to respond to IL-2, IL-4, IL-7, IL-9 and IL-15, causing severe abnormalities in both T- and B-cell functions. However, their B cells could proliferate and produce IgE following stimulation through CD40 in the presence of IL-4 or IL-13, indicating that both IL-4 and IL-13 can mediate their biological effects through the IL-4–IL-13R complex in the absence of a functional common c chain (Matthews et al., 1995; Izuhara et al., 1996; Matthews et al., 1997). In addition, macrophages from cc/ mice respond to both IL-4 and IL-13 by upregulating MHC class II and inhibiting nitric oxide (NO) production (Andersson et al., 1997). IL-4 and IL-13 signaling in the absence of cc has also been observed on renal cell carcinoma (RCC), glioblastoma and endothelial cells that apparently express high levels of IL-13Ra2, and possibly low levels of IL-13Ra1 and IL-4Ra. IL-4 may not be able to compete for IL-13 binding on these cells (Debinski et al., 1995, 1996; He et al., 1995; Schnyder et al., 1996; Obiri et al., 1995, 1997; Murata et al., 1998b).

IL-13 SIGNAL TRANSDUCTION Binding of IL-13 to the IL-13R a1 chain, leads to its dimerization with the IL-4Ra chain and initiation of the signal transduction cascade. A strong similarity is observed in the activation of signal transduction pathways between stimulation of cells with IL-4 and IL-13 since the IL-4Ra chain is one of the signal transducing receptor components. IL-13, like IL-4, activated the JAK-1 and Tyk-2 kinases and induced tyrosine phosphorylation of the IL-4Ra chain and the 170-kDa insulin receptor substrate-2/IL-4 induced phosphotyrosine substrate (IRS-2)/4PS (Sun et al., 1995), which, in its phosphorylated state forms a docking site for the Src homology domain (SH2) containing the 85-kDa subunit of PI3 kinase, in lymphohematopoietic cells (Keegan et al., 1995; Lefort et al.,

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1995; Smerz-Bertling and Duschl, 1995; Wang et al., 1995; Welham et al., 1995). Activation of PI-3K is the main intracellular signal transduction pathway responsible for the IL-4- or IL-13-induced reduction of transepithelial resistance in epithelial cells (Ceponis et al., 2000). IL-13 did not induce activation of the JAK-3 kinase which associates with the cc chain of the IL-4R complex following IL-4 binding (Keegan et al., 1995; Welham et al., 1995). Phosphorylation of the IL-4Ra chain following binding of IL-13 to the IL-13R complex leads to the recruitment, phosphorylation, dimerization and nuclear translocation of STAT-6 (signal transducers and activators of transcription) and activation of IL-4/IL-13-responsive genes such as the Ige promoter in a cc–JAK-3-independent manner (Izuhara et al., 1996). The involvement of STAT-6 in IL-13 signaling is also demonstrated in STAT-6/ mice where IL-13 was unable to induce morphological changes, MHC class II up-regulation and NO inhibition in peritoneal macrophages (Takeda et al., 1996). IL-13 also induces phosphorylation of the IL-4Ra chain and activation of JAK-2 and STAT-6 in a cc-independent manner in nonhematopoietic cells such as endothelial cells and colonic or ovarian carcinomas and fibroblasts (Palmer-Crocker et al., 1996; Murata et al., 1996, 1997, 1998). IL-13 also induces phosphorylation of IRS1 and activation of PI3-kinase in the human epithelial cell line HT-29 resulting in inhibition of iNOS expression (Wright et al., 1997). IL-4 and IL-13 were also able to induce phosphorylation of STAT-3 in keratinocytes and lung fibroblasts (Wery-Zennaro et al., 1999; Doucet et al., 2000). The protein tyrosine phosphatase Shp-1 is implicated as a negative regulator of the IL-4/IL-13-activated Jak–Stat pathway leading to termination of signaling (Haque et al., 1998). Whether IL-13 like IL-4 leads to activation of c-fes or a c-fes-like protein has not yet been reported, but both cytokines induce the expression of lck in human monocytes (Musso et al., 1994).

IL-13 PRODUCTION IL-13 is produced by several cell types including T cells, NK cells (Hoshino et al., 1999b), basophils, eosinophils, mast cells, interdigitating cells and dendritic cells (de Saint-Vis et al., 1998; Johansson et al.,

2000). In mouse T cells, IL-13 production is restricted to CD4 TH2 and CD8 T-H2 cells. In contrast, IL-13 is not only produced by human CD4 TH2 cells, but also by TH0 and CD8 T cells, and, unlike IL-4, by naive CD45RA T cells and TH1 cells, albeit at lower levels (de Waal Malefyt et al., 1995). However, all T cells that produce IL-4, produce IL-13 when studied at the single cell level (Jung et al., 1996). Due to its activity of skewing towards TH2 responses, IL-4 stimulates the production of IL-13. IL-25 is a novel cytokine belonging to the IL-17 family and also a potent inducer of TH2 responses (Fort et al., 2001). IL-25 induces high levels of IL-13 expression by dendritic cells. TCR activation of naive CD4CD45RA T cells from peripheral blood in the absence of IL-4 resulted in T cells producing IL-13, however, addition of IL-4 could enhance the percentage of IL-13-expressing cells whereas IL-12 reduced this (Brinkmann and Kristofic, 1995; Jung et al., 1996). IL-13 production was also observed in IL-4/ mice following challenge with Onchocerca volvulus antigens (Pearlman et al., 1996). These results indicate that IL-4 is not absolutely required for the induction of IL-13-producing cells. The kinetics of IL-13 production by activated T cells differs significantly from that of IL-4. IL-4 mRNA appears 2–4 h after activation, and it is almost undetectable after 12 h, whereas IL-13 mRNA can still be observed 48 h after T-cell activation, suggesting that IL-13 is produced for significantly longer periods of time after antigen-specific T-cell activation. This was also observed in mice in vivo following administration of goat- anti-mouse IgD antibodies (Kricek et al., 1995). The activation requirements for IL-13 induction are less stringent than those necessary for IL-4 induction in that activation of protein kinase C alone or a rise in intracellular Ca is sufficient to allow IL-13 mRNA expression in human T-cell clones (de Waal Malefyt et al., 1995). Interestingly, TCR engagement of antiCD28 and PMA-activated T cells resulted in a decrease of IL-13 production, which could be reversed by cyclosporin A (CsA). However, CsA did inhibit IL-13 production by T cells following activation by PHA or CD3 activation. These results indicate a complex regulation of IL-13 with possible involvement of the NFATp transcription factor, since NFATp/ mice showed unexpectedly enhanced immune reponses and changes in cytokine production revealing its possible role as a transcription suppressor (Hodge et al., 1996).

THE CYTOKINES AND CHEMOKINES

EFFECTS OF IL - 13 ON B LYMPHOCYTES

The production of IL-13 by NK and T cells could be enhanced by IL-18 (Hoshino et al., 1999a). IL-13 is also produced by Epstein–Barr virus (EBV)transformed B-cell lines (Fior et al., 1994; de Waal Malefyt et al., 1995), B-cell lymphomas (Emilie et al., 1997), keratinocytes (Michel et al., 1994), mast cells (Burd et al., 1995; Marietta et al., 1996; Pawankar et al., 1997) and basophils (Gibbs et al., 1996; Li et al., 1996). Production of IL-13 by mast cells and basophils following cross-linking of FceRI could contribute to initiation and maintenance of allergic responses (Gauchat et al., 1993; Pawankar et al., 1995; Jaffe et al., 1996; Marietta et al., 1996; Kobayashi et al., 1998).

IN VITRO BIOLOGICAL ACTIVITIES OF IL-13 Based on the composition of IL-13 receptor complexes and the requirement of the IL-4Ra chain for signal transduction, it is not surprising that IL-13 shares many of its biological activities with IL-4. However, we were unable to demonstrate binding of 125 I-labeled IL-13 to human PHA blasts or T-cell clones and failed to demonstrate any biological activities of IL-13 on T cells, indicating that IL-13 lacks the T-cell stimulatory or inhibitory activities of IL-4, presumably through a lack of IL-13 binding protein expression (Zurawski et al., 1993; de Waal Malefyt et al., 1995). In contrast to IL-4, IL-13 could not support the proliferation of activated T cells or induce the expression of CD8a on CD4-positive T cells (de Waal Malefyt et al., 1995). In addition, IL-13 was unable to drive the differentiation of naive CD4 cord blood T cells towards a TH2 phenotype (Sornasse et al., 1996). However, IL-13 may indirectly affect T-cell functions and T-cell differentiation through its down-regulatory effects on the production of IL-12 and IFN-a, which direct TH1 development. IL-13 also lacked activities on mouse T cells.

EFFECTS OF IL-13 ON B LYMPHOCYTES The effects of IL-13 on human B cells are largely similar to those of IL-4, but IL-13 is slightly less (two- to five-fold) potent than IL-4. IL-13 does not have

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additive or synergistic effects with IL-4 when both cytokines are added at optimal concentrations. IL-13 modulates the phenotype of normal human B cells. It up-regulates the expression of CD23, CD71, CD72, sIgM, CD80, CD86 and MHC class II molecules on purified human B cells (Punnonen et al., 1993; McKenzie et al., 1993b; Defrance et al., 1994). The IL-13-induced up-regulation of CD23 is observed on only a subpopulation of B cells. IL-13 has growth promoting activity on normal B cells stimulated by anti-IgM or anti-CD40 mAbs (Aversa et al., 1993; Cocks et al., 1993; McKenzie et al., 1993b; Minty et al., 1993a), but it inhibits IL-2induced proliferation of chronic lymphocytic leukemia B cells (Chaouchi et al., 1996) and the proliferation of pre-B cells induced by stromal cells and IL-7 (Renard et al., 1994). IL-13 enhanced survival of mouse B cells, leading to production of higher IgM levels but failed to up-regulate CD23 or induce class switching (Lai and Mosmann, 1999; Poudrier et al., 2000). IL-13 enhances the production of IgM, IgG and IgA, and induces IgG4 and IgE synthesis by human B cells cultured in the presence of activated CD4 T cells, anti-CD40 mAbs or CD40 ligand (L) transfectants (Aversa et al., 1993; Cocks et al., 1993; Punnonen et al., 1993). IL-13 also induced proliferation and IgM, total IgG, IgG4, and IgE synthesis when total fetal BM cells or highly purified surface (s) mu, CD10, CD19 fetal B cells were cultured in the presence of antiCD40 mAb or activated CD4 T cells (Punnonen and de Vries, 1994). Even highly purified s mu, CD10, CD19 pre-B cells co-cultured in the presence of IL-13 and activated cloned CD4 T cells and IL-7 could be induced to secrete IgG4 and IgE, although IL-13 did not enhance CD23, CD40 and HLA-DR expression on cultured s mu pre-B cells in the absence of other stimuli, nor could it induce germline e transcription by itself, suggesting that IL-13 alone, unlike IL-4 alone, does not activate pre-B cells and that IL-13R are expressed later during B-cell differentiation than IL-4R (Punnonen and de Vries, 1994; Punnonen et al., 1995). IL-13, like IL-4, induced germline e transcription, a prerequisite for switching to successful IgE production. IL-13-induced germline e induction, IgE switching and IgE production is enhanced by antiCD40 signaling, TNFa and IL-10, and inhibited by IFNc, IFNa and TGFb (Cocks et al., 1993; Punnonen et al., 1993; Ezernieks et al., 1996). Activation of the

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germline e promoter is mediated by PKC delta and PKC zeta which modulate threonine phosphorylation and transcriptional activity of PU.1 in a STAT-6independent manner (Ikizawa et al., 2001). Although both IL-4 and IL-13 induce IgE synthesis by human B cells in vitro, the relative contribution of the two cytokines to IgE synthesis under physiological situations remains to be determined. Neutralization of either IL-4 or IL-13 activities in supernatants of activated allergen-specific TH2 cells with anti-IL-4 or antiIL-13 mAbs, indicated that IL-4 is the dominant cytokine in inducing IgE synthesis. However, IgE production induced by supernantants of TH1 or CD8 T-cell clones could be completely blocked by antiIL-13 mAbs, indicating that IL-13 may be the dominant cytokine inducing IgE synthesis in situations where IL-4 is absent or present at low levels, e.g. during the initiation of allergic responses (Essayan et al., 1996; Levy et al., 1997a; Punnonen et al., 1997). This notion is supported by the observations that IL-13 is also produced by naive T cells. Several lines of indirect evidence suggest that both IL-4 and IL-13 are required for optimal induction of human IgE synthesis. Both IL-4 and IL-13 are up-regulated in the lungs of asthmatic patients after allergen challenge and in atopic dermatitis, indicating that both cytokines play a role in the regulation of allergic inflammatory responses (Huang et al., 1995a; Hamid et al., 1996; Kotsimbos et al., 1996a; Kroegel et al., 1996a; Tengvall Linder et al., 1996; Miadonna et al., 1997). In addition, IL-4deficient mice produce low levels of IgE in vivo, following viral or parasite infections, indicating that IL-4-independent IgE synthesis occurs in these mice (von der Weid et al., 1994; Noben-Trauth et al., 1996).

EFFECTS OF IL-13 ON MONOCYTES The effects of IL-13 on monocytes/macrophages and endothelial cells are similar to those of IL-4. IL-13 modulates the phenotype of both human and murine monocytes/macrophages. IL-13 up-regulates the expression of various adhesion molecules on monocytes including CD11b, CD11c, CD18, CD29 and CD49e (VLA-5) (de Waal Malefyt et al., 1993). This may contribute to the changes in morphology induced in monocytes/macrophages such as homotypic aggre-

gation, strong adherence and the development of long cytoplasmic processes. IL-13 up-regulates the expression of MHC class II molecules on human monocytes as well as that of CD80 and CD86, the ligands of CD28 on T cells, leading to an enhanced capacity to stimulate alloantigen-specific T-cell responses. Long-term culture of monocytes and macrophage precursors in the presence of IL-13 and GM-CSF leads to the differentiation of monocytes into dendritic cells and inhibition of monocyte proliferation (Piemonti et al., 1995; Sakamoto et al., 1995; Romani et al., 1996). IL-13 acts as a chemoattractant for human monocytes (Magazin et al., 1994). Interestingly, IL-13 enhanced the production of MDC by monocytes and dendritic cells. Since MDC preferentially attracts TH2 cells, this effect of IL-13 (and IL-4) may contribute to an amplification loop of polarized TH2 responses (Andrew et al., 1998; Bonecchi et al., 1998). IL-13 enhanced the expression of CXCR1 and CXCR2 on monocytes and dendritic cells making them responsive to IL-8 by chemotaxis and production of superoxide (Bonecchi et al., 2000). In addition to these immunostimulatory properties, IL-13 also has important immuno suppressive and antiinflammatory activities. IL-13 inhibits synthesis of IL-1a, IL-1b, IL-6, IL-12, TNF-a, the chemokines IL-8, MIP-1a, MIP-1b and MCP-3 and the chemokine receptor CXCR4 by LPS-activated monocytes, synovial fluid and alveolar macrophages (Doherty et al., 1993; Minty et al., 1993a, 1993b; Cosentino et al., 1995; Hart et al., 1995; Yanagawa et al., 1995; Berkman et al., 1996; Yano et al., 1996; Wang et al., 2001). Furthermore, IL-13 enhances the production of the IL-1 receptor antagonist and the release of the decoy IL-1RII, molecules that both posses antiinflammatory properties by antagonizing IL-1 activities (Colotta et al., 1994, 1996; Muzio et al., 1994; Vannier et al., 1996). However, IL-13 decreased the glucocorticoid receptor binding affinity of LPSactivated monocytes, resulting in a decreased sensitivity of these cells to the immunosuppressive effects of dexamethasone on IL-6 production (Spahn et al., 1996). Although IL-13 inhibits the production of IL-12 by LPS-activated monocytes, overnight priming of monocytes with IL-13 prior to activation with LPS or SAC increased the production of IL-12 (D’Andrea et al., 1995; Bullens et al., 2001). IL-13 induces expression of 15-lipoxygenase, which

THE CYTOKINES AND CHEMOKINES

EFFECTS OF IL - 13 ON OTHER CELL TYPES

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catalyzes the formation of 15-S HETE and lipoxin A4, mediators that antagonize proinflammatory leukotrines (Nassar et al., 1994). IL-13 also upregulates the leukotrine LTD(4) receptor resulting in an enhanced chemotactic responsiveness (Thivierge et al., 2001). In addition, IL-13 inhibits the formation of PGE-2 from arachidonic acid through the inhibition of cox-2 induction by LPS-stimulated monocytes (Endo et al., 1996) and osteoclasts, which results in inhibition of IL-1-induced bone resorption (Onoe et al., 1996). IL-13 inhibits the production of nitric oxide by LPSactivated mouse macrophages and mesangial cells, and inhibits superoxide anion production by human monocytes (Doherty et al., 1993; Doyle et al., 1994; Sozzani et al., 1995; Saura et al., 1996; Wright et al., 2000). Inhibition of NO production resulted in an enhanced iron uptake by murine macrophages, which contributed to the downregulation of their effector functions (Weiss et al., 1997). IL-13 increases the expression of mannose receptors on mouse and human monocytes and macrophages, which will result in the elimination of proteins bearing terminal mannosyl ligands, such as lysosomal hydrolases and plasminogen activators (Doyle et al., 1994; DeFife et al., 1997). In addition, enhanced expression of mannose receptors is involved in human macrophage fusion and formation of foreign body giant cells (DeFife et al., 1997). IL-13, like IL-4, down-regulates tissue factor expression on LPS-activated monocytes and thus inhibits procoagulant activity (Herbert et al., 1993; Del Prete et al., 1995; Ernofsson et al., 1996). Finally, IL-13 downregulates the expression of Fc c receptors, CD16, CD32 and CD64 on human monocytes, but induces expression of the low-affinity FceRII (CD23) (de Waal Malefyt et al., 1993; McKenzie et al., 1993b). Down-regulation of CD64 expression correlated with a decreased ADCC activity (de Waal Malefyt et al., 1993).

et al., 1995) and IL-13 induces the production of soluble VCAM leading to stimulation of angiogenesis (Fukushi et al., 2000). Co-stimulation of endothelial cells by CD40 engagement in the presence of IL-13 increased expression of VCAM and P-selectin (Kotowicz et al., 2000). Eosinophils accumulate at sites of allergic inflammation, and play a crucial role in the pathogenesis of lung inflammation and in lung epithelial cell destruction in asthmatic patients. Consequently, the ability of IL-13, like that of IL-4, to induce VCAM-1 and P-selectin expression on endothelial cells and the subsequent interactions of VCAM-1 and P-selectin on endothelial cells with a4b1 integrins and PSGL1 on eosinophils, which leads to enhanced eosinophil recruitment, further emphasizes the importance of these cytokines in the pathogenesis of allergic and asthmatic responses (Ying et al., 1997; Woltmann et al., 2000). In addition, IL-13 induced the expression of eotaxin-3, a chemoattractant for eosinophils, by endothelial cells and dermal fibroblasts (Shinkai et al., 1999; Hoeck and Woisetschlager, 2001), but inhibited the expression of Rantes by airway smooth muscle cells and endothelial cells (Marfaing-Koka et al., 1995; John et al., 1997) Furthermore, IL-13 acts directly on eosinophils and induced expression of CD69 on eosinophils, as well as prolonged survival of eosinophils (Luttmann et al., 1996). IL-13 also enhanced survival of eosinophils by up-regulation of GM-CSF expression by bronchial epithelial cells (Nakamura et al., 1996). IL-13 inhibited the expression of E selectin by TNFa or IL-1-activated endothelial cells thereby controlling the rolling adhesion and leukocyte emigration of neutrophils (Etter et al., 1998). In addition, IL-13 inhibited the production of fractalkine by activated vascular endothelial cells leading to diminished recruitment of NK cells and TH1 T cells and thus limiting the amplification of polarized type I responses (Fraticelli et al., 2001) and favoring type 2 responses.

EFFECTS OF IL-13 ON ENDOTHELIAL CELLS AND EOSINOPHILS

EFFECTS OF IL-13 ON OTHER CELL TYPES

IL-13 induces VCAM-1 expression on human umbilical vein endothelial cells, which results in adhesion of eosinophils to these cells (Sironi et al., 1994; Bochner

As discussed, IL-13 inhibits the production of proinflammatory cytokines, chemokines and NO by human monocytes and alveolar macrophages but the effects of IL-13 on other cell types can be variable.

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IL-13 induced the production of IL-1RA, IL-1RII and IL-8 by human neutrophils (Girard et al., 1996). IL-13 also activated immediate–early gene expression (c-fos) and up-regulated ICAM-1 expression on human mast cells (Nilsson and Nilsson, 1995). Furthermore, IL-13 enhanced the production of IL-6 by human microglial cells and keratinocytes (Derocq et al., 1994; Sebire et al., 1996). IL-13 enhanced the IL-1induced production of IL-8 by an epithelial cell line and induced neutral endopeptidase (CD13) expression on monocytes, renal carcinoma cell lines and tubular renal epithelial cells (de Waal Malefyt et al., 1993; Riemann et al., 1995; Kolios et al., 1996). IL-13 also enhanced TNF-induced expression of complement C3 and inhibited factor B by dermal fibroblasts (Katz et al., 1995) but inhibited IL-8 and PGE-2 production by synovial fibroblasts (Seitz et al., 1996). IL-13 interacts directly with epithelial cells, fibroblasts, and smooth muscle cells in the lungs (Kraft et al., 2001; Laoukili et al., 2001; Laporte et al., 2001; Webb et al., 2001; Venkayya et al., 2002). Therefore, the activity of IL-13 does not require the presence of T cells or B cells (Grunig et al., 1998; Fort et al., 2001). The direct interaction of IL-13 with epithelial cells, fibroblasts and smooth muscle cells has also been demonstrated using primary human cells isolated from the lungs. These cells respond to IL-13 stimulation with a profound change in the gene-expression profile (Doucet et al., 1998; Kobayashi et al., 1998; Striz et al., 1999; Fujisawa et al., 2000; Lee et al., 2001b; Richter et al., 2001). It is remarkable that IL-13-induced changes in gene expression are virtually non-overlapping in the three airway cell types examined (epithlelial cells, fibroblasts, smooth muscle cells) (Lee et al., 2001b). IL-13 inhibited the production of fibrinogen by Hep G2 cells, which might contribute to its anti-inflammatory activities (Vasse et al., 1996). IL-13 also affects hematopoiesis. IL-13 increased proliferation of mouse Lin Sca bone marrow cells in combination with stem cell factor and G-CSF or IL-11, leading to formation of macrophage colonies (Jacobsen et al., 1994). Furthermore, IL-13 promotes megakaryocyte formation from CD34 human cord blood cells (Xi et al., 1995) . These effects of IL-13 were confirmed by continuous infusion of IL-13 in Balb/c mice, which led to splenomegaly and extramedullary hematopiesis due to expansion of erythroid and

megakaryocyte colonies as well as a monocytosis (Lai et al., 1996). As mentioned previously, IL-13 inhibited GM and M colonies formation from CD34 precursors from human bone marrow or cord blood (Sakamoto et al., 1995).

IN VIVO BIOLOGICAL ACTIVITIES OF IL-13 IL-13 is a powerful cytokine that is critically involved in protective immunity, regulating immune responses, and immune response-induced pathology.

PROTECTIVE IMMUNITY IL-13 has been shown to be a critical component of immunity that heals and protects from helminth infections (Barner et al., 1998; McKenzie et al., 1998a; Bancroft et al., 1998, 2000; Urban et al., 1998, 2000a, 2000b, 2001; Finkelman et al., 1999; Artis et al., 1999b; Finkelman and Urban, 2001; Shea-Donohue et al., 2001). IL-13 is required for the expulsion and protection from infections with certain gastro-intestinal nematodes. Protection is mediated by effects on hematopoietic and non-hematopoietic cells in the intestinal mucosa (Urban et al., 2001) that result in goblet cell hyperplasia (McKenzie et al., 1998a; Khan et al., 2001a), increased mucosal permeability, reduced absorption, increased secretion, nervemediated effects in mucosal function (Shea-Donohue et al., 2001), mast cell responses that induce worm expulsion (Urban et al., 2000b), enhanced expression of TNFa that promotes worm expulsion (Artis et al., 1999a), and increased contractility of the intestinal smooth muscle (Khan et al., 2001b).

REGULATION OF IMMUNE RESPONSES IL-13, like IL-4 and IL-10, down-regulates and controls inflammation induced by excessively activated macrophages (Mulligan et al., 1997; Lentsch et al., 1999; Watson et al., 1999; Matsukawa et al., 2000; Woods et al., 2000; Chabaud et al., 2001; Morita et al.,

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2001; Relic et al., 2001). An example is rheumatoid arthritis, where IL-4, IL-13 and IL-10 inhibit the production of inflammatory mediators and cellular changes in inflamed joints (Woods et al., 2000; Chabaud et al., 2001; Morita et al., 2001; Relic et al., 2001). IL-13 inhibited IL-1b and TNFa production by synovial fluid macrophages from patients with rheumatoid arthritis or from co-cultures of RA synoviocytes and peripheral blood monocytes (Chomarat et al., 1995; Hart et al., 1995; Isomaki et al., 1996). In vivo, IL-13, delivered through a single injection of CHO cells which were genetically engineered to express it, reduced severity and incidence of collageninduced arthritis (CIA) in DBA/1 mice, which correlated with a reduced production of TNFa in the spleen (Bessis et al., 1996). This same approach was also beneficial in autoimmune models. IL-13 suppressed the development of experimental autoimmune enchephalomyelitis (EAE) as assesed by a reduction in mean duration, severity and incidence of the disease. Although this is primarily a T-cell disease, the antiinflammatory effects of IL-13 on macrophages/ monocytes seem to ameliorate the pathology (Cash et al., 1994). IL-13 inhibited the poke weed mitogen (PWM)-induced production of IL-1, IL-6 and TNFa by monocytes isolated from patients with inactive IBD, but not from patients with active IBD. However, in combination with IL-10, both IL-13 and IL-4 synergized in inhibiting the production of proinflammatory cytokines even from these patients (Kucharzik et al., 1996; Kucharzik et al., 1997). Interestingly, IL-13, IL-4 and IL-10 have different potential for inhibiting certain disease manifestations. For example, IL-13 and IL-4, but not IL-10, enhance the growth of synoviocytes by inhibiting apoptosis of these cells (Relic et al., 2001). In the lungs, IL-13 inhibits inflammation induced by TNFa (Watson et al., 1999) and by the deposition of IgG immune complexes (Mulligan et al., 1997; Lentsch et al., 1999). IL-13 is also a potent inhibitor of excessive inflammation in endotoxin-mediated sepsis (Muchamuel et al., 1997; Baumhofer et al., 1998; Matsukawa et al., 2000). In IgG immune complexmediated disease as well as in inflammation due to release of endotoxin, IL-10 is as potent or even more potent than IL-13 in inhibiting excessive release of cytokines and chemokines (Berg et al., 1995; Mulligan et al., 1997).

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IMMUNE RESPONSEINDUCED PATHOLOGY Chronic helminth infections While the overall role of IL-13 is to protect from nematode infection, IL-13 is also critical for the pathological changes seen in chronic infection (Chiaramonte et al., 1999a, 1999b, 2001; Jankovic et al., 1999). The pathological changes include the production of high levels of IgE, the development of granulomas in the lungs (Chiaramonte et al., 1999b) and liver (Chiaramonte et al., 1999a; Jankovic et al., 1999), and liver fibrosis (Chiaramonte et al., 1999a, 2001; Fallon et al., 2000).

Allergic lung diseases Most, if not all, of the IL-13-induced mechanisms that protect against nematode infections cause asthmalike disease when triggered by inhaled allergens.

Role of IL-13 in mouse models IL-13 is the major mediator of the asthma phenotype (airway hyperreactivity, over-production of mucus, airway eosinophilia, peribronchial fibrosis (Grunig et al., 1998; Wills-Karp, 1998, 1999; Cohn et al., 1999; Corry, 1999; Zhu et al., 1999; Blease et al., 2001a; Ford et al., 2001; Lukacs et al., 2001b; Tomkinson et al., 2001; Yang et al., 2001; Venkayya et al., 2002). Blockade of IL-13 reduces airway hyperreactivity, goblet cell hyperplasia, airway eosinophilia, and, in addition, alveolar inflammation. Although IL-4 is capable of eliciting similar changes in the lungs (Grunig et al., 1998), blockade of IL-13 in asthma models inhibits airway hyperreactivity and goblet cell hyperplasia to a much larger extent when compared with blockade of IL-4 (Corry, 1999; Blease et al., 2001b; Tekkanat et al., 2001; Walter et al., 2001). The inhibition of the asthma phenotype has been observed in many different mouse strains using different antigens as well as different IL-13 blockers. IL-13 blockade also inhibits the signs of asthma in guinea pigs (Morse et al., 2002). A novel cytokine, IL-25, that induces the expression of TH2 cytokines (including IL-4, IL-5 and IL-13) in nonT cells, is capable of eliciting lung inflammation with many of the characteristics of the asthma phenotype (Fort et al., 2001). IL-13, but not IL-4 or IL-5, is the

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major effector of lung lesions in mice exposed to IL-25. In addition to the asthma phenotype, overexpression of IL-13 in the lungs induces emphysema (Zheng et al., 2000). Although IL-13-mediated lung injury is often dependent on signaling through the IL-4Ra (Grunig et al., 1998; Cohn et al., 1999; Tomkinson et al., 2001; Yang et al., 2001), IL-13 has also been shown to induce airway hyperreactivity independently of IL-4Ra or STAT6 (Mattes et al., 2001; Blease et al., 2002). IL-13 has an attenuated role in lung inflammation induced by mixed T-cell responses (TH1 and TH2 present at the same time) (Ford et al., 2001). Blockade of IL-13 significantly reduced goblet cell hyperplasia in mice which developed a mixed T-cell response, although to a lesser extent than in mice which developed a polarized TH2 response. There was a trend towards reduction of airway hyperreactivity. There was no change in airway eosinophilia and alveolar inflammation. IL-13-induced changes in the lungs are dependent on the activation of the GATA3 system (Ray and Cohn, 1999; Zhang et al., 1999; Das et al., 2001; Kishikawa et al., 2001). Other TH2 cytokines, including IL-9, also play a role in IL-13-induced airway disease, either by eliciting the expression of IL-13 in the lungs (Temann et al., 2002), or by directly mediating goblet cell hyperplasia and airway hyperreactivity (Kung et al., 2001). However, the IL-13-mediated, allergeninduced changes can occur independently of IL-9 (McMillan et al., 2002). IL-13-induced airway eosinophilia is a result of the induction of chemokines (eotaxin) in combination with the production of IL-5 in the course of the TH2 response (Li et al., 1999; Foster et al., 2001; Pope et al., 2001). IL-13 also induces the expression of TGFb1 which is critical for peribronchial fibrosis (Lee et al., 2001a; Lukacs et al., 2001a) and metalloproteinases which are critical for the development of emphysema (Zheng et al., 2000). The molecular pathways that lead to IL-13-induced goblet cell hyperplasia and airway hyperreactivity have not been clearly delineated but may include IL-9, epidermal growth factor receptor, eosinophils and neutrophils (Kung et al., 2001; Shim et al., 2001; Singer et al., 2002).

IL-13 disease association in individuals with asthma IL-13 has been detected at increased levels in samples from the airways and blood of asthmatic patients (Huang et al., 1995b; Kotsimbos et al., 1996b; Jaffar et al., 1996, 1999; Kroegel et al., 1996b; Humbert et al., 1997; Naseer et al., 1997; Bodey et al., 1999; Devouassoux et al., 1999; Prieto et al., 2000; Krug et al., 2001; Oldfield et al., 2001). Increased IL-13 levels have also been seen in skin samples from individuals with atopic dermatitis (Koning et al., 1997; Leung, 1997). The increased production of IgE in atopic patients may also strongly depend on IL-13 (Levy et al., 1997b; Van der Pouw Kraan et al., 1998; Tang et al., 2001).

Polymorphisms of the IL-13 gene It has been known for some time that asthma has a genetic component, as there are families with increased occurrence of the disease. The analysis of the IL-13 (Pantelidis et al., 2000) and the IL-13 receptor (Ahmed et al., 2000) genes has revealed polymorphisms in the coding region and in the promoter region. IL-13 gene polymorphisms have been associated with asthma, atopy (increased IgE levels), or atopic dermatitis in several ethnic groups from Asia, Europe and the United States (van der Pouw Kraan et al., 1999; Graves et al., 2000; Heinzmann et al., 2000; Liu et al., 2000; Howard et al., 2001). A recent Dutch study demonstrated that the association with asthma was even higher in individuals who simultaneously had polymorphisms in the IL-4Ra and IL-13 genes (Howard et al., 2002). This is surprising because the IL-4Ra and the IL-13 genes are located on different chromosomes (chromosomes 16 or 5q, respectively). Therefore, the increased association of asthma with combined IL-4Ra and IL-13 gene polymorphisms indicates functional synergy.

Autoimmune diseases Many autoimmune diseases are thought to be the result of excessive TH1 responses, and TH2 cytokines like IL-4, IL-13 and IL-10 are thought to be protective (reviewed in Isomaki and Punnonen, 1997; Zaccone et al., 1999; Duda et al., 2000; Young et al., 2000; Scola

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et al., 2002). Recent studies have challenged this paradigm and found that TH2 responses can in fact mediate autoimmune diseases, antibody-mediated autoimmune diseases (Romagnani, 2000), as well as cell-mediated autoimmune diseases (Theofilopoulos et al., 2001). Examples of autoimmune diseases with a possible contribution of IL-13 to the pathogenesis include Graves’ Disease (Yamada et al., 2000) and bullous pemphigoid (Teraki et al., 2001).

Tumors IL-13 is secreted by lymphoma cell lines and may be an autocrine growth factor. Cutaneous T-cell lymphoma lines as well as Hodgkin’s and Reed–Sternberg tumor cells (that are thought to originate from germinal center B cells) secrete IL-13 (Kapp et al., 1999; Fiumara et al., 2001; Oshima and Puri, 2001b; Skinnider et al., 2001; Nielsen et al., 2002). IL-13 made by the Hodgkin’s lymphoma cells contributes to STAT6 phosphorylation (Skinnider et al., 2002) and thus induces the signals for cell proliferation (Kapp et al., 1999). IL-13 is also expressed in pancreatic cancer (Crnogorac-Jurcevic et al., 2001). IL-13 inhibits the growth of other types of tumors, for example, of gliomas and renal cell carcinomas. In those instances, expression of the IL-13Ra2 chain protects these cells from growth inhibition exerted by IL-13 (Bernard et al., 2001). In contrast, the expression at high levels of the IL-13Ra2 chain in human breast and pancreatic cancer lines has been shown to inhibit tumor growth in vivo in a neutrophil-dependent manner (Kawakami et al., 2001a). IL-13 also has indirect effects on tumor growth by affecting the immune response. This effect appears to be largely inhibitory. IL-13 (but not by IL-4), most probably made by NK–T cells, inhibits the immune response that protects from tumor recurrence (Terabe et al., 2000). IL-13 is being evaluated for use as a vehicle to deliver cytotoxic drugs to tumor cells. For this purpose, the expression at high levels of the IL-13Ra2 chain by certain tumors, coupled with the high affinity of IL-13 to the IL-13Ra2 is utilized (Debinski and Gibo, 2000; Husain and Puri, 2000; Husain et al., 2001; Nash et al., 2001; Oshima and Puri, 2001b).

CONCLUSIONS IL-13 is made by many different cell types, and the distribution of its receptors is widespread. Owing to the shared signaling components, IL-13 and IL-4 have many overlapping functions. However, in vivo, IL-13 has roles that are distinct from IL-4 (e.g. protection from some helminth infections, asthma phenotype, activity on tumor growth). It is not clear why IL-13 and IL-4 have so clearly distinguishable functions in vivo. One possibility is differential bio-availability of the cytokines, or a differential distribution of the different receptor components. The in vivo effects of IL-13 have been well documented in the mouse as well as in other experimental and domestic animals. The role of IL-13 in human physiology and pathology remains unknown. While IL-4 is the major cytokine to induce TH2 responses, IL-13 may be required for the full induction of TH2 responses as well as IgE production (Barner et al., 1998; McKenzie et al., 1998b; Matthews et al., 2000; McKenzie, 2000; Fallon et al., 2001). However, this phenomenon could be due in part to the genetic linkage of the IL-4 and IL-13 genes (Loots et al., 2000), which results in diminished transcription of the IL-4 gene when linked to an IL-13 gene showing diminished function (Guo et al., 2001). IL-13 shares with IL-4 the ability to induce IgE production in human B cells and to regulate the activity of macrophages. However, the most significant function of IL-13 which distinguishes this cytokine is its ability to regulate the functions of non-hematopoietic cells, like epithelial cells, smooth muscle cells and fibroblasts, particularly on mucosal surfaces. Finally, therapeutic inhibition of IL-13 could be an important aspect of the treatment of allergeninduced diseases such as asthma and of excessive pathology in chronic helminth infections. Both disease complexes affect millions of people worldwide. IL-13 inhibition could be attempted at different levels, including inhibiting IL-13 using antibodies or soluble IL-13Ra2 complexes, or inhibiting both IL-13 and IL-4 by blocking the IL-4Ra or STAT6 (Donaldson et al., 1998, 2000). The availability of blocking reagents for therapeutic interventions in human disease will allow us to draw final conclusions on the biological role of IL-13 in health and disease.

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ACKNOWLEDGEMENTS DNAX Research Inc is supported by Schering-Plough corporation.

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18 Interleukin-15 and 21 Michael R. Shurin1, Irina L. Tourkova1, Holger Hackstein1,2 and Galina V. Shurin1 1

University of Pittsburgh Medical Center, Pittsburgh, PA, USA 2

Justus-Liebig University Giessen, Giessen, Germany

Adversity has the effect of eliciting talents, which in prosperous circumstances would have lain dormant Horace

INTRODUCTION Since the cloning of interleukin-15 (IL-15) in 1994, there have been many reports expanding the central biologic effects and mechanisms of action of this cytokine on T cells, dendritic cells and NK cells. Although IL-15 shares activities with IL-2 and utilizes the common b and c subunits of the IL-2R, it became evident that these cytokines exert differential effects on many cell populations. The IL-15 gene is not expressed in T cells, which are the primer producers of IL-2, but rather expression of its gene has been detected in placenta, skeletal muscle, kidney, lung, heart, myeloid progenitor cells and dendritic cells. IL-15 acts on various cells of the immune system, including T and B lymphocytes, NK cells, monocytes, eosinophils and circulating neutrophils. IL-15 stimulates NK cells, T cells and B cells to proliferate, secrete cytokines, and exhibit increased cytolytic activity or produce antibody. It also stimulates phagocytes, maintains mast cells and regulates migration of The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

activated/memory T cells. IL-15 is involved in protection against different viral and bacterial infections regulating not only innate immunity but also adaptive immunity. There is increasing evidence to suggest that IL-15 plays a pivotal role in protective immune responses, allograft rejection and the pathogenesis of various autoimmune and chronic immunoinflammatory diseases. IL-21, first described by (Parrish-Novak et al., 2000) is a novel cytokine, structurally and functionally related to IL-2, IL-4 and IL-15. IL-21 was discovered by employing cell lines expressing the IL-21 receptor (IL-21R) as a functional assay for ligand detection and subsequent cloning. IL-21R, a novel class I cytokine receptor, was identified in sequence databases by using gene prediction algorithms and was cloned by two independent groups (Ozaki et al., 2000; ParrishNovak et al., 2000). The discovery of IL-21 adds another member to the ever-growing list of cytokines that have potent effects on different populations of lymphoid cells (Vosshenrich and Di Santo, 2001). Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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Initial characterization of the IL-21R complex suggests that IL-21 may belong to the cytokine family whose receptors share the common c chain (cc). However, questions about IL-15 and the IL-15 receptor (IL-15R) as well as IL-21 and IL-21R expression in different cell types and the role of IL-15 and IL-21 in immune homeostasis are still to be resolved.

IL-15: STRUCTURE AND GENOMIC ORGANIZATION IL-15 was first identified by two groups based on its ability to stimulate proliferation of the IL-2-dependent T-cell line CTLL-2 in the presence of anti-IL-2 antibody. Using this strategy, the factor was identified in the supernatants of the simian kidney epithelial cell line, CV-1 EBNA, molecularly cloned, and designated IL-15 (Grabstein et al., 1994). The same factor isolated from the conditioned medium of a human T-cell leukemia virus I-induced cell line HuT-102, was called IL-T (Bamford et al., 1994; Burton et al., 1994). Human IL-15 was subsequently cloned from the bone marrow stromal cell line, and comparison between the simian and human cDNAs reveals 97% sequence homology within the coding regions. Murine IL-15 exhibits 73% sequence homology with human IL-15. The open reading frame of the human IL-15 cDNA is 486 bp in length and is flanked by a 316-bp 5 non-coding region and a 400-bp 3 noncoding region. The gene consists of nine exons (1–8 and 4a) and eight introns. The murine IL-15 gene locus is approximately 34 kb in length and contains seven introns and eight exons, of which, exons 2 and 4–8 code for the mature protein. The IL-15 gene was mapped to chromosome 4q31 in human and chromosome 8 in the mouse (Anderson et al., 1995a; Krause et al., 1996). The bovine IL-15 sequence was cloned from abomasal (gastric) lymph node mRNA by enzymatic amplification of cDNA using human primers proximal to and including the translation start and stop sites. The open reading frame is 486 bp in length, and the proposed protein sequence shows 78.4% and 73.5% similarity with that predicted for the human andmousesequences,respectively(Canals etal.,1997). IL-15 is a 14–15-kDa glycoprotein and a member of the 4a-helix bundle family of cytokines along with IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and IL-9 (Grabstein

et al., 1994). These four helices are arranged in the up–up–down–down configuration and are connected by three loops. IL-15 consists of 114 amino acids and has two cysteine disulfide crosslinkages and three asparagines residues that in two cases are sites for Nlinked glycosylation (Grabstein et al., 1994). Although IL-15 binds to the b and c subunits of the IL-2R, it exhibits no amino acid sequence homology with IL-2. The existence of two isoforms of IL-15 has been also demonstrated: one with a short (SSP) and another with a long signal peptide (LSP) (Plate 18.1). Both isoforms of IL-15 with LSP and SSP signal peptides were identified in different murine and human tissues with a different tissue distribution pattern (Meazza et al., 1997; Onu et al., 1997; Tagaya et al., 1997; Nishimura et al., 1998). In contrast to most signal peptides that are encoded by a single exon, IL-15 leader sequence isoforms are encoded by at least two exons. The LSP is encoded by exons 3–5 of the human IL-15 gene, whereas the SSP is encoded by exon 5 as well as by an additional 119-nucleotide (nt) sequence termed exon 4a located in intron 4 (Kurys et al., 2000). Thus, the two signal peptides share 11 amino acids encoded by exon 5. The presence of exon 4a disrupts the LSP sequence by inserting a premature termination codon and then provides an alternate initiation codon. PCR cycle sequencing of the larger transcript revealed the mouse homolog of the alternatively spliced exon A as it is known from the human IL-15 gene (Prinz et al., 1998). Analysis of the corresponding mouse IL-15 gene region shows that the larger IL-15 transcript contains a 5 sequence of exon 5 while the shorter transcript uses an internal splice acceptor site. The mouse exon 5A segment has a length of 136 nt (17 nt longer than the human exon A). It contains five in-frame stop codons at its 5 end and a new translation initiation site at its 3 end. This new start site is surrounded by a favorable Kozak consensus sequence suggesting a more efficient translation rate (Prinz et al., 1998). Further translational control by stem–loop binding factors is inferred by a predicted RNA stem–loop structure around the start site. Insertion of exon 5A would lead to an IL-15 polypeptide with a shortened leader sequence of 26 amino acids, as compared with the 48 amino acid leader sequence encoded by the transcript lacking exon 5A. Thus, the final IL-15 protein of the two splice variants is identical; different leader sequences could, however, lead to differences

THE CYTOKINES AND CHEMOKINES

IL - 15: SOURCES

in the intracellular sorting, processing and/or secretion of IL-15 (Prinz et al., 1998).

IL-15: SOURCES Many different cell types express either the soluble or membrane-bound form of IL-15, although IL-15 is probably mainly produced by activated macrophages (Carson et al., 1995) and muscle cells (Table 18.1). For instance, in the Maeurer et al. (1999) study, increased IL-15 secretion was observed in IL-4/ granulocyte–macrophage colony-stimulating factor (GM-CSF)-activated antigen-presenting cells (APCs) compared with unstimulated macrophages. Interestingly, immunocytological detection of intracellular IL-15 revealed that infection with different mycobacterial species resulted in different staining patterns of anti-IL-15-immunoreactive material in APCs. In addition, IL-15 transcripts were constitutively expressed by freshly isolated skin dendritic cells, Langerhans cells (Blauvelt et al., 1996). Human cultured CD14 monocyte-derived dendritic cells also express low levels of IL-15 mRNA, which are significantly augmented by CD40 ligation (Pirtskhalaishvili et al., 2000). CD40L-mediated stimulation of human peritoneal mesothelial cells has been also shown to result in IL-15 release (Basok et al., 2001). Freshly isolated keratinocytes, immortalized keratinocytes (HaCaT cells) and cultured keratinocytes all constitutively express both IL-15 protein and mRNA (Blauvelt et al., 1996; Ruckert et al., 2000). The authors proposed that keratinocyte-derived IL-15 may be important during the generation of skin inflammatory and immune responses by attracting T cells to epidermis and subsequently enhancing the activation of TH1 T cells within epidermis. However, Han et al. (1999) have also investigated the levels of IL-15 and its mRNA produced by epidermal and cultured keratinocytes and found that normal keratinocytes did not constitutively express IL-15 in the epidermis, but in culture began to produce the cytokine. Some epidermal keratinocytes express IL-15 in inflammatory conditions associated with infiltration of neutrophils and eosinophils. Furthermore, TNFa-stimulated dermal fibroblasts express membrane-bound IL-15, which enhances proliferation of activated T cells (Rappl et al., 2001). Unstimulated

433

fibroblasts, however, accumulate IL-15 in the cytoplasm and no IL-15 can be detected in the culture supernatant. Cytokines and growth factors have been suggested as playing important roles in uterine function. Using differential gene expression in human endometrium between the proliferative and the secretory phases, Okada et al. (2000) have identified IL-15 as an upregulated transcription product during the secretory phase in comparison with the proliferative phase. RTPCR analysis confirmed these results and revealed that the most abundant expression of IL-15 mRNA during the menstrual cycle was in the midsecretory phase. Additionally, in the first trimester of pregnancy, the expression of IL-15 mRNA in the decidua was significantly higher than that in the chorionic villi (Okada et al., 2000). Placental tissues have also been shown to release IL-15 into the culture medium (Agarwal et al., 2001). Thus, midtrimester amniotic fluid cytokines, including IL-15, may reflect the function of the maternal immune system in the maternal–fetal interface and thus be predictive of preeclampsia. In fact, the level of secretion by term placental tissues was much higher than that by first trimester tissues. The presence of labor at term resulted in a further increase in placental IL-15 production. The levels of both IL-15 mRNA and protein, however, were significantly reduced in the preeclamptic placental tissues. In contrast, Heikkinen et al. (2001) detected no difference in IL-15 concentrations in amniotic fluid at 14–16 weeks of gestation between women with normal pregnancies and from those who subsequently developed severe preeclampsia. Similarly, Searle et al. (2000) detected no IL-15 in normal midtrimester amniotic fluid and suggested that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto–placental unit. Thus, although these data suggest an important role for IL-15 in human pregnancy and menstrual cycle, additional studies are required to characterize a functional significance of IL-15 in pregnancy. Rhabdomyosarcoma cell lines express and secrete IL-15. Interestingly, wild-type p53 transduction in human rhabdomyosarcoma cells induces the downmodulation of both IL-15 production and IL-15 receptor expression (De Giovanni et al., 1998). Other tumor cells and cell lines, including T-cell lymphoma,

THE CYTOKINES AND CHEMOKINES

434

INTERLEUKIN - 15 AND 21

TABLE 18.1 Cellular expression of IL-15 mRNA and/or protein Tissue or cell types

Note

References

Monocytes/macrophages, monocytic cell lines (MONO-MAC-6, THP-1 and U937

RT-PCR Membrane-bound forms

Carson et al., 1995 Musso et al., 1999 Quaranta et al., 1999

Macrophages

Increased IL-15 secretion was observed in IL-4/GM-CSF-activated APC compared with unstimulated macrophages

Doherty et al., 1996; Maeurer et al., 1999

Skeletal muscle

Protein

Quinn et al., 1995

Thymic epithelial stromal cells

RT-PCR

Leclercq et al., 1996

Freshly isolated keratinocytes, immortalized keratinocytes (HaCaT cells), and cultured keratinocytes

RT-PCR

Blauvelt et al., 1996; Han et al., 1999

Langerhans cells

RT-PCR

Blauvelt et al., 1996

Cultured dendritic cells

RT-PCR DC were generated from adherent PBMC-derived monocytes

Blauvelt et al., 1996; Jonuleit et al., 1997; Pirtskhalaishvili et al., 2000

Murine splenic dendritic cells

Protein and mRNA

Liu et al., 2000; Mattei et al., 2001

Melanoma cell lines

RT-PCR

Azzarone et al., 1996; Barzegar et al., 1998

Myeloma cell lines and freshly purified myeloma cell isolated from patient

RT-PCR

Hjorth-Hansen et al., 1999

Rhabdomyosarcoma cell lines Rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma

Protein and mRNA mRNA

Lollini et al., 1997; De Giovanni et al., 1998

Acute lymphoblastic leukemia cells

RT-PCR

Kebelmann-Betzing et al., 2001

Cutaneous T-cell lymphoma cells

RT-PCR

Dobbeling et al., 1998

Human lung carcinoma cells

A549 cells constitutively expressed IL-15 mRNA which could be up-regulated by stimulation with TNFa or IL-1b

Stoeck et al., 2000

Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG glioma KG-1-C glioma and NTera2 teratocarcinoma

RT-PCR

Satoh et al., 1998

Squamous cell carcinoma cell line HSC-5

IFNc slightly increased the IL-15 protein and mRNA levels in a dose-dependent fashion

Han et al., 1999

Renal cell carcinoma (RCC), small cell lung carcinoma (SCLC), glioblastoma, neuroblastoma, mesothelioma cells and in EBV-transformed B lymphocytes

RT-PCR

Trinder et al., 1999

THE CYTOKINES AND CHEMOKINES

IL - 15: SOURCES

435

TABLE 18.1 Continued Tissue or cell types

Note

References

Colon

Multiprobe ribonuclease protection assay in C57BL/5 and Min mice

Bassonga et al., 2001

Murine small intestinal and colonic epithelial cells

RT-PCR

Meijssen et al., 1998

Human cortical tubular epithelial cells

IL-15 expression was analyzed by RT-PCR, ELISA and bioactivity

Weiler et al., 1998; Weiler et al., 2001

Thyroid

By RT-PCR, in vivo gene expression of IL-15 was studied in multinodular goiter (MNG), Graves’ disease (GD), and Hashimoto’s thyroiditis (HT)

Ajjan et al., 1997

Rat intestinal epithelial cells

IL-15 synthesis was detected on day 1 after an oral inoculation of L. monocytogenes in rat in vivo

Reinecker et al., 1996; Hirose et al., 1998

Peritoneal mesothelial cells

IFNc up-regulated IL-15 mRNA levels and protein secretion in a dose-dependent manner. IL-15 may serve as a mediator of T cell-regulated immune and inflammatory response during peritonitis

Hausmann et al., 2000; Basok et al., 2001

Human dermal and umbilical vein endothelial cells

Expression of IL-15 mRNA and protein can be up-regulated by UVB

Mohamadzadeh et al., 1996

Placenta

Placental tissues releases IL-15 into the culture medium

Agarwal et al., 2001

Human endometrium and deciduas Human endometrial stromal cells

DNA array and RT-PCR RT-PCR and ELISA

Okada et al., 2000; Kitaya et al., 2000; Okada et al., 2000

Bone marrow stromal cells

Express the IL-15 transcript, and supernatants from long-term cultures contain IL-15 protein

Mrozek et al., 1996

Retinal pigment epithelial cells

RT-PCR demonstrated expression of both IL-15 and IL-15Ra/IL-2Rbc, which may play an important role in ocular immune and inflammatory responses by stimulating infiltrated T cells and retinal pigment epithelial cells via paracrine and autocrine loops, respectively

Kumaki et al., 1996

Human fetal astrocytes and microglia

Low levels of IL-15 expression at both the mRNA and protein level were increased by treatment with IL-1, IFNc or TNFa

Lee et al., 1996

Human cerebral and cerebellar tissues

RT-PCR

Satoh et al., 1998

Human peripheral nerve and skeletal muscle

RT-PCR

Satoh et al., 1998

THE CYTOKINES AND CHEMOKINES

436

INTERLEUKIN - 15 AND 21

melanoma, myeloma, acute lymphoblastic leukemia, lung carcinoma, renal cell carcinoma, neuroblastoma, glioma, glioblastoma, retinoblastoma, teratocarcinoma and mesothelioma cells (Azzarone et al., 1996; Dobbeling et al., 1998; Satoh et al., 1998; Hansen et al., 1999; Trinder et al., 1999; Stoeck et al., 2000; Leroy et al., 2001; Kebelmann-Betzing et al., 2001) express IL-15 mRNA or protein. IL-15 is also produced by human cortical tubular epithelial cells (Ajjan et al., 1997), thyroid (Weiler et al., 1998). Hanisch et al. (1997) have shown that IL-15, its specific receptor molecule, IL-15Ra, and the signal-transducing receptor subunits, IL-2Rb and IL-2Rc, are constitutively present in various regions of the developing and adult mouse brain. They further demonstrated that IL-15 and the components for IL-15Ra/ IL-2Rbc are expressed by microglia. At doses of 0.1–10 ng ml1, IL-15 affected functional properties of these cells, such as the production of nitric oxide, and supported their growth in culture, suggestive of a role as an autocrine growth factor (Hanisch et al., 1997). In addition, low levels of IL-15 expression by unstimulated human fetal astrocytes and microglia could be augmented by treatment of astrocytes with IL-1, IFNc, or TNFa at both the mRNA and protein level (Lee et al., 1996). Treatment of microglia with IFNc and lipopolysaccharide (LPS) similarly increased IL-15 expression in microglia. Thus microglial IL-15 may play an important role in the CNS and may participate in certain CNS and neuroendocrine functions previously ascribed to IL-2. Finally, the expression of IL-15 mRNA was also identified in the human cerebral and cerebellar tissues and peripheral nerves (Satoh et al., 1998).

REGULATION OF IL-15 EXPRESSION There are several major differences in the mechanisms controlling synthesis and secretion of IL-2 and IL-15. IL-2 is synthesized by activated T cells and is predominantly controlled at the levels of mRNA transcription and stabilization, whereas the widely distributed IL-15 has a much more complex control of expression with regulation at the levels of transcription, translation, and intracellular trafficking

(Bamford et al., 1996; Tagaya et al., 1996a; Nishimura et al., 2000; Kurys et al., 2000). Meazza et al. (1997) have shown that human cancer cell lines of different histotypes express two IL-15 amplification products: a 524-bp band corresponding to the IL-15 mRNA found in macrophages, and another of 643 bp corresponding to an alternatively spliced mRNA including a 119-bp alternative exon. Although IL-15 could be detected intracellularly in some tumor cells by confocal microscopy, it was undetectable in the supernatant of tumor cell lines expressing either one or both of the mRNA isoforms, indicating that IL-15 was not secreted. The authors suggested that a poor efficiency of natural signal peptides may represent one of the mechanisms involved in the control of IL-15 secretion. It has been proposed that the dominant control of IL-15 expression is posttranscriptional, i.e. at the levels of message translation and intracellular protein trafficking with a further demonstration that IL-15 expression is posttranscriptionally regulated by multiple elements, including the 10 upstream AUGs of the 5 UTR, a 48 amino acid signal peptide (long signal peptide (LSP)) and the carboxy-terminus of the mature protein (Waldmann et al., 1998). Furthermore, Pereno et al. (1999) have reported the existence of different nuclear localization and intracellular trafficking of IL-15 and IL-15Ra in cells expressing the two isoforms. They speculated that an intercellular microcirculation of IL-15, not detectable with ELISA kits, but displaying a role as an antiapoptotic factor is able to induce the deflection of the TNFR-associated factor 2 (TRAF) to IL-15Ra. These data point to a juxtacrine mechanism of action of IL-15 and suggest a role for IL-15/IL-15Ra in the regulation of apoptosis. A number of findings suggest different trafficking of the two IL-15 isoforms and multiple mechanisms controlling IL-15 secretion (Gaggero et al., 1999). Although signal peptides classically function to target the endoplasmic reticulum (ER), it has been reported that the SSP–IL-15 isoform does not enter the ER and is not secreted, but rather is stored intracellularly and appears in nuclear and cytoplasmic components (Plate 18.1). In contrast, the LSP isoform enters multiple intracellular trafficking pathways leading to different destinations. In one pathway, LSP–IL-15 is relatively inefficiently translocated into the ER and

THE CYTOKINES AND CHEMOKINES

IL - 15 R : STRUCTURE AND DISTRIBUTION

therefore remains unprocessed in the cytoplasm, where the unglycosylated protein is degraded in proteasomes. In a second pathway, LSP–IL-15 enters the ER and is partially processed to yield a proposed 19amino acid 3-element of the LSP linked to the mature glycosylated protein. This alternatively cleaved IL-15 is not secreted into the medium in this form. The third pathway of LSP–IL-15 trafficking is the classical one and involves translocation of LSP–IL-15 into the ER, where the LSP is fully processed, and the protein is N-glycosylated and ultimately secreted as a 17-kDa cytokine by the classical Golgi route (Kurys et al., 2000).

IL-15R: STRUCTURE AND DISTRIBUTION A number of early studies suggested that IL-15 utilized the b and c chains of the IL-2R, and that these are essential for IL-1-mediated signal transduction. However, several lines of evidence have indicated the existence of an additional, IL-15-specific receptor component (Cosman et al., 1995). An IL-15 binding chain was identified on a murine T-cell clone, and direct expression cloning was used to isolate the corresponding cDNA. The predicted structure of this protein shows sequence similarity to the IL-2Ra chain (Giri et al., 1995a). Transfection of this cDNA into a murine, IL-3-dependent myeloid cell line, 32D-01, conferred IL-15 binding and, together with transfection of the IL-2Rb chain, rendered the cells responsive to IL-15 stimulation. This experiment confirmed that the IL-15 binding chain is part of the IL-15R, and it is designated as the IL-15Ra subunit (Cosman et al., 1997). Thus, the IL-15 receptor is a heterotrimeric complex, which is composed of the IL-2Rb and the common cc chains in combination with a unique a chain. The IL-15Ra chain has a strong sequence homology to the IL-2Ra chain and confers highaffinity binding to the IL-15R. However, the murine IL-15Ra alone bound IL-15 with a 1000-fold higher affinity than that seen with IL-2Ra and IL-2: Kd  10 pM and 10 nM, respectively (Anderson et al., 1995; Giri et al., 1995b). The murine and human IL-15Ra chains were subsequently cloned, characterized and reported to be

1

2

3

437

4 4a

5

6

7

IL-15 gene

8

Exons

5'UTR

3'UTR

1 2 3 4 5 6 7 LSP (48 aa)

IL-15 (114 aa)

N

C 3,4,5

cytoplasmic unglycosylated form

5'UTR

5,6,7,8 translocation into the ER

SSP (21 aa)

IL-15 (114 aa)

4a,5

5,6,7,8

8

C

N

translocation into the ER

N-glycosylation N-glycosylation rapid degradation by proteosomes

3'UTR

2 3 4 4a 5 6 7

8

intracellular (nuclear and cytoplasmic components)

IL-15 precursor isoforms

IL-15 mRNA proteins IL-15 intercellular microcirculation

alternative Signal cleavage peptide is (19 aa peptide) fully processed secretion

FIGURE 18.2 Structure and distribution of IL-15Ra. highly homologous to their IL-2Ra counterparts (Figure 18.2). The full-length human IL-15Ra is a type I transmembrane protein with a signal peptide of 32 amino acids, an extracellular domain of 173 amino acids, a transmembrane part of 21 amino acids, a 37-amino acid cytoplasmic tail and several N- and O-linked glycosylation sites (Dubois et al., 1999). Unlike IL-2Rb and IL-2Rc, IL-2R and IL-15Ra chains are not members of the hemopoietin receptor family, but instead contain in their extracellular parts conserved domains called the ‘sushi domain’ (type I glycoprotein or GP-1 motif) and are defined as protein-binding motifs. IL-2Ra contains two such domains while IL-15Ra contains only one. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines, which form two disulfide bonds. These two disulfide bonds are essential to maintain the tertiary structure of the protein and are crucial for the binding and function of IL-15a chain (Wei et al., 2001). This unique structure of IL-2 and IL-15 receptors caused them to be placed in a new receptor family. The IL-15Ra and IL-2Ra genes have a similar intron–exon organization and are closely linked in both human and murine genomes. The identification of eight different IL-15Ra transcripts resulting from exon-splicing mechanisms within the IL-15Ra gene has recently been reported (Dubois et al., 1999). Two main classes of transcripts were distinguished that do or do not (D2 isoforms) contain the exon 2-coding

THE CYTOKINES AND CHEMOKINES

438

INTERLEUKIN - 15 AND 21

sequence. Both classes were expressed in numerous cell lines and tissues at comparable levels, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single Nglycosylation site as well as extensive O-glycosylation. Whereas IL-15Ra bound IL-15 with high affinity, D2IL-15Ra was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ra was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15/IL-15Ra complex at the nuclear level. In contrast, D2IL-15Ra was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain plays an important role in receptor posttranslational routing (Dubois et al., 1999). The distribution of expression of IL-15Ra is much wider than that of IL-2Ra, suggesting a broader range of cellular targets for IL-15 (Anderson et al., 1995b). Expression of all eight IL-15Ra isoforms was demonstrated in a wide variety of tissues, including PBMC, cell lines, liver, brain and intestine, and appears to be augmented in response to environmental/stress stimuli and infectious agents. In mast cells, however, IL-15 uses a distinct receptor system different from that used in T and NK cells. Although mast cells lack IL-2Rb and do not respond to IL-2, they proliferate in response to IL-15. Using transfectants of these cells with a cytoplasmic-truncated mutant of cc, it has been demonstrated that IL-15 signaling in mast cells does not involve cc. Mast cells express a distinct 60–65-kDa IL-15R molecule, designated IL-15RX (Tagaya et al., 1996b).

IL-15R: SIGNAL TRANSDUCTION Since IL-2 and IL-15 share signaling common chains (b and cc), they induce similar signaling pathways in various cell types. IL-15 induces tyr phosphorylation of the p75IL-2Rb and p64IL-2Rc subunits (Adunyah et al., 1997). IL-2/15Rb chain is associated with JAK1 and cc chain is physically and functionally associated with the JAK3 tyrosine kinase. These molecular pairs may be considered as the trigger of the signal-

ing cascades, inducing the activation of JAK1 and 3 upon heterodimerization with a cytokine-specific receptor component. JAK1, JAK3 and other tyrosine kinases, phosphorylate the receptor, thereby creating docking sites for signaling molecules (Demoulin and Renauld, 1998). Among them, phosphatidylinositol-3 kinase (PI)-3 kinase and downstream effectors play a central role in the signaling processes involved in proliferation and inhibition of apoptosis for every c chain-interacting cytokine. Other important mediators – signal transducers and activators of transcription (STAT) transcription factors – regulate the expression of specific genes that IL-2 and IL-15 activate. The current paradigm is that activated JAKs phosphorylate receptor subunits and subsequently proteins with Src homology 2 (SH2) or phosphotyrosine-binding domains bind the phosphorylated receptors allowing signal propagation. IL-2–IL-15 stimulation can lead to the activation of STAT3 and STAT5, as well as the PI-3kinase and the Ras/mitogenactivated protein kinase (MAPK) pathways (Johnston et al., 1995; Karnitz and Abraham, 1995). Mast cell IL15RX complex recruits JAK2 and STAT5, instead of JAK1/3 and STAT3/5 that are activated in T cells (Tagaya et al., 1996b). Interestingly, anti-IL-15 neutralizing monoclonal antibody treatment resulted in down-regulation of cc chain and disruption of cc/JAK3 interaction in normal myeloid cells but had no effect in leukemic progenitors (Carayol et al., 2000). SHP-2 (Src homology 2 domain containing phosphatase 2) has been previously identified as an important intermediate in IL-2-dependent MAPK activation (Gadina et al., 1998). Furthermore, it was demonstrated that Gab2, a recently identified adapter molecule, is the major SHP-2 and PI-3 kinase-associated 98 kDa protein in normal, IL-2- or IL-15-activated lymphocytes (Gadina et al., 2000). Phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2Rb chain and only IL-2 and IL-15, but not other cc cytokines induce Gab2 phosphorylation. Interestingly, Gab2 levels are regulated by T-cell activation, and resting T cells express little Gab2. Taken together, these results suggest that Gab2 may be a key substrate in IL-2 and IL-15 signaling that serves to couple a limited number of cytokine receptors to MAPK activation. Thus, Gab2 may be one additional molecule by which cytokines can achieve signaling specificity.

THE CYTOKINES AND CHEMOKINES

IL - 15: BIOLOGICAL ACTIVITIES

IL-15: BIOLOGICAL ACTIVITIES IL-15 shares many biologic functions with IL-2 but also exhibits unique effects. IL-15 has a broad spectrum of biologic activities on different types of cells and tissues due to a wide distribution of IL-15 receptor complexes (Table 18.2).

439

Regulation of lymphocyte development Cytokine receptor signaling plays an essential role in the early stages of lymphocyte development. Signals through various cytokine receptors including IL-15R are known to promote the expansion and survival of uncommitted progenitor cells as well as their

TABLE 18.2 Biological activities of IL-15 Effects

Notes

References

Activation of NK cells

Cytotoxicity Cytokine production IFNc, GM-CSF, TNFa

Carson et al., 1994

soluble and membrane bound TNFa TRAIL Chemokine production MIP-1a, MCP-1a, RANTES Survival Stimulation of T cells

Proliferation, including antigen-specific proliferation Cytokine production IFNc production in CD4 and CD8 T cells IL-4 Inducer of CC-, CXC- and C-type chemokines MMP-9 production Redistribution of adhesion molecules Up-regulation of IL-2Ra and down-regulation of IL-15Ra Stimulation of intestinal intraepithelial lymphocytes to proliferate and produce IFNc

Stimulation of B cells

Activation of neutrophils

Stimulation and proliferation of B cells activated with anti-IgM Stimulation of proliferation and immunoglobulin production by HIV-1-treated B cells Stimulation of leukemic B cells, but not resting normal B lymphocytes Increase in phagocytosis Protects from apoptosis antimicrobial function Chemokine production IL-8 synthesis and NFjB activation

THE CYTOKINES AND CHEMOKINES

Carson et al., 1994 Gosselin et al., 1999 Caron et al., 1999 Kayagaki et al., 1999 Bluman et al., 1996; Chang et al., 2000 Carson et al., 1997 Grabstein et al., 1994 Hasan et al., 2000 Bonig et al., 1999 Borger et al., 1999 Perera et al., 1999 Constantinescu et al., 2001 Nieto et al., 1996 Kumaki et al., 1996 Sancho et al., 1999 Hirose et al., 1998

Armitage et al., 1995 Kacani et al., 1999 Trentin et al., 1996 Girard et al., 1996 Musso et al., 1998 Cassatella and McDonald, 2000 McDonald et al., 1998

440

INTERLEUKIN - 15 AND 21

TABLE 18.2 Continued Effects

Notes

References

Regulation of dendritic cells

Protection from tumor-induced apoptosis Up-regulation of IL-15Ra expression Up-regulation of expression of the Bcl-2 family of proteins

Pirtskhalaishvili et al., 2000 Tourkova et al., 2002 Pirtskhalaishvili et al., 2000

Regulation of macrophages

Expression of IFNc Enhance in superoxide production and antifungal activity Induction of TNFa

Agostini et al., 1999 Vazquez et al., 1998

Stimulation of mast cells

Stimulation of proliferation Production of IL-4

Masuda et al., 2000 Masuda et al., 2001

Myoblast (mouse C2 skeletal myogenic cell line skeletal myogenic and primary fetal bovine cultures)

Stimulation of differentiated myocytes and muscle fibers to accumulate increased amounts of contractile proteins Regulation of muscle fiber hypertrophy

Quinn et al., 1997; Quinn et al., 1995

Effect on keratinocytes

Protection from apoptosis No effect on IL-6 and IL-8 secretion No effect on proliferation

Ruckert et al., 2000

Alleva et al., 1997

Fibroblasts

Murine fibroblast cell line L929

Bulfone-Pau et al., 1999

Intestinal epithelial cells

Down-regulation of IL-8 and MCP-1 production

Lugering et al., 1999

Human umbilical vein endothelial cells

IL-15 can regulate endothelial cell function and thereby facilitates entry of activated T lymphocytes into inflammatory sites

Estess et al., 1999 Oppenheimer-Marks et al., 1998

Tumor cells

Growth or viability factor for cutaneous T-cell lymphoma-derived cell lines Block of apoptosis and induction of proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells Stimulation of proliferation of peripheral blood leukemic cells from adult T-cell leukemia patients

Dobbeling et al., 1998

migration to the appropriate microenvironment and subsequent differentiation into B, T or NK cells (Baird et al., 1999). That the signal transduction pathways used by the cytokines IL-2 and IL-15 are identical would suggest that these cytokines have redundant roles in lymphoid development; instead, IL-2 is the guardian of thymus-derived T-cell homeostasis, while IL-15 promotes extrathymic development of T and NK cells (DiSanto, 1997). For instance, Leclercq et al. (1996) provided direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential

Hjorth-Hansen et al., 1999

Mori et al., 1998

T/NK progenitors: addition of low concentrations of IL-15 to fetal thymic organ culture resulted in an increase of both TCRab and TCRcd T cell subpopulations; in contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCRcd cells. High concentrations of IL-15 blocked TCRab development and shifted differentiation towards NK cells, whereas high concentrations of IL-2 similarly induced development into NK cells, but the cell number was four-fold lower than that observed in IL-15 cultures.

THE CYTOKINES AND CHEMOKINES

IL - 15: BIOLOGICAL ACTIVITIES

Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2Rcc do not. Further, treatment of mice with an antibody directed against IL-2Rb results in a loss of virtually all of the NK-cell compartment. These data further confirm that a cytokine other than IL-2, namely IL-15, is important for NK-cell development and survival in vivo (Carson et al., 1997). Thus, IL-15 is required for NK-cell differentiation and, in addition, stimulates expression of at least one of the MHC-specific inhibitory receptor families expressed by NK cells (Raulet, 1999). Interestingly, defects in IL-2R–1L-15Rb expression can lead to a unique NK-deficient SCID immunophenotype: an infant boy had a significantly reduced expression of the IL-2R–IL-15Rb chain in his peripheral blood mononuclear cells (PBMC), typical clinical features of SCID and was found to lack NK cells in his peripheral circulation (Gilmour et al., 2001). Elutriated human CD34 precursor cells, grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15, differentiated into NK cells expressing both CD56 and CD7. These in vitrodifferentiated CD56 NK cells displayed cytolytic activity against the HLA class I target K562 (Carayol et al., 1998). In addition, IL-15 induced the proliferation and maturation of highly positive CD56 NK cells. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15. Interestingly, IL-15 and SCF fail to induce NK differentiation and proliferation of CD34 hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although both normal and leukemic CD34 cells display comparable expression of the c-kit or IL-15 receptor subunits (Carayol et al., 2000). The authors also demonstrated the existence in both normal and leukemic CD34 cells of constitutive production of bioactive IL-15 that does not lead to NK differentiation. However, only leukemic progenitors express the membrane-bound IL-15, which might serve as a hallmark of leukemic progenitors. Interestingly, the development of dendritic cells from human CD34 hematopoietic precursors cultured for 2–4 weeks with IL-15 alone has recently been reported (Bykovskaia et al., 1999). Dendritic cells generated with IL-15 have typical morphologic, immunocytochemical, phenotypic, and functional characteristics of mature dendritic cells. These results

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were later confirmed by others demonstrating that monocytes cultured for 6 days with GM-CSF and IL-15 differentiate into CD1aHLA-DRCD14 dendritic cells and agents such as LPS, TNFa, and CD40L induce maturation of these dendritic cells to CD83 cells (Mohamadzadeh et al., 2001).

Regulation of NK cells The cytotoxic activity of NK cells can be modulated by a variety of cytokines, including IL-12, IFNc, and IL-15. For instance, Gosselin et al. (1999) have studied the potential of IL-15 to act as a modulator of NK cellmediated antiviral defense and indicated that IL-15 can curtail infections by three human herpesviruses: Herpes simplex virus type 1, Epstein–Barr virus and human herpesvirus 6. The antiviral activity of IL-15 was dose-, time- and NK cell-dependent. IL-15treated NK cells showed an increased killing potential against a variety of cells, including virus-infected target cells. Overnight treatment of freshly isolated NK cells with IL-15 augmented their binding to cultured endothelial cells in vitro, especially to resting endothelial cells (Allavena et al., 1997). IL-15activated NK cells bound to resting and TNF-activated endothelial cells by use of LFA-1/ICAM-1 and VLA-4/ VCAM-1 adhesion pathways. Other in vitro studies indicate that monocyte-derived IL-15 may be an important determinant of IFNc production by NK cells (Carson and Caligiuri, 1998). IL-15-triggered synthesis of IFNc from NK cells could act in both autocrine and paracrine fashion to modulate NK cytolytic potential. Stimulation with IL-15 upregulates TNFa mRNA, soluble TNFa and membraneassociated TNFa expression in NK cells suggesting several mechanisms of NK-cell activation and increased cytotoxicity induced by IL-15 (Caron et al., 1999). IL-15-stimulated NK cells have also been shown to produce a variety of other cytokines and chemokines, including macrophage inflammatory protein-1a (MIP-1a) (Bluman et al., 1996), macrophage chemotactic protein-1a (MCP-1a) (Chang et al., 2000a), IFNc (Gosselin et al., 1999) and TRAIL (Kayagaki et al., 1999). In addition, IL-15 plays an important role not only in NK-cell development and activation (Mrozek et al., 1996) but also in NK-cell survival. For instance, Carson et al. (1997) have demonstrate that resting human NK cells express IL-15Ra

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mRNA, and, furthermore, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 days in the absence of serum. Taken together, these results suggest that the IL-15 response of the host to viral infection and the subsequent NK-cell activation represent an important effector mechanism of the innate immune surveillance of the host against viral infections (Fawaz et al., 1999). In conclusion, IL-15 is both a growth, differentiation, activation and survival factor for NK cells and, thus, can contribute to the establishment of an antiviral state in three ways: first by increasing the cytolytic activity of NK cells, second by stimulating the synthesis and secretion of IFNc, and third by prolongation of NK cell survival.

Regulation of T lymphocytes IL-15 is a potent T-cell stimulating factor which has been demonstrated to have various effects on T cells. Jonuleit et al. (1997) tested DC supernatants for chemokinetic and chemotactic activities for T cells in a checkerboard filter assay and reported that this activity was blocked almost completely by addition of an anti-IL-15 antibody. Interestingly, protein release was triggered by phagocytic activity. Thus, DCderived IL-15 has chemotactic and chemokinetic activities for T cells, suggesting a role for IL-15 as an attractant of T cells during the initial DC–T cell interaction. Extending these observations, Perera et al. (1999) have shown that IL-15 is a potent inducer of CC-, CXC- and C-type chemokines in T lymphocytes. In addition, they demonstrated that IL-15 induces CC chemokine receptors, but not CXC chemokine receptors, in a dose-dependent manner. IL-15 also induces IFNc production by T cells (Bonig et al., 1999). Furthermore, DC-derived IL-15 induces T cells with augmented antigen-specific lysis, and increases the yield of antigen-specific IFNc-producing T cells (Kuniyoshi et al., 1999). IL-15 induced IFNc synthesis in both CD4 and CD8 T cells while IL-4 production was increased only in CD4 T cells (Borger et al., 1999). Together, these findings suggest that the proinflammatory effects of IL-15, at least in part, may be due to the induction of chemokines and their receptors in T cells. While naive T cells appear to require constant activation of TCR by self antigens for homeostasis, mem-

ory T cells have no such requirement. The numbers of CD8 memory T cells in animals are controlled by a balance between IL-15 and IL-2 since the division of memory CD8 T cells required IL-15 and was markedly increased by inhibition of IL-2 (Ku et al., 2000). Furthermore, Zhang et al. (1998) have recently shown that, unlike IL-2, IL-15 closely mimics the effects of type I IFN in causing strong and selective stimulation of memory phenotype CD44hiCD8 (but not CD4) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rb on CD8 cells than on CD4 cells. Furthermore, IL-15, like IL-2, has been demonstrated to up-regulate expression of IL-2Ra on human T and B cells, but rapidly down-regulates IL-15Ra (Kumaki et al., 1996b). This leads to decreased responsiveness to IL-15 as measured by induction of JAK3 tyrosine phosphorylation, and suggests a mechanism by which IL-15 may co-operate with IL-2 at the initiation of an immune response and enhance subsequent IL-2 responsiveness during T-cell expansion. IL-2 induces or inhibits T-cell apoptosis in vitro, depending on T-cell activation, whereas IL-15 inhibits cytokine deprivation-induced apoptosis in activated T cells (Waldmann et al., 2001). These and other observations suggest that both cytokines can differentially regulate T cells, e.g. T-cell functions relevant to the control of cell cycle progression and apoptosis, and/or that they can stimulate different T-cell subsets (Bulfone-Paus et al., 1997a). For instance, apoptosis induced by anti-Fas, anti-CD3, dexamethasone and/ or anti-IgM in activated human T and B cells in vitro can be inhibited by IL-15 (Bulfone-Paus et al., 1997a). Furthermore, the recent data assign a novel function to the previously reported antiapoptotic activity of IL-15, namely, the capacity to redirect the T-cell response to partial stimulation from clonal deletion to anergy (Dooms et al., 2000). In fact, differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, Li et al. (2001) have demonstrated that IL-15 is a critical growth factor in initiating T-cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via down-regulation of the cc expression. Decreased cc expression on cycling T cells reduced

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sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Thus, these data documented that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo. IL-15 also facilitates the growth of epithelial cd T cells (Edelbaum et al., 1995) and proliferation and survival of cd T cells in antigen-stimulated cultures of PBMC (Elloso et al., 1996, 1998). IL-15 has been further shown to activate proliferation and cytokine production in intraepithelial lymphocytes, which resemble memory cells and are located next to epithelial cells that produce IL-15 (Ebert, 1998). Interestingly, Chu et al. (1999) found that both IL-15 and IL-2 supported growth of the restimulated cd T cells, but exerted different effects on their survival; quantitation of apoptotic cells showed more cell death in the IL-2 group than in the IL-15 group. Cell death was associated with diminished levels of Bcl-xL protein over time in the IL-2 group whereas the level was sustained in the IL-15-treated group.

Regulation of B lymphocytes IL-15 regulates both proliferation and antibody production by activated B cells. For instance, IL-15 costimulates proliferation of B cells activated with IgM or phorbol ester, but has no stimulatory effect on resting B cells (Armitage et al., 1995). In combination with recombinant CD40L, IL-15 induced polyclonal IgM, IgG1 and IgA secretion, but did not cause production of IgG4 or IgE. Similar results were obtained by Trentin et al. (1996), demonstrating that IL-15 stimulated the proliferation of freshly isolated leukemic B cells, but not resting normal B lymphocytes; the latter being able to grow in the presence of IL-15 only after in vitro preactivation with phorbol myristate acetate. In addition, IL-15 has been reported to stimulate proliferation and immunoglobulin production by B cells co-incubated with inactivated HIV-1 (Kacani et al., 1999).

Effect on dendritic cells and macrophages Activation of dendritic cells and macrophages by infectious agents results in secretion of IL-12, which subsequently induces IFNc synthesis by different types of cells including dendritic cells and macro-

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phages. In turn, IFNc affects macrophages to increase IL-12 secretion and to produce nitric oxide (NO), which eradicates infection agents. It has recently been shown that in IL-15-deficient mice but not in IL-2deficient mice, production of IL-12, IFNc and NO by dendritic cells and macrophages was severely impaired (Ohteki et al., 2001). This shows that the IL-15–IL-15R interaction is critical in early activation of antigen-presenting cells and plays an important role in the innate immune system. IL-15 itself was able to activate dendritic cells, as in vivo or in vitro exposure of splenic dendritic cells to IL-15 resulted in an up-regulation of co-stimulatory molecules, markedly increased production of IFNc by dendritic cells and an enhanced ability of dendritic cells to stimulate Ag-specific CD8 T cell proliferation (Mattei et al., 2001). Although human alveolar macrophages express the IL-15 binding proteins (IL-15Ra and cc) at resting conditions, they do not express IL-15 mRNA. However, a 24 hour stimulation with IL-15 induces the expression of IFNc and IL-15 itself, suggesting a role for this cytokine in the activation of the pulmonary macrophage pool during inflammation (Agostini et al., 1999). Interestingly, whereas high IL-15 concentrations enhanced proinflammatory (i.e. TNFa, IL-1 and IL-6) and antiinflammatory (i.e. IL-10) cytokine production by two- to six-fold, extremely low IL-15 concentrations (picomolar to attomolar range) markedly and selectively suppressed proinflammatory, but not antiinflammatory, cytokine production two- to four-fold in macrophages (Alleva et al., 1997). In addition, Varquez et al. (1998) have reported the ability of IL-15 to enhance superoxide production and antifungal activity of human monocytes: After 18 and 48 hours of treatment with IL-15, human elutriated monocytes manifested enhanced superoxide production in response to either phorbol myristate acetate or opsonized Candida albicans blastoconidia. Additionally, human monocytes showed enhanced killing activity against C. albicans after 18 hours of incubation with IL-15, but this treatment did not enhance the ability of these cells to phagocytose the organism. We have recently demonstrated that stimulation of dendritic cells with IL-15 prior to their co-incubation with prostate cancer cells resulted in a significant increase in dendritic cell survival in the tumor microenvironment (Pirtskhalaishvili et al., 2000).

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Furthermore, activation of dendritic cells with these cytokines was also accompanied by increased expression of the antiapoptotic protein Bcl-xL in dendritic cells, suggesting a possible mechanism involved in dendritic cell protection from apoptotic death. We have also shown that transduction of human dendritic cells with the IL-15 gene increases their resistance to prostate cancer-induced apoptosis (Tourkova et al., 2002). Protection of IL-15-transfected human dendritic cells from prostate cancer-induced apoptosis was associated with maintenance of expression of the antiapoptotic protein Bcl-2. Interestingly, this effect was probably mediated by an autocrine upregulation of expression of IL-15Ra, since dendritic cells transduced with the IL-15 gene express a significantly higher level of IL-15Ra compared with nontransfected or control-transfected dendritic cells and the up-regulation of IL-15Ra expression on dendritic cells was abrogated by the addition of anti-IL-15 neutralizing antibody (Tourkova et al., 2002). Interestingly, autocrine/paracrine effect of IL-15 on IL-15Ra expression on dendritic cells was opposite to its effect on T cells or lymphocytes suggesting the involvement of different intracellular mechanisms of regulation of IL-15Ra chain in different immune cells.

Effect on neutrophils IL-15 was initially observed to induce cytoskeletal rearrangements, enhance phagocytosis, increase the synthesis of several cellular proteins, and delay apoptosis in neutrophils. For instance, Girard et al. (1996) have observed that IL-15, in contrast to IL-2, after addition to neutrophils induces important morphological cell shape changes that are typical of activated neutrophils. Furthermore, phagocytosis of opsonized sheep red blood cells was significantly increased by IL-15 but not by IL-2. However, similar to IL-2, IL-15 did not modulate the oxidative burst response. Finally, the authors found that IL-15 delayed apoptosis of neutrophils more efficiently than IL-2 when evaluated by both microscopic observations and flow cytometry procedures. Recently, IL-15 has been found to elicit other functional responses in neutrophils such as chemokine production (Cassatella and McDonald, 2000). Neutrophils synthesize and release IL-8 in response to IL-15, but not to IL-2 (McDonald et al., 1998). More-

over, a nuclear factor jB (NFjB) DNA-binding activity was enhanced in nuclear extracts of IL-15-treated neutrophils, which further emphasizes the potential relevance of this cytokine to immune and inflammatory processes.

Regulation of mast cells It has been reported that IL-15 uses a distinct receptor system in mast cells. Although mast cells lack IL-2Rb and do not respond to IL-2, they proliferate in response to IL-15 and produce functionally active IL-4 (Masuda et al., 2000). In addition, it has been demonstrated that IL-15 prevents mouse mast cell line MC/9 and bone marrow-derived mast cell apoptosis induced by growth factor withdrawal or anti-Fas antibody treatment. IL-15 increased mRNA and protein levels of an antiapoptotic protein Bcl-xL in these cells, which was mediated by STAT6 (Masuda et al., 2001). Thus, IL-15 may play an important role in some allergic diseases by increasing mast cell numbers and inducing their IL-4 secretion. Asthma, in which mast cells and TH2 cells are dominant, may be one of them. However, more studies are required to understand the functional significance of IL-15RX-mediated effects in mast cells since no developmental defects of mast cells were reported in IL-15 knockout mice (Kennedy et al., 2000).

Effect on non-immune cells Cultured keratinocytes stimulated with IL-15 significantly increased their intracellular as well as cell surface-bound FasL expression in a time- and dosedependent manner (Arnold et al., 1999). Moreover, enhanced FasL expression on stimulated keratinocytes induced apoptosis in co-cultured Fas T cells, demonstrating that up-regulated FasL was functionally active (Arnold et al., 1999). Thus, these results demonstrate the important regulatory role of IL-15controlled Fas–FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes. In addition, since human keratinocytes both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Ra protein on the cell surface, it is likely that they can employ IL-15 for juxtacrine

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signaling (Ruckert et al., 2000). While IL-15 exerted no significant effect on keratinocyte proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced keratinocyte apoptosis in vitro. Unlike IL-2, IL-15 also binds to endothelial cells with high affinity since endothelial cells have been found to express the IL-15Ra chain and the IL-2/15Rb and common c chains (Angiolillo et al., 1997). The expression of hyaluronan on primary endothelial cells and microvascular endothelial cell lines is induced by IL-15, whereas IL-2 has no such activity (Estess et al., 1999). Moreover, intraperitoneal administration of IL-15 in the absence of other exogenous proinflammatory stimuli allows extravasation of superantigenstimulated T cells into this site in vivo in a CD44-dependent manner. The results suggest that IL-15 can regulate endothelial cell function and thereby enables a CD44-initiated adhesion pathway that facilitates entry of activated T lymphocytes into inflammatory sites. IL-15 can also perform a novel activity to stimulate the differentiation of osteoclast progenitors into preosteoclasts, which cannot be replaced by IL-2 but may use components in common with IL-2R to mediate its effects (Ogata et al., 1999).

IL-15 and skeletal muscles Quinn et al. (1995) have proposed a potentially important role for IL-15 in skeletal muscle. Indeed, IL-15 can stimulate differentiated myocytes and muscle fibers to accumulate increased amounts of contractile proteins (Quinn et al., 1995). In addition, IL-15 stimulates muscle-specific myosin heavy chain accumulation by differentiated myocytes and muscle fibers in culture and mouse skeletal myoblast differentiation, suggesting that IL-15 may play a role in skeletal muscle fiber growth in vivo (Quinn et al., 1997). However, in vivo administration of IL-15 did not alter muscle mass or muscle protein content, and resulted in a significant alteration of both muscle protein synthesis and degradation (Carbo et al., 2001). Recent data of Carbo et al. (2000) give new insights into the mechanisms by which IL-15 prevents muscle protein wasting in cancer cachexia. It has been demonstrated that IL-15 treatment partly inhibits skeletal muscle wasting in a rat ascites hepatoma

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model decreasing protein degradative rates to values even lower than those observed in non-tumorbearing animals. These alterations in protein breakdown rates were associated with an inhibition of the ATP–ubiquitin-dependent proteolytic pathway (Carbo et al., 2000). Thus, these data suggest the use of IL-15 in the treatment of pathological states characterized by tissue protein hypercatabolism, particularly in skeletal muscle such as cancer cachexia.

In vivo effects of IL-15 Investigating the differences between IL-2 and IL-15 in pharmacokinetics and biodistribution, Kobayashi et al. (2000) found that only IL-2 showed specific binding to a2-macroglobulin, which may be the reason that IL-2 displays a delayed blood clearance when compared with IL-15. Upon injection of these cytokines into mice, they observed that IL-15 accumulated significantly more than IL-2 in kidney, spleen and bone, the tissues that express IL-15Ra but not IL-2Ra. While IL-15 did not cause changes in either muscle mass or muscle protein content, it induced significant changes in the fractional rates of both muscle protein synthesis and degradation, with no net changes in protein accumulation when injected in vivo in rats. Additionally, IL-15 administration resulted in a 33% decrease in white adipose tissue mass and a 20% decrease in circulating triacylglycerols; this was associated with a 47% lower hepatic lipogenic rate and a 36% lower plasma VLDL triacylglycerol content. The decrease in white fat induced by IL-15 was in adipose tissue. No changes were observed in the rate of lipolysis as a result of cytokine administration. These findings indicate that IL-15 has significant effects on both protein and lipid metabolism, and suggest that this cytokine may participate in reciprocal regulation of muscle and adipose tissue mass (Carbo et al., 2001). Since IL-15 displayed better target-specific accumulation and more rapid clearance from the circulation than did IL-2, it can be considered to be a novel and unique therapeutic agent. For instance, in vivo, anti-Fas-induced lethal multisystem apoptosis in mice was suppressed by an IL-15–IgG2b fusion protein (Bulfone-Paus et al., 1997b). Only IL-15, but not IL-2, offered complete protected from lethal hepatic failure (Bulfone-Paus et al., 1997b). A rat model with

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dose-limiting toxicity profiles that are similar to those observed in patients treated with Irinotecan (CPT-11, a chemotherapeutic agent that is active in the treatment of a variety of solid tumor malignancies), was used to evaluate the role of IL-15 in the modulation of the therapeutic selectivity of CPT-11 in normal rats and rats bearing advanced colorectal cancer (Cao et al., 1998). IL-15 offered complete and sustained selective protection against CPT-11-induced delayed diarrhea and lethality. IL-15 also moderately potentiated the antitumor activity of CPT-11 in rats bearing advanced colorectal cancer. In addition, in vivo administration of IL-15 augments anti-toxoid IgG (IgG1, IgG2, IgG2b) responses well above (up to 10-fold) those achieved with liposomal toxoid alone (Gursel and Gregoriadis, 1997). It has also been reported that IL-15 is a stimulator of angiogenesis in vivo; when injected s.c. into nude mice, IL-15 consistently induced neovascularization.

IL-15 TRANSGENIC AND KNOCKOUT ANIMALS As mentioned above, IL-15 is required for lymphocyte homeostasis and the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms. Recently, a transgenic mouse overexpressing IL-15 by eliminating the posttranscriptional checkpoints was engineered and characterized (Fehniger et al., 2001). IL-15 transgenic mice have early expansions of NK and CD8 T lymphocytes. Later, these mice develop fatal lymphocytic leukemia with a T–NK phenotype. Interestingly, these data provide additional evidence that leukemia, like other cancers, can arise as the result of chronic stimulation by a proinflammatory cytokine (Fehniger et al., 2001). Since at least two types of IL-15 mRNA isoforms are generated by alternative splicing, Nishimura et al. (2000) constructed two groups of transgenic mice, using originally described IL-15 cDNA with a normal exon 5 (normal IL-15 transgenic mice) and IL-15 cDNA with an alternative exon 5 (alternative IL-15 transgenic mice). Normal IL-15 transgenic mice constitutionally produced a significant level of IL-15 protein and had markedly increased numbers of memory-type CD44hi or CD8 T cells. These mice showed resistance to Salmonella infection accompa-

nied by enhanced IFNc production. On the other hand, a large amount of intracellular IL-15 protein was detected but only minimal secreted cytokine in alternative IL-15 transgenic mice. Although most of the T cells developed normally in the alternative IL-15 transgenic mice, they showed impaired IFNc production upon TCR engagement. The alternative IL-15 transgenic mice were susceptible to Salmonella accompanied by impaired production of endogenous IL-15 and IFNc (Nishimura et al., 2000). Thus, two groups of IL-15 transgenic mice may provide information concerning the different roles of IL-15 isoforms in the immune system in vivo. For instance, Ishimitsu et al. (2001) found that eosinophilia and TH2-type cytokine production in the airways were severely attenuated in OVA-sensitized IL-15 transgenic mice following OVA inhalation. IL-15 transgenic mice preferentially developed TC1 responses mediated by CD8 T cells after OVA sensitization, and in vivo depletion of CD8 T cells by anti-CD8 antibody aggravated the allergic airway inflammation in IL-15 transgenic mice following OVA inhalation. Interestingly, the adoptive transfer of CD8 T cells from OVA-sensitized IL-15 transgenic mice into normal mice before OVA sensitization suppressed TH2 response to OVA in the normal mice, suggesting that overexpression of IL-15 in vivo suppresses TH2-mediated allergic airway response via induction of CD8 T cell-mediated TC1 response (Ishimitsu et al., 2001). Mice genetically deficient in IL-15 (IL-15/ mice) were also recently generated using gene-targeted oblation (Kennedy et al., 2000). IL-15/ mice displayed marked reductions in numbers of thymic and peripheral NK–T cells, memory phenotype CD8 T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes. The reduction but not the absence of these populations in IL-15/ mice probably reflects an important role for IL-15 for expansion and survival of these cells. IL-15/ mice lack NK cells, again indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Importantly, specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15, suggesting that IL-15 administration represents a powerful means by which to elucidate further the biologic roles of this cytokine. Despite their immunological defects, IL-15/ mice remained healthy when maintained under specific pathogen-free conditions.

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However, IL-15/ mice are likely to have compromised host defense responses to various pathogens, as they are unable to mount a protective response to challenge with vaccinia virus (Kennedy et al., 2000). These data confirm the critical role of IL-15 in the development of specific lymphoid lineages. Finally, IL-15Ra null (IL-15Ra/) mice have recently been generated in an effort to understand the role of IL-15Ra in immune development and function (Lodolce et al., 1998). IL-15Ra/ mice are markedly lymphopenic despite largely normal T- and Blymphocyte development. This lymphopenia is due to decreased proliferation and decreased homing of IL-15Ra/ lymphocytes to peripheral lymph nodes. In addition, these mice are also deficient in NK cells, NK–T cells, CD8 T lymphocytes and TCRcd intraepithelial lymphocytes. Furthermore, memory phenotype CD8 T cells are selectively reduced in number (Lodolce et al., 1998). Thus, IL-15Ra has pleiotropic roles in immune development and function, including the positive maintenance of lymphocyte homeostasis. In fact, it has recently been reported that Syk kinase physically and functionally associates with the IL-15Ra chain in B cells and upon association, the activated Syk kinase phosphorylates the IL-15Ra chain as well as phospholipase Cc and rescues B cells from C2 ceramide-induced apoptosis (Bulanova et al., 2001). It has also been shown that IL-15 signals in several tumor cells lines which express no or only marginal levels of IL-2R b and c chains (Stevens et al., 1997; Bulfone-Paus et al., 1999). Together, these results suggest that in spite of general opinion, IL-15Ra chain can transduce a signal even in the absence of the b and/or c chains.

IL-15 AND DISEASES Autoimmune diseases and inflammation The serum levels of IL-15 correlate with the titer of autoantibody in patients with pemphigus vulgaris, a rare dermatosis of autoimmune origin (Ameglio et al., 1999). Interestingly, increased serum levels of IL-15 were detected in other dermatoses with an altered immune response, including bullous pemphigoid, pemphigus erythematosus, psoriases and systemic

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lupus erythematosus (D’Auria et al., 1999). It is likely that increase in IL-15 is non-specific in dermatological diseases and that its pleiotropic effects might be in agreement with some features of these different pathologies (D’Auria et al., 1999). In fact, it has been recently reported that IL-15 protects human keratinocytes from apoptotic death (Lindner et al., 1998; Ruckert et al., 2000). Together with the role of IL-15 in sustaining chronic immune reactions, this raises the question of whether a reduction of keratinocyte apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low keratinocyte apoptosis in the epidermis. Importantly, compared with non-lesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15 (Ruckert et al., 2000). Therefore, antagonizing the inhibitory effects of IL-15 on keratinocyte apoptosis may deserve evaluation as a novel therapeutic strategy in psoriasis. Further studies need to be conducted to clarify the involvement of IL-15 in the pathogenesis of different skin diseases with autoimmune compound. Evaluating patients with atopic eczema, which is characterized by persistence of infiltrating T lymphocytes in the dermis, Orteu et al. (2000) demonstrated that dysregulation of normal T-cell apoptosis may contribute to the pathogenesis and chronicity of atopic eczema. The authors speculated that the expression of IL-15 in cutaneous lesions may contribute to excessive T-cell survival which leads to the persistence of inflammation in patients with atopic eczema. IL-5 plasma levels were also significantly elevated in patients with an acquired aplastic anemia, a disorder with a stem cell defect and a few immune abnormalities (Dirksen et al., 1998). Clinical features of active rheumatoid arthritis such as joint swelling and pain may be caused by an increased level of IL-15. It has been shown that IL-15 is present in the synovium of patients with rheumatoid arthritis and may recruit and activate synovial T cells in the relative absence of IL-2 (McInnes et al., 1996; Thurkow et al., 1997). Importantly, synovial T lymphocytes produce TNFa upon stimulation with IL-15 and induce TNFa production by macrophages, suggesting an important role of IL-15 for regulation of inflammation within the synovium (McInnes et al.,

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1997; Sebbag et al., 1997). Freshly isolated synovial T cells did not express significant levels of CD154, whereas IL-15 significantly increased the expression of CD154 on synovial and peripheral blood T cells from patients. IL-2 had minimal effects (Mottonen et al., 2000). Furthermore, IL-15 induced extensive proliferation in synovial T cells. Additional studies indicate that fibroblast-like synoviocytes are an important source of IL-15 in rheumatoid arthritis joints and confirm that IL-15 may be responsible for the local T-cell activation and expansion in the presence of deficient IL-2 production by T cells (Harada et al., 1999). On other hand, the production of IL-15 and tissue-degrading enzymes in rheumatoid synovitis is T-cell dependent, since elimination of tissueinfiltrating T cells in the synovial grafts in NOD-SCID mice engrafted with rheumatoid synovial tissue resulted in a marked decline in the production of IL-15, MMP-1 and MMP-2 mRNA (Klimiuk et al., 1999). It is also possible that IL-15 mediates its effects in inflammation in part through MMP-9. In fact, IL-15, but not IL-12, induces MMP-9 in PBMC and in T cells (Constantinescu et al., 2001). In systemic lupus erythematosus (SLE), dysregulation of apoptosis and various cytokines have been observed and implicated in the pathogenesis of the disease. Serum IL-15 levels in 20 SLE patients were determined in a study of Park et al. (1999) to assess the relationship between IL-15 and disease activity. The IL-15 levels in SLE patients were significantly higher than those of controls (5.38 ± 4.89 versus 1.04 ± 1.26 pg ml1). IL-15 levels did not correlate with the disease activity of SLE. Interestingly, in vitro IL-15 led to a significant increase in Bcl-2 and a reduction in PBMC apoptosis rates, which was even more pronounced in SLE patients when compared with healthy volunteers (Graninger et al., 2000). Antiapoptotic cytokine signaling may thus significantly influence the deregulation of cell death in SLE lymphocytes. Moreover, immunohistochemical analyses revealed membrane IL-15 on dermal fibroblasts in discoid lupus erythematosus skin lesions whereas no membrane IL-15 was found on the surface of fibroblasts in healthy skin (Rappl et al., 2001). Pashenkov et al. (1999) have analyzed spontaneous expression of IL-15 by PBMC from patients with multiple sclerosis (MS) and demonstrated that IL-15positive PBMC were elevated in patients with MS

when compared with healthy controls. The elevation of IL-15-expressing PBMC was restricted to patients with chronic progressive MS and was not observed in patients studied during the relapsing–remitting phase of MS. The numbers of IL-15-expressing PBMNC correlated with the duration and disability of MS suggesting that IL-15 expression by PBMC is upregulated in the chronic stage of MS. Similar data indicating that IL-15 mRNA expression is upregulated in MS were also obtained by Kivisakk et al. (1998) further suggesting a role for proinflammatory cytokines in the pathogenesis of MS. Studying duodenal biopsy specimens from celiac patients cultured in vitro for 24 h with or without IL-15, Maiuri et al. (2000) have evaluated epithelial expression of Ki67, FAS and transferrin receptor by immunohistochemistry and apoptosis by TUNEL technique and concluded that IL-15 is involved in the modulation of epithelial changes in celiac disease. Finally, expression of IL-15 has been reported for a variety of other chronic inflammatory diseases, including inflammatory bowel diseases, sarcoidosis, ulcerative colitis and chronic active hepatitis, suggesting that IL-15-mediated regulation of inflammation may be of more general importance (Agostini et al., 1996; Kirman and Nielsen, 1996; Kakumu et al., 1997; Kirman et al., 1998; Sakai et al., 1998). For instance, a dysregulated local immune reaction with unbalanced cytokine expression seems essential in inflammatory bowel disease (IBD), i.e. ulcerative colitis (UC) and Crohn disease (CD). Evaluating colonic biopsies from 24 UC, 18 CD and 12 controls for IL-15 expression, Vainer et al. (2000) demonstrated that IL-15 concentrations were higher in CD patients (34 pg mg1 tissue; 24–53) when compared with controls (20 pg mg1 tissue; 15–21; P  0.001) while being 22 pg mg1 tissue (15–32) in UC. Similarly, isolated lamina propria mononuclear cells from IBD patients but not from controls produced IL-15 when stimulated with LPS or IFNc (Liu et al., 2000). Moreover, lamina propria T cells from IBD patients were more responsive to IL-15 when compared with controls. IL-15 alone without a primary T-cell stimulus induced IFNc and TNF production by isolated IBD T cells, especially by T cells from patients with Crohn disease (Liu et al., 2000). Together, these data indicate that IL-15 is overexpressed in the inflamed mucosa in IBD and that IL-15 enhances local T cell activation, proliferation

THE CYTOKINES AND CHEMOKINES

IL - 15 AND DISEASES

and proinflammatory cytokine production by both T cells and macrophages. It is possible that treatment directed against IL-15 may have therapeutic potential in IBD (Liu et al., 2000).

Cancer Since IL-15 induces proliferation and promotes cell survival of human T and B lymphocytes, NK cells and neutrophils, it is possible that IL-15 may also play a role in some hematological malignancies. In fact, Tinhofer et al. (2000) have reported the constitutive expression of a functional IL-15R in six of six myeloma cell lines and in CD38hiCD45lo plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, IL-15 transcripts was detected in all six myeloma cell lines, and IL-15 protein in four of six cell lines and also in the primary plasma cells of eight of 14 multiple myeloma patients. Importantly, blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis (Tinhofer et al., 2000). These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner. Furthermore, leukemic cells from patients with chronic B-cell malignancies, including B-cell chronic lymphocytic leukemia and hairy cell leukemia, have been shown to proliferate in response to IL-15 regardless of in vitro preactivation, in contrast with normal B cells which required preactivation with polyclonal mitogens or other stimuli (Trentin et al., 1997). This peculiar IL-15 responsiveness distinguishes malignant B cells from normal B lymphocytes. The findings that IL-15 mRNA can be detected in Sézary syndrome PBMC and that the IL-15 protein is detected in skin sections from cutaneous T-cell lymphoma (CTCL) patients suggest that IL-15 may play an important role in the biology of CTCL (Dobbeling et al., 1998). Similarly, Leroy et al. (2001) reported differences in IL-15 protein expression between normal human skin, atopic dermatitis and psoriasis on the one hand, and parapsoriasis and CTCL on the other. IL-15 protein expression was not detected in normal human skin, atopic dermatitis or psoriasis, but was detected, at low levels (and in a few patients at higher levels), in epidermal keratinocytes in parapsoriasis,

449

mycosis fungoides and Sézary syndrome. Thus, induction of keratinocyte IL-15 expression appears to be a feature of CTCL. Remarkably, there was some evidence for a stage-dependent increase during mycosis fungoides progression; slight overexpression in early stage MF, when only few tumor cells are detectable within the infiltrates, whereas marked overexpression was found in more advanced lesions, which are characterized by a higher density of malignant cells (Asadullah et al., 2000). Considering the significant overexpression of IL-15 and its biological capacities it is likely that this cytokine contributes to tumor development and might be involved in growth and skin homing of CTCL cells. A potential role for IL-15 in HTLV-I-associated diseases such as adult T-cell leukemia has also been suggested (Azimi et al., 1998). In fact, adult T-cell leukemia cells have been shown constitutively to express the complete form of IL-15R including the a chain, and that these cells proliferate in response to exogenous IL-15 (Yamada and Kamihira, 1999). Since the mRNA of IL-15 is ubiquitous and is detected in many tissues and cells, it is possible that IL-15R stimulation is involved in the development and progression of adult T-cell leukemia. Similarly, it has been shown that IL-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 as well as freshly isolated myeloma cells (Hjorth-Hansen et al., 1999). The possible involvement of IL-15 in nonhematologic malignancies has not been systematically evaluated as yet. Since a number of different tumor cell lines has been shown to produce IL-15, it is likely that IL-15 might be an autocrine growth factor for some tumor cells. However, Lissoni et al. (1998) studying 40 patients with solid tumors, 24 of whom had metastatic disease, reported no significant difference in serum levels of IL-15 between cancer patients and controls. Moreover, the serum levels of IL-15 found in metastatic cancer patients were not significantly different from those found in patients with limited disease, suggesting that IL-15 secretion is substantially within the normal range in cancer patients, both in early and advanced disease. It is also possible that monocytes obtained from cancer patients produced lower levels of IL-15 contributing to immunosuppression of cancer patients (Merendino et al., 2000).

THE CYTOKINES AND CHEMOKINES

450

INTERLEUKIN - 15 AND 21

Viral infections Recently, PBMC from 21 HIV-infected adults and 24 HIV-seronegative healthy individuals were isolated and cultured to determine the effect of escalating doses of IL-15 (0, 1, 10, 100, 1000 ng mL1) on apoptosis (Chang et al., 2000b). The results revealed that IL-15 reduced lymphocyte apoptosis in HIV patients in a dose-dependent manner, decreased the level of apoptosis after 3 and 5 days of culture in HIV patients, and increased down-regulated expression of Bcl-2 in HIV patients. In vitro replacement of IL-15 has been shown to enhance immunity in HIV-1-infected lymphocytes. Naora and Gougeon (1999a) found that IL-15 and IL-2 differed in their ability to induce proliferation, enhance survival and control apoptosis of CD45RO and CD45RO T-cell populations of HIVinfected individuals. When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 in activating and stimulating proliferation of CD4CD45RO, CD8CD45RO and CD8CD45RO cells, but failed to be more effective than IL-2 in reducing apoptosis. Poor activation of CD4CD45RO cells by IL-15 and IL-2 appeared to be attributable to low expression of the b chain utilized by both cytokines. However, IL-15 was more effective than IL-2 in enhancing survival of the CD4CD45RO population, suggesting a greater protective effect of IL-15 for naive CD4 T cells, which are preferentially lost in HIV-infected individuals. Alveolar macrophages of patients with HIV infection, but not control donors, express IL-15, IFNc, and IL-15R (a and c subunits) suggesting an up-regulation of co-stimulatory molecules B7 and CD5 co-ligand CD72, which are involved in the antigen presentation of macrophages (Agostini et al., 1999). Additional stimulation of these macrophages with IL-15 results in further up-regulation of CD80 and CD86 expression, suggesting that IL-15 might play an important role in TC1-mediated defense mechanisms taking place in extravascular tissues of patients with HIV infection. Furthermore, IL-15 has been shown to induce a dose-dependent elevation of proliferation and immunoglobulin production by B cells incubated in the presence of heat-inactivated HIV-1 (Kacani et al., 1999). These findings suggest that during the late stages of AIDS, when monocytes/macrophages become the major site of viral production, IL-15 may

promote polyclonal B-cell activation and hypergammaglobulinemia, which are frequently associated with HIV infection. Finally, IL-15 can induce LAK cell activity in HIV-seropositive patients and can stimulate IFNc production from PBMC of some donors (Lucey et al., 1997). IL-15 also stimulated levels of HIV production from PBMC, which was similar to or moderately lower than those obtained with IL-2, depending on cytokine concentration. It was also proposed that IL-15 may have differential effects on latent and acute HIV infection, and its ability to stimulate HIV production may depend on cell activation (BayardMcNeeley et al., 1996). Thus, the use of IL-15 may be dictated by the clinical state of the patient. The CD56CD16 NK-cell population plays a crucial role in eliminating virus-infected cells and is diminished in HIV-infected individuals. IL-15 can induce LAK cell activity in HIV-seropositive patients and can stimulate IFNc production from PBMC of some donors. IL-15 stimulates levels of HIV production from PBMC, which are similar to or moderately lower than those obtained with IL-2, depending on cytokine concentration. In addition, IL-15 enhances NK activity and antibody-dependent cellular cytotoxicity (ADCC) of mononuclear cells (MNCs) from HIV children and their mothers (Lin et al., 1998). When used at equivalent concentrations in vitro, IL-15 was more potent than IL-2 as a growth factor for CD56 CD16 cells and also CD4 and CD8 T cells (Naora and Gougeon, 1999b). Analysis of cell survival indicated that IL-15 was also more potent than IL-2 as a survival factor for CD56 cells by virtue of its greater ability to up-regulate bcl-2 expression. P-glycoprotein may play a role in the mechanism of increased NK cell activity in HIV-infected individuals after IL-15 stimulation (Chang et al., 2000b). The immunotherapeutic potential of IL-15 appears to be superior to that of IL-2 with regard to expanding NK cell populations in HIV-infected individuals, but needs to be weighed against less vigorous increases in T-cell populations (Naora and Gougeon, 1999a). Furthermore, polymorphonuclear leukocyte (PMN) dysfunction has been reported in HIV-infected patients; chemotaxis and fungicidal activity of unprimed PMN was significantly lower in patients with untreated HIV infection compared with controls (Mastroianni et al., 2000). After incubation with IL-15, a significant increase in PMN chemotaxis and fungicidal activity was found; more-

THE CYTOKINES AND CHEMOKINES

IL - 15 - BASED IMMUNOTHERAPIES

over, IL-15 induced a significant reduction in the number of apoptotic HIV PMN. Thus, IL-15 is an important cytokine in the activation of the functional properties of HIV PMN by delaying apoptosis and enhancing chemotaxis and fungicidal activity. It has recently been reported that HSV-1-mediated enhancement of NK activity could be abrogated by neutralizing anti-IL-15 antibodies but not antibodies for other cytokines, including IL-2, IL-12, IFNc, IFNa, and TNFa (Ahmad et al., 2000). Anti-CD122 antibody, which blocks signaling through the IL-2Rb chain and therefore neutralizes the effects of IL-15 (and IL-2), also abrogates this enhancement. Furthermore, HSV-1 increased the expression of IL-15 mRNA and protein in HSV-1-infected PBMC cultures. The neutralization of IL-15 in co-cultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production (Ahmad et al., 2000). These results thus strongly suggest a role for IL-15 in the HSV-1-mediated increase of NK activity and suppression of HSV-1 replication.

Other diseases Analyzing the release of vascular permeability factor (VPF) by ConA-stimulated PBMC in patients with minimal-change nephrotic syndrome and patients with IgA nephropathy, Matsumoto and Kanmatsuse (1999) reported that IL-15 augmented VPF release and thus, may be important in the pathophysiology of nephrotic syndrome. T-cell lymphotropic virus type I (HTLV-1) is the etiologic agent of the neurologic disease myelopathy/ tropical spastic paraparesis (HAM/TSP) that has clinical characteristics similar to those of multiple sclerosis (MS). The PBMC obtained from HAM/TSP patients undergo spontaneous proliferation in the absence of addition of any exogenous cytokines in an ex vivo culture (Azimi et al., 2000). It has been shown that IL-15 expression is elevated in HAM/TSP PBMC when compared with that of normal donors and that this elevation was due to Tax trans-activation of its promoter and induction of NFjB transcription factors (Azimi et al., 2000). Thus, HTLV-1 infection of T cells results in the production of both IL-2 and IL-15, growth factors that support the proliferation of T cells.

451

IL-15-BASED IMMUNOTHERAPIES Growing lines of evidence clearly indicate that IL-15 may influence immunologic abnormalities in HIV infection, cancer and autoimmune diseases, and provide an experimental basis for IL-15 immunotherapy. In part, these effects are mediated by its ability to activate T and NK cells, prevent apoptosis of immune cells by suppressing the down-modulation of Bcl-2, and regulate cytokine production and TH1/TH2/TH3 balance. Since IL-15 has recently been detected in the synovium of patients with rheumatoid arthritis and in tissue and organ allografts, it has led to speculation that IL-15 may contribute to the development of rheumatoid arthritic and graft rejection and its possible therapeutic application was evaluated in animal models. Ruchatz et al. (1998) reported that the administration of a soluble fragment of IL-15Ra profoundly suppressed the development of collagen-induced arthritis in mice. This effect was accompanied by a marked reduction in antigen-specific proliferation and IFNc synthesis by spleen cells and a significant reduction in serum levels of anti-collagen antibody in treated mice. In addition, it has recently been reported that soluble IL-15Ra injected at the beginning of transplantation significantly prolonged the survival of allogenic heart grafts in mice (Smith et al., 2000). These data directly demonstrate that antagonists of IL-15 may have therapeutic potential in a wide range of diseases. Di Carlo et al. (2000) reported that MHC class Inegative human SCLC cell line N592, engineered to express a modified IL-15 cDNA, secreted biologically active IL-15 (300–500 pg ml1) and was capable of boosting T-cell proliferation and NK activity in vitro. The effect of IL-15 gene transfer on natural immunity in vivo was assessed by xenotransplants in nude mice, displaying a significant delay in tumor growth and a marked recruitment of NK cells at the site of the tumor. The potent recruitment of NK cells mediated by IL-15 gene transfer suggests its possible therapeutic use in tumors lacking MHC class I. Similar results were reported for the in vivo growth in nude mice of a prostate cancer cell line, PC-3, genetically engineered to produce IL-15 (Suzuki et al., 2001). Furthermore, in mice, IL-15 producing genetically modified MethA

THE CYTOKINES AND CHEMOKINES

452

INTERLEUKIN - 15 AND 21

fibrosarcoma cells underwent complete rejection, in a response characterized by massive infiltration of CD4 T cells and neutrophils (Hazama et al., 1999). Moreover, rechallenged parental cells were also rejected in association with CD8 T-cell infiltration, demonstrating that IL-15-secreting tumor cells can stimulate local and systemic T cell-dependent immunity and therefore may have a potential role in cancer therapy. Similar data were reported by Tasaki et al. (2000), who observed that subcutaneous murine colon adenocarcinoma tumor cells transfected with IL-15 regressed spontaneously in contrast with tumors of wild-type cells. The mice which had eliminated IL-15-transfected tumors rejected wild-type cells when subsequently challenged. The survival of the mice which had been inoculated intraperitoneally with Colon 26/IL-15 cells was significantly prolonged compared with that of mice injected with non-transfected cells. Subtherapeutic doses of IL-15 potentiated the antitumor effects of intratumoral administration of IL-12. IL-12–IL-15 combination induced complete tumor regressions in 50% of mice, although IL-12 alone led to considerable delay of tumor development without achieving curative responses (Lasek et al., 1999). Similar results were obtained in a model in which tumorbearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown stimulation of both NK activity and specific anti-melanoma cytotoxic effector cells in tumor-draining lymph nodes. The strong antitumor effect of the IL-12  IL-15 combination also correlated with a high serum level of IFNc in the treated mice. Furthermore, intrapleural administration of IL-15 significantly prolonged the survival time of mice after an intrapleural inoculation of MethA fibrosarcoma cells enhancing the activities of NK and CD8 T cells in the thoracic exudate cells (Kimura et al., 2000). In a study of Gamero et al. (1995), PBMC of patients newly diagnosed with metastatic melanoma were incubated with different doses of recombinant human IL-15 and tested against autologous tumor

cells, LAK sensitive cell lines (i.e. FMEX and Daudi), as well as the NK-sensitive cell line K562. The effect of IL-15 was found to be both time and dose dependent. LAK and not CTL activity in patients’ PBMC was detected by the inability of anti-CD4, CD8 and MHC class I antibodies to block lysis of autologous tumor and FMEX melanoma cells, suggesting that LAK activity can be generated from melanoma patients’ PBMC in the presence of IL-15 to lyse autologous tumor cells in a non-MHC-restricted manner. Similarly, it has been shown that IL-15 like IL-2, is capable of stimulating the growth of tumor-infiltrating T lymphocytes with antitumor activity (Lewko et al., 1995). Chenetal. (2000) have also studied the role of IL-15, as compared with IL-2, in generating CTL from the malignant effusions of cancer patients. IL-15 was found to have at least an equivalent, if not higher, activity to IL-2 in terms of lymphocyte proliferation and generation of CTL from effusion-associated lymphocytes. An interesting conclusion was made by Takeuchi et al. (2001), who investigated the effect of IL-15 on modulation of type-1/type-2 balance in addition to nonMHC-restricted killer induction at the tumor site. IL-15 induced significant killer activity in MNC in malignant pleural effusions as well as those in peripheral blood. Pleural MNC produced more IFNc (type-1 cytokine) by incubation with IL-15 or IL-2 than blood MNC. Moreover, IL-4 and IL-5 (type-2 cytokines) production by pleural MNC was observed only by incubation with IL-2, but not with IL-15 (Takeuchi et al., 2001). Since TH1/TH2 misbalance has been well documented in cancer (Shurin et al., 1999), these observations suggest that IL-15 has potent activity to restore type-1/type-2 balance in addition to NK-cell activation and stimulation. In summary, in addition to the better-known cytokines IL-2 and IL-12, IL-15, whose antitumor potential was first described in 1995 (Munger et al., 1995), should be considered as a new antitumor cytokine activating NK, CTL and LAK cells with a mechanism of action similar to that of IL-2. Finally, IL-15 might also serve as a potent therapeutic agent in some bacterial and viral infections (Yoshikai and Nishimura, 2000). For instance, Maeurer et al. (2000) have shown that IL-15 is able to enhance survival of M. tuberculosis-infected mice, accompanied by a qualitatively different cellular immune response in spleen cells as reflected by

THE CYTOKINES AND CHEMOKINES

INTERLEUKIN - 21

increased TNFa and decreased IFNc secretion in response to M. tuberculosis-infected antigenpresenting cells. Moreover, in vivo administration of recombinant IL-15 protected normal mice from HSV-2-induced lethality, accompanied by increases in numbers of NK cells and HSV-2-specific TH1 cells (Tsunobuchi et al., 2000). Taken together with data demonstrating that IL-2–IL-15Rb-deficient mice were susceptible to systemic HSV-2 infection compared with their heterozygous littermates, these results suggest that IL-15, using the IL-2–IL15Rb chain, plays an important role in mounting protective immunity during the course of systemic HSV-2 infection. In conclusion, IL-15 has an effect on different cells associated with host defense, immunity and inflammation, supporting its central role in orchestrating multiple aspects of effector functions in immunity and inflammation (Perera, 2000). IL-15 may play an important role in antitumor, antiviral and antibacterial immunity. A better understanding of cellular and molecular biology of IL-15 and its role in human diseases will open a new avenue for the potential clinical utility of IL-15 as a therapeutic agent or target. Additional preclinical and clinical trials are required to evaluate the therapeutic potential of IL-15 for a number of diseases.

hIL-21 mIL-21 hIL-15 hIL-4 hIL-2 hIL-21 mIL-21 hIL-15 hIL-4 hIL-2 hIL-21 mIL-21 hIL-15 hIL-4 hIL-2

453

INTERLEUKIN-21 IL-21 protein IL-21 is encoded by a gene located on human chromosome 4q26–q27, forming a gene cluster with IL-2 and IL-15. Its 489-bp cDNA encodes a polypeptide precursor of 162 amino acids with a mature protein of 131 amino acids and a predicted relative molecular mass of 15 K (GenBank accession No. AF254069) (Parrish-Novak et al., 2000). Based on overall sequence homology and two conserved cysteine residues, IL-21 is considered to be most closely related to IL-15 (Figure 18.3). The 122 amino acid mature murine IL-21 protein (GenBank accession No. AF254070) shares 50% sequence homology with human IL-21 (Figure 18.3).

IL-21 receptor The IL-21R gene is located on human chromosome 16p11 in close proximity ( 70 kb) to the structurally related IL-4Ra chain gene (Figure 18.4). The cDNA encodes a 538 amino acid protein with a predicted molecular mass of 60 kDa (Ozaki et al., 2000; ParrishNovak et al., 2000) (Figure 18.4). As determined by

--------MRSSPGNMERIVICLMVIFLGTLVHKSSSCGODRHMIRMRQLIDIVDQLKNYVNDLV ---------------MERTLVCLVVIFLGTVAHKSSPCGPDRLLIRLRHLIDIVEQLKIYENDLD **** *:: * *:: MRISKPHLRSISIQCYLCLLLNSHFLTEAG??VFJLGCFSAGLPKT3ANWVNVISDLKKI-3DLI ------------------MGLTSQLLPP?FFLLACAGNFVHGHKCD-ITLQEIIKTLNSLTEQKT ----------------MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLONILNGIN PEF-----------------LPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKR PEL-----------------LSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIICLVAQLRR : :: : *: :: : * *: * QSMHIDAT------------LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL LCTELTVTDI----------FAASKNTTE----KETFCRAATVLRQFYSHHEKDTRCLGATAQQF NYKN------PKLTRMLTFKFYMPKKATE---LKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL KPPSTNAGRRQKHRL------TCPSCDSYEKK--PPKEFLERFKSLLCKMIHQHLSSRTHGSEDS RLPARRGGKKQKHIA------KCPSCDSYEKR--TPKEFLERLKWLLQKMIHQHLS : * * : :* *: : ::::* : ***: : ANNSLSSNGNVIESG-------CKECEELEEK--NIKEFLQSFVHIVQMFINTS HRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS ISNINVIVLGLKGSE------TTFMCE-YADETATIVEFLNRWITFCQSIISTL

FIGURE 18.3 Alignment of human and murine IL 21 with related cytokines. Identities (colon) or similarities (asterisk) between either human or murine IL-21 and human IL-15 are indicated. Mature N termini are indicated by open boxes. Potential N-linked glycosylation sites are underlined. Reprinted by permission from Nature (Parrish-Novak et al., 2000). Copyright (2000) Macmillan Magazines Ltd. THE CYTOKINES AND CHEMOKINES

454

INTERLEUKIN - 15 AND 21

-27kb

A

-39kb

** B

IL-4R alpha

NILR

human mouse

MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKD MPRGPVAALLLLILHGAWSCLDLTCYTDYLWTITCVLETRSPNPSILSLTWQDEYEELQD **** .*.****:*:*.*.* **.****** *: *:** . :** *:*****:****:*

60 60

human mouse

EATSCSLHRSAHNATHATYTCHMDVF?FMADDIFSVNITDQSCNYSQECGSFLLAESIKP QETFCSLHRSGHNTIHIWYTCHMRLSQFLSDEVFIVNVTDQSGNNSQECGSFVLAESIKP : * ******.**:** ***** : :*::*::* **.****** *******:*******

120 120

human mouse

APPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDFWAVSPRRKLISVDS APPLNVTVAFSGRYDISWDSAYDEPSNYVLRGKLQYELQYRNLRDPYAVRPVTKLISVDS ***:****:***:*:*** * *::*: *:*:*********** **:** * *******

180 180

human mouse

RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLL RNVSLLPEEFHKDSSYQLQVRAAPQPGTSFRGTWSEWSDPVIFQTQAGEPEAGWDPHMLL *.****** **:*****:*****.* **:*::**************: * : **:**:**

240 240

human mouse

LLLLVIVFIPAFWSLKTHPLWRLWKKIWA-VPSPERFFMPLYKGCSGDFKKWVGAPFTGS LLAVLIIVLVFMG-LKIHLPWRLWKKIWAPVPTPESFFQPLYREHSGNFKKWVNTPFTAS ** ::*:.: : ** * ********* **:** ** ***: **:*****.:***.*

299 299

human mouse

SLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSG SIELVPQSSTTTSALHLS--LYPAKEKKFPGLPGLEEQLECDGMSEPGHWCIIPLAAGQA *:** * *. ..*:*.: * . * *. *:* * . : .: * * ...

359 357

human mouse

GSAYSEERDRFYGLVSIDTVTVLDAERPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLL VSAYSEERDRPYGLVSIDTVTVGDAEGLCVWPCSCEDDGYPAMNLDAGRESGPNSEDLLL ********************** *** *.************::**** *..*. ** **

419 417

human mouse

DAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAG VTDPAFLSCGCVSGSGLRLGGSPGSLLDRLRLSFAKEGDWTADPTWRTGSPGGGSESEAG :..:.******... ***. *******: .:*. **:.. .* **** ******

479 477

human mouse

SPLSGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS SPPG-LDMDTFDSGFAGSDCGSPVET------DEGPPRSYLRQWVVRTPPPVGSGAQSS ** . **********.****.**** ************** .** ..*.*:*

538 529

FIGURE 18.4 The IL-21 receptor and IL-4RA genes are adjacent, and human and murine IL-21 receptor genes are highly related. (A) Schematic showing two overlapping BAC clones that together span the IL-21R (NILR) gene. The BAC clone positioned to the left contains all IL-21 receptor coding exons; the one on the right contains at least two non-coding exons of the IL-21R gene (asterisks) and the IL-4Ra gene. Marked distances are approximate. (B) Alignment of deduced human and murine IL-21R amino acid sequences. Asterisks, colons and periods indicate identical amino acids and conservative and semiconservative substitutions, respectively. Reproduced with permission from Ozaki et al. (2000). Copyright (2000) National Academy of Sciences, USA. Western blotting, the IL-21R protein is 80–100 kDa glycoprotein in accordance with the presence of several putative glycosylation sites in the extracellular domain (Ozaki et al., 2000). The exon–intron organization of the IL-21R gene reveals the presence of two non-coding exons (1a and 1b) that are alternatively spliced to exon 2, which contains the ATG translation start site (Ozaki et al., 2000) (Table 18.3). Furthermore, one alternative form of exon 2 that has an extended 5sequence has been identified (Ozaki et al., 2000). The mouse IL-21R cDNA shares 62% amino acid homology with the human gene and encodes a 529

amino acid protein (Ozaki et al., 2000; Parrish-Novak et al., 2000). The four cysteine residues and the ‘WSXWS’ motif, both typical of class I cytokine receptors, are conserved in human and mouse IL-21R. IL-21R is structurally most closely related to IL-2Rb and IL-4Ra.

IL-21R signaling The expression pattern of IL-21R and its ligand IL-21 strongly suggests that it plays an important role as a signaling molecule in the lymphohematopoietic sys-

THE CYTOKINES AND CHEMOKINES

INTERLEUKIN - 21

455

TABLE 18.3 Genomic structure of IL-21Ra Exon

Exon length bp

GenBank accession No.b

Location in genomec

Splice acceptord

Splice donord

Intron length bp

1ae

202

AC004525

36742–36541

Not applicable

ACAGAAGgtaattc

27 425

e

1b

120

AC004525

36058–35939

Not applicable

TCGGATGgtaagag

26 823

f

1

99

AC002303/ (AC004525)

26245–26398/ (9203–9050)

Not applicable

CAGGGAGgtaagtg

4226

2

65

AC002303/ (AC004525)

26334–26398/ (9115–9050)

cttgcagGCCCGTG

CAGGGAGgtaagtg

4226

3

103

AC002303/ (AC004525)

30625–30727/ (4823–4721)

cctccagGCTGGGG

TTACCTGgtaagta

3038

4

200

AC002303/ (AC004525)

33766–33965/ (1685–1484)

cttgaagGCAAGAC

GAGAGCAgtgagta

5274

5

155

AC002303

39240–39394

caccaagTCAAGCC

GGCTGTGgtgagga

1425

6

178

AC002303

40820–40997

ctggcagAGTCCGA

TCAGAGGgtaggtt

457

7

100

AC002303

41455–41554

ttcccagAGTTAAA

TGTGGAGgtgaggc

780

8

82

AC002303

42285–42366

ccctcagGCTATGG

CTTCAAGgtgagct

730

9

1540

AC002303

44812–46351

ctcacagAAATGGG

Not applicable

a

Reproduced with permission from Ozaki et al. Proc. Natl Acad. Sci. USA 2000; 97, 11439–11444. Copyright (2000) National Academy of Sciences, USA. b AC004525 sequence is in reverse orientation with respect to the IL-21R transcript. c Exon location is given with the numbering of AC004525 and AC002303. d Intronic sequence is in lower case. e The 5 boundaries of exons 1a and 1b are the 5 end of cDNA clones and have not been proven to be the transcription initiation site. f Exon 1 is identical to exon 2 except that it extends further 5. Its 5 boundary has not been mapped.

tem. High amounts of IL-21R transcripts were only detectable by Northern blots in murine or human lymphoid tissues (thymus, spleen, lymph node) (Ozaki et al., 2000; Parrish-Novak et al., 2000). Faint signals were detectable in lung and small intestine tissue, probably related to the presence of lymphocytes (Ozaki et al., 2000). IL-21R-cell surface expression is detectable by flow cytometry on human peripheral blood CD23 B lymphocytes, CD56 NK cells, but not on CD3 or CD14 cells. However, in another study, IL-21R mRNA was detectable in two HTLV-1transformed T-cell lines, indicating that activated T cells can express IL-21R (Ozaki et al., 2000). In contrast to the broad expression of IL-21R in lymphoid tissues, no expression of its ligand IL-21 was detected in normal tissues, indicating a much more restricted expression pattern for IL-21 than for its receptor. Significant amounts of IL-21 mRNA are inducible in human peripheral CD4, but not CD8 T cells by

PMA/ionomycin or anti-CD3/CD28 monoclonal antibody stimulation (Parrish-Novak et al., 2000). Because of the structural similarity of IL-21 to IL-2, IL-4 and IL-15 and of IL-21R to IL-2Rb and IL-4Ra, it was hypothesized that the common c chain (CD132) was also a subunit of the functional IL-21R complex. It has been demonstrated that IL-21 bound to the IL-21R expressed on cc-deficient ED4015 cells but was unable to transduce intracytoplasmic signals (Asao et al., 2001). The authors further showed that IL-21 activated JAK1, 3 and STAT1, 3, 5 signaling when a common c chain-transfected ED4015 cell line was used. Moreover, chemical crosslinking experiments revealed a direct binding of IL-21 to the common c chain (Asao et al., 2001). These data demonstrate that the cc is a functional subunit of the IL-21R complex. It has been suggested that IL-21R associates with JAK1 and activates STAT5 after IL-21R homodimerization (Ozaki et al., 2000).

THE CYTOKINES AND CHEMOKINES

456

INTERLEUKIN - 15 AND 21

Biologic effects of IL-21 IL-21 co-stimulates CD40L-induced proliferation of B cells, but conversely, completely blocks antiIgM/IL-4-mediated B-cell proliferation, indicating an important immunoregulatory function of IL-21 in humoral immunity (Parrish-Novak et al., 2000). Although competition for a shared receptor subunit – such as cc – might be responsible for this observation, such competition cannot fully explain the result as both IL-21 and IL-4 appear capable of stimulating B-cell growth (Vosshenrich and Di Santo, 2001). Along these lines, it would be interesting to see whether IL-21 and IL-4 would cross-inhibit anti-CD40mediated B-cell stimulation, or if IL-21 can act as a positive co-stimulator for anti-IgM-activated B cells in the absence of IL-4 (Vosshenrich and Di Santo, 2001). With respect to T cells, IL-21 was found to co-stimulate anti-CD3-induced proliferation and to synergize with IL-2 and IL-15 to promote T-cell expansion. Interestingly, IL-21 co-stimulated antiCD3-activated human naive CD45RA T cells but not CD45RO memory cells (Parrish-Novak et al., 2000). In addition, IL-21 supports IL-15-induced expansion of CD56 NK cells from bone marrow progenitor cells. Importantly, IL-21 effectively promotes NK-cell maturation and lytic activity in FLT3L plus IL-15-stimulated bone marrow progenitor cell cultures. In line with these data, IL-21 was also found to enhance the effector function of mature human NK cells, however, the overall effects were weaker than those of IL-2 or IL-15 (Parrish-Novak et al., 2000). The unique role for IL-21 as a cc-dependent cytokine in NK-cell differentiation could help to explain why mice deficient in IL-15 signaling have reduced NK cells, while ccdeficient mice completely lack this lymphoid subset (Vosshenrich and Di Santo, 2001). The essential biologic roles of IL-21 in vivo should be more clearly elucidated through the generation of mice deficient in this cytokine and its receptor.

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19 Interleukin-16 Kevin C. Wilson, David M. Center and William W. Cruikshank Boston University School of Medicine, Boston, MA, USA

Education is a progressive discovery of our own ignorance. Will Durant

INTRODUCTION Interleukin-16 (IL-16) was initially described in 1982 as the first T-cell chemoattractant (Center and Cruikshank, 1982; Cruikshank and Center, 1982). Originally designated lymphocyte chemoattractant factor (LCF), the protein was identified in the supernatants of mitogen- or antigen-stimulated human peripheral blood mononuclear cells. Since its initial description, details about its synthesis, structure, and bioactivity have been elucidated. In this chapter, we review current data regarding IL-16 gene structure, synthesis, protein structure, biochemical characteristics and cellular origin. IL-16’s interaction with its receptor (CD4), its bioactivities, and its role in HIV infection and inflammation are also discussed. Furthermore, we address the most recent data about the N-terminal domain of pro-IL-16 and its putative role in regulating lymphocyte cell cycle progression, as well as a second family member, neuronal IL-16. The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

SYNTHESIS Gene structure Located on human chromosome 15q26.1–3 (Kim, 1999) and murine chromosome 7 D2–D3 (Bannert et al., 1999a), the IL-16 gene encodes a large precursor protein, pro-IL-16 (Baier et al., 1997), from which mature IL-16 is generated. The single-copy gene contains seven exons and six introns (Bannert et al., 1999b). Comparison of the gene sequence across species including the human, mouse and feline reveals high sequence homology (84%) (Keane et al., 1998; Leutenegger et al., 1998; Bannert et al., 1999b). Bannert et al. (1999b) have demonstrated that the promoters of both the human and murine genes contain two CAAT box-like motifs and three binding sites for GA-binding protein (GABP) transcription factors Plate 19.1 (see Plate section). Both genes lack a TATA box. Two of the motifs make up a dyad symmetry element. After GABPa and GABPb complex with the dyad symmetry Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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element, the co-activator CREB binding protein/p300 binds to GABPa and induces the IL-16 gene promoter.

Transcription In T lymphocytes the gene is transcribed into mRNA of approximately 2.6 kb (Baier et al., 1997), which undergoes alternate splicing. Its half-life is 2 h (Laberge et al., 1995). The message is constitutively expressed and present in 95% T lymphocytes (Wu et al., 1999). Under non-disease conditions IL-16 mRNA is limited almost exclusively to lymphatic tissue (Baier et al., 1997; Keane et al., 1998). The IL-16 message is detected in other cell types in association with inflammation, for example, epithelial cells obtained from the airways of asthmatics (Laberge et al., 1997), or the footpads of mice undergoing a delayed type hypersensitivity reaction (Yoshimoto et al., 2000). In epithelial cells, the IL-16 message is not expressed without stimulation but is inducible by a variety of stimulants (Arima et al., 1999). A similar profile exists for mast cells (Rumsaeng et al., 1997). In B cells (Kaser et al., 2000) and fibroblasts (Sciaky et al., 2000) the message is constitutive and not inducible.

Translation and post-translational modifications Translation results in a 631-amino acid precursor protein, pro-IL-16 (Baier et al., 1997). Pro-IL-16 resides in the perinuclear space (Zhang et al., 2000) where it is enzymatically cleaved at serine residue 511 (Baier et al., 1997; Zhang et al., 1998) by caspase 3 (Zhang et al., 1998) Plate 19.2 (see Plate section). Following cleavage, the C-terminal domain of pro-IL-16 is secreted from the cell as bioactive, or mature, IL-16. In CD8 T cells, caspase-mediated cleavage is constitutive (Wu et al., 1999). In contrast, in CD4 T cells, caspase-mediated cleavage follows antigenic stimulation of lymphocytes (Bannert et al., 1999a; Wu et al., 1999), a process that is accelerated by co-stimulation with CD28 stimulation (Wu et al., 1999). Secretion of IL-16 from stimulated CD4 T cells correlates with cleavage of pro-caspase 3, suggesting that caspase processing of pro-IL-16 regulates the rate at which IL-16 is secreted (Wu et al., 1999).

STRUCTURE, BIOCHEMICAL CHARACTERISTICS AND SEQUENCE Although IL-16 is a secreted protein, it does not contain a consensus secretory leader sequence, suggesting that the endoplasmic reticulum is not involved. Zhou et al. (1999) determined that when the N-terminal 20 amino acids of IL-16 were deleted, there was reduced capacity for secretion. Conversely, when a secretory leader sequence was added, secretion was enhanced. Thus, the N-terminus of IL-16 appears important for its secretion; however, the exact mechanism remains unknown. Bioactivity is detected only after autoaggregation into homodimers or tetramers (Center and Cruikshank, 1982; Cruikshank and Center, 1982). Because CD8 T cells and mast cells contain intracellular bioactive IL-16 (Laberge et al., 1995; Rumsaeng et al., 1997), autoaggregation must occur intracellularly. In vitro studies have demonstrated extracellular autoaggregation of IL-16 monomers suggesting that they may occur in vivo. The core structure of each IL-16 monomer contains a PDZ domain (Muhlhahn et al., 1998). Typically, PDZ proteins are intracellular proteins that associate with other intracellular proteins via interaction of the conserved G–L–G–F sequence (Woods and Bryant, 1995). IL-16 has not been shown to associate with any other proteins and therefore the PDZ domains may facilitate autoaggregation. As PDZ proteins are typically intracellular, IL-16 may be the first example of a secreted PDZ protein. The amino- and carboxytermini of IL-16 lie outside the core structure (Muhlhahn et al., 1998). Peptide studies have identified function in association with the C-terminal sequence. Synthetic peptides corresponding to the carboxy-terminus sequence, Arg106 to Ser109, inhibited the chemotactic activity of IL-16, as did antibodies directed against this sequence. When alanine was substituted for Arg107 in the synthetic oligopeptide, inhibition was lost, suggesting that Arg107 is essential for IL-16-induced chemotactic activity. These data suggest that the bioactive site on IL-16 is located in the C-terminal end, distinct from the PDZ domain. The sequence, structure and function of IL-16 are highly conserved in all species examined. Simian proIL-16 and human pro-IL-16 are approximately 96%

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homologous. When simian IL-16 (secreted) is compared with human IL-16, homology is 98%, and simian-derived IL-16 has demonstrated the same bioactivities on human and simian T cells as human IL-16 (Baier et al., 1995; Lee et al., 1998). Similarly, murine pro- and mature IL-16 demonstrate high homology and functional cross-reactivity with the human protein (Keane et al., 1998). In fact there is functional cross-reactivity and antibody neutralization for all species investigated to date, which includes rat, sheep, bovine and feline IL-16.

CELL SOURCES Initially identified as the major chemoattractant factor produced by CD8 T cells (Laberge et al., 1995; Laberge et al., 1996), IL-16 was later shown to be produced by numerous cell types including CD4 T cells, mast cells, eosinophils, dendritic cells, bronchial epithelial cells, B cells and fibroblasts (Table 19.1). CD8 T cells stimulated by mitogen or antigen require 12–24 h before IL-16 is detected in the cell supernatants (Center et al., 1996; Wu et al., 1999). IL-16 is not detected after treatment with either a transcription or translation inhibitor, indicating that the IL-16 secreted in response to mitogens is a result of de novo IL-16 synthesis. In contrast, when CD8 T cells are stimulated by histamine or serotonin, IL-16 is detected in the cell supernatants within 1–4 h (Berman et al., 1984; Laberge et al., 1996), and transcription or translation inhibitors do not impair IL-16 secretion. These data suggest that in CD8 T cells two pathways for IL-16 expression may exist. Preformed bioactive IL-16 is stored in vesicles responsive to secretagogues such as vasoactive amine stimulation and

new IL-16 is synthesized and released following T-cell receptor activation. CD4 T cells contain a constitutive message and pro-IL-16 but no bioactive IL-16. The cells must be stimulated for 18–24 h before bioactive IL-16 is detected in the supernatant as a result of caspase 3 processing of pro-IL-16 (Wu et al. 1999). The initial observation that airway epithelial cells can produce IL-16 was reported by Bellini et al. (1993). In their studies, bronchial epithelial cells were obtained by bronchoscopic biopsy from normal, atopic asthmatic and atopic non-asthmatic volunteers, and cultured for 48 h with or without histamine. Only the supernatants from the cell cultures obtained from asthmatics contained IL-16 and were chemotactic for T lymphocytes. Both chemotactic activity and IL-16 titer were increased when the bronchial epithelial cells were stimulated with histamine. Subsequent studies have revealed that the TH2 cytokine IL-9 can stimulate IL-16 production by airway epithelial cells (Little et al., 2001). The most recently identified cell source is the fibroblast. Fibroblasts obtained from non-diseased tissue do not constitutively generate IL-16, however, fibroblasts obtained from diseased tissue such as the synovium of patients with rheumatoid arthritis (RA) have been shown to produce IL-16 (Franz et al., 1998; Blaschke et al., 2001). Franz et al. (1998) have compared patients with rheumatoid arthritis to patients with non-rheumatoid arthritis or osteoarthritis (OA). They found that the synovial lining contained IL-16 message with an increase of IL-16 protein contained in the synovial fluid of patients with RA, which was not detected in the fluid from OA patients. Fibroblasts from all tissues generate IL-16 in response to IL-1b, TNFa and leukoregulin (Sciaky et al., 2000).

TABLE 19.1 Cells of origin for IL-16 Cells

Message

Pro-IL-16

IL-16 bioactivity (preformed)

CD8 T cell CD4 T cell Mast cell Eosinophil Dendritic cell Epithelial cell Fibroblast

Constitutive and inducible Constitutive Constitutive and inducible Constitutive Constitutive Inducible Constitutive

Constitutive Constitutive Constitutive Constitutive Constitutive Inducible Inducible

Present Absent Present Present Absent Absent Absent

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INTERACTION OF IL-16 WITH ITS RECEPTOR CD4 Expression of CD4 is an absolute requirement for all T-cell responses to IL-16. This was first demonstrated in studies in which murine T-cell hybridoma cells were transfected with the cDNA encoding human CD4. CD4 expression imparted IL-16 responsiveness in previously unresponsive cells as measured by induction of second messengers and cell migration (Cruikshank et al., 1991; Ryan et al., 1995). The physical interaction between IL-16 and CD4 has been shown by IL-16 purification from lysates using recombinant soluble CD4 affinity chromatography and by immunoprecipitation (Cruikshank et al., 1994). The IL-16 binding site on CD4 has recently been determined. Comparison of the human CD4 amino acid sequence with several different species revealed the D4 domain to be the most conserved. Using peptide inhibition, the CD4 binding site for IL-16 was mapped to the D4 domain and the active site to the Trp345QCLLSer350 sequence within the D4 domain (Liu et al., 1999). Mutational analyses suggest that both leucines347,348 are essential for induced signaling but not for binding. It does not appear that a co-receptor is necessary to elicit cellular signaling in T cells, however, this may not be the case for all cell types. Recent reports have suggested that alternate or co-receptor signaling may be present for both murine monocytes (Mathay et al., 2000) and human dendritic cells (Stoitzner et al., 2001). In T cells, IL-16 binding to CD4 has been shown to decrease the chemotaxis induced by macrophage inflammatory protein-1b (MIP-1b) binding to CCR5 (Mashikian et al., 1999) and stromalderived factor-1a (SDF-1a) (Van Drenth, 2000) which binds to CXCR4. IL-16 desensitization of both of these chemokine receptors is suggestive of a co-receptor relationship, especially in light of data indicating physical coupling of CCR5 and CXCR4 with CD4 (Xiao et al., 1999). IL-16 is active in multimeric form suggesting that cross-linking of CD4 is necessary for IL-16-induced intracellular signaling (Cruikshank et al., 1994). Further observations supporting this include requirement of oligomerization of CD4 for optimization of MHC class II-dependent cell activation (Konig et al., 1995; Sakihama et al., 1995), and cross-linking of CD4 by multimeric IL-16 results in the generation of sev-

eral second messengers not detected by monovalent binding (Ryan et al., 1995). In lymphocytes and monocytes, detectable increases in intracellular calcium, inositol-(1,4,5)-triphosphate (IP3), and phosphorylation of CD4 and p56lck are observed within minutes after stimulation with aggregated IL-16 (Cruikshank et al., 1987; Ryan et al., 1995). In T cells, IL-16-induced signals are dependent on the interaction of CD4 with p56lck. This was identified by transfecting L3T4-murine hybridoma T cells with CD4 constructs that prevent or allow for interaction with p56lck. IL-16-induced signals were detected only when constructs that allowed for interaction with p56lck were used (Ryan et al., 1995). Interestingly, p56lck constructs lacking the catalytic SH1 kinase domain functioned normally, however, all signaling was lost when the adapter SH3 domain was deleted. This suggests that a migratory signal induced by an IL-16–CD4 interaction is not transmitted by the enzymatic activity of the SH1 domain, but rather by the SH3 domain.

BIOACTIVITY IL-16 induces a variety of bioactivities depending upon the CD4 cell type that is stimulated. Specifically, it is involved in chemotaxis, cell proliferation and cellular differentiation. In addition, IL-16 can regulate antigen-induced TCR signaling. Although initially characterized as a chemoattractant specifically for CD4 T cells (Berman et al., 1985), it was later determined that IL-16 is a potent chemoattractant for all peripheral immune cells expressing CD4 including eosinophils (Rand et al., 1991), monocytes (Cruikshank et al., 1987) and dendritic cells (Kaser et al., 1999). Unlike most chemokines, IL-16 does not require prior activation of the cell to exert its chemotactic properties on lymphocytes (Cruikshank et al., 1994, 1996a). In addition, IL-16 is a competence growth factor, capable of stimulating cell cycle progression in CD4 T cells. Priming CD4 T cells with IL-16 results in cell surface expression of IL-2Ra and b (Parada et al., 1998) in association with progression from G0 to G1 in the cell cycle. However, cell proliferation does not occur until the cells are stimulated with IL-2 or IL-15 (Parada et al., 1998). Stimulation of peripheral blood mononuclear cells with IL-16 followed by IL-2 results in a 1000-fold increase in the

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IL - 16 AND HIV INFECTION

number of CD4CD25CD29CD45RO T cells (Parada et al., 1998). Similar but less dramatic results are seen when IL-16 and IL-2 are used to stimulate HIVinfected cells (Parada, unpublished observation). In cells that have lost regulated cell growth, IL-16 can act as a complete growth factor. CD4 T cell lymphomas increase their growth rate when stimulated by IL-16 (Cruikshank et al., 1996b). In addition, cells obtained from patients with Mycosis fungoides, a common cutaneous T-cell lymphoma, have been shown to have increased IL-16 expression (Blaschke et al., 1999). IL-16 stimulation also influences B-cell differentiation. Szabo et al. (1998) noted that stimulation of murine bone marrow cells with IL-16 resulted in differentiation of CD4 pro-B cells into pre-B cells, increased expression of RAG-1, and expansion of the number of pre-B cells in the bone marrow. Interaction of CD4 with the ligands HIV-1 gp120 or anti-CD4 antibody can induce second messengers (Merrill et al., 1989; Juszczak et al., 1991; Chirmule et al., 1994) and cellular responses (Merrill et al., 1989; Wahl et al., 1989; Clouse et al., 1991) such as chemotaxis (Kornfeld et al., 1988). Binding of these ligands to CD4 also inhibits antigen-induced cellular activation through TCR–CD3 (Chirmule et al., 1990; Cefai et al., 1992). IL-16 similarly inhibits antigen-induced TCR signaling, as indicated by its inhibitory effect on a mixed lymphocyte reaction (Theodore et al., 1996) or anti-CD3 stimulation (Cruikshank et al., 1996c). Point mutated IL-16 is incapable of signaling through CD4 but can still inhibit TCR signaling (Nicoll et al., 1999). This suggests that the interaction with CD4 alone and not signaling through CD4 facilitates the inhibitory activity. The mechanism by which IL-16–CD4 binding stimulates many cellular responses while inhibiting antigen-dependent stimulation via TCR is unclear. One hypothesis is that IL-16 contributes to antigenindependent non-clonal recruitment and priming of CD4 cells in an inflammatory process. The ability of IL-16 selectively to inhibit chemokine-induced migration would serve to define further the recruited cell population. Although the recruited cells would be responsive to cytokine stimulation (IL-2, IL-15), they would be refractory to antigen-specific activation. The effect would be to increase the number of cells recruited to an inflammatory focus and to increase further the number of viable cells by simultane-

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ously reducing their susceptibility to antigen-specific induced cell death (Cruikshank et al. 2000).

IL-16 AND HIV INFECTION Binding of IL-16 to CD4 cells results in suppression of human immunodeficiency virus (HIV-1) and simian immunodeficiency virus (SIV) infection and replication. Unlike RANTES, MIP-1a, and MIP-1b, IL-16 binds to CD4 at an epitope distinct from HIV-1 (Liu et al., 1999) and therefore does not alter HIV-1 binding and internalization. Rather, the inhibitory effect of IL-16 appears to be at the level of transcriptional regulation. Maciaszek et al. (1997) reported that IL-16 pretreatment of CD4 lymphoid cells suppressed HIV-1 promoter activity 60-fold. This effect required sequences within the core enhancer, but was not due to the down-regulation of the binding activity of transcription factors such as NF-jB. It appears that IL-16 stimulation activates a transcriptional repressor that functions through sequences within or immediately adjacent to the core enhancer. In fact, cells transfected with the bioactive portion of IL-16 are resistant to HIV-1 infection (Zhou et al., 1997, 1999). In addition, Truong et al. (1999) reported that IL-16 not only inhibits viral replication but also prevents viral entry in dendritic cells. Both Idziorek et al. (1998) and Lee et al. (1998) have reported that IL-16 is capable of inhibiting HIV even when it is added after infection. While the antiviral activity of IL-16 is not as effective as many agents currently used in clinical trials, it may be useful for HIV therapeutics as an adjunct to IL-2 for immune reconstitution. IL-16 stimulation results in up-regulation of IL-2Ra, imparting IL-2 responsiveness to CD4 lymphocytes. Thus, IL-16 treatment would be expected to increase the IL-2R population, potentially decreasing the required amount of IL-2, thereby reducing the risk of IL-2 toxicity. Preliminary studies have indicated that peripheral blood mononuclear cells obtained from HIV-1 individuals, cultured with both IL-16 and IL-2, results in an increase in a homogeneous CD4 T-cell population. In addition, cells from some patients demonstrated renewed antigen responsiveness (Parada, unpublished observation). Clinically, a pattern has developed correlating HIV disease progression with systemic levels of IL-16. IL-16 levels are low in

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patients with AIDS (Scala et al., 1997), but rise dramatically after treatment with indinavir (Bisset et al., 1997), concomitant with a rise in CD4 counts. Interestingly, IL-16 levels are within the normal range in long-term non-progressors (Bisset et al., 1997).

IL-16 IN INFLAMMATION Initial characterization of IL-16 bioactivity, induction of cell migration and priming T cells for proliferation, would suggest a pro-inflammatory cytokine. As such, IL-16 has been detected at sites of inflammation in association with a variety of diseases, such as rheumatoid arthritis (RA), asthma, sarcoidosis, inflammatory bowel disease, delayed-type hypersensitivity (DTH) and Graves’ disease. While under certain conditions IL-16 may function as a proinflammatory cytokine, recent data have suggested that IL-16 can also act in an immunomodulatory fashion. Identifying a role for IL-16 in inflammation has focused on diseases characterized by CD4 infiltrates. Asthma was the first disease to be directly associated with IL-16 production. Bellini et al. (1993) identified IL-16 in cultures of histamine-stimulated primary epithelial cells obtained from asthmatics, but not from normal individuals. Cruikshank et al. (1995) challenged asthmatic individuals with antigen resulting in detection of IL-16 in the bronchoalveolar lavage fluid 4 h post-challenge. IL-16 could not be detected in normal or atopic non-asthmatic individuals. Subsegmental histamine challenge of asthmatics, but not non-asthmatics, also resulted in the elaboration of IL-16 (Mashikian et al., 1998). When biopsies obtained from asthmatics were assessed for immunohistochemical staining and in situ hybridization, IL-16 message and protein was detected in the airway epithelium and infiltrating CD4 cells (Laberge et al., 1997). There was significant correlation between the amount of protein and message and the number of infiltrating CD4 cells. Non-asthmatics had little detectable IL-16 protein or message. The role of IL-16 in asthmatic inflammation is becoming clearer. Intratracheal instillation of IL-16 in a murine model of asthma has indicated a significant reduction in airway hyper-reactivity as well as a reduction in lung inflammation (unpublished observations). These findings

are currently being confirmed using IL-16 transgenic and knockout animals. In another animal model, Klimiuk et al. (1999) demonstrated that transfer of CD8 T cells in a murine model of RA reduces IFN-c, IL-1b, and TNF-a levels by more than 90% and significantly reduces CD4 T cell infiltrates, effects that were blocked by co-administration of anti-IL-16. However, the role of IL-16 in inflammation appears to be disease specific. Immunohistochemical staining of sarcoidosis-associated granulomas from the lymph node and lung reveals high levels of IL-16 staining, with greatest prevalence in the perivascular areas of lymphocyte accumulation. IL-16 is abundant in the bronchoalveolar lavage fluid of patients with sarcoidosis and tuberculosis (Berman, unpublished observations). Using an animal model of DHT, Yoshimoto et al. (2000) demonstrated that anti-IL-16 antibody treatment significantly reduced granuloma formation in murine footpads. In patients with Crohn disease, colonic tissue sections show increased IL-16 protein and message compared with uninvolved colonic tissues from the same patients or normal controls. In an animal model of Crohn disease, anti-IL-16 antibody treatment significantly reduced all parameters of inflammation (Keates et al., 2000).

THE N-TERMINAL DOMAIN OF PRO-IL-16 Pro-IL-16 is a 631-amino acid precursor molecule that is cleaved by caspase 3, permitting secretion of the 121 C-terminal peptide identified as mature IL-16 (Plate 19.3) (see Plate section). While the biochemical characteristics and functions of mature IL-16 have been intensely studied, relatively little is known about the pro-piece of IL-16 following cleavage. We have recently begun to look at the structure and function of the residual N-terminal domain of proIL-16. Using fluorescence immunochemistry, Zhang et al. (2000) localized pro-IL-16 to the cytoplasm, primarily in the perinuclear region. Following cleavage by caspase 3, the N-terminal domain was found primarily in the nucleus, indicating cleavage-dependent translocation. Sequence analysis of pro-IL-16 indicated the presence of a consensus bipartite nuclear translocation domain (NLS), two PDZ domains, a

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cdc2 enzymatic substrate site, a casein kinase II (CK II) substrate site and a SH3-binding domain. Initial studies identified that mutation of the NLS prevented nuclear translocation. In light of its nuclear localization and the identification of several potential cell cycle-related kinase substrate sites the N-terminal domain may be important in contributing to the regulation of the lymphocyte cell cycle. Along those lines, Zhang et al. (2000) have demonstrated that the presence of pro-IL-16 in the nucleus is associated with increased G0/G1 cell cycle arrest. This effect is not detected when NLS-mutated constructs of IL-16 are used. The physiologic importance of these findings has yet to be determined. In addition, as many cell types are capable of generating IL-16 the potential exists for pro-IL-16 to contribute to the regulation of cell cycle progression in a variety of cell types.

NEURONAL IL-16 Kurschner and Yuzaki (1999) identified a neuronal form of IL-16. Neuronal IL-16 (NIL-16) message and protein is found only in neurons of the cerebellum and hippocampus. The protein contains 1322 amino acids with the C-terminus identical to lymphocyte-derived pro-IL-16. Similar to lymphocyte IL-16, NIL-16 is processed by caspase 3 resulting in secretion of mature IL-16. The N-terminus, however, is unique as it contains four PDZ domains and interacts selectively with a variety of neuronal ion channels (Kurschner and Yuzaki, 1999). The presence of both PDZ domains and the interaction with ion channels is typical of several neuronal intracellular scaffolding proteins. It is hypothesized that NIL-16 may have two roles in the nervous system; extracellularly, mature IL-16 acts to activate CD4 neuronal cells; and intracellularly, proIL-16 may function to anchor ion channels to the membrane. In summary, IL-16 is a multi-functional cytokine generated by a variety of cell types and which functions are restricted to surface expression of CD4 (Table 19.2). While IL-16 may have a role in homeostasis it is dramatically up-regulated at sites of inflammation where it appears to have the capacity to function both as a pro-inflammatory and an

TABLE 19.2 Summary of IL-16 properties and functions Properties 1. Promoter contains two CAAT box motifs and three GA-binding protein binding sites 2. Promoter is activated by binding of GABPa and GABPb to a dyad element 3. IL-16 message is usually constitutively expressed 4. Initial synthesis is as a precursor molecule, proIL-16, cleaved by caspase 3 5. Mature IL-16 is secreted, pro-IL-16 translocates into the nucleus 6. Autoaggregation into homotetramers is required for bioactivity 7. IL-16 is highly conserved across species 8. The D4 domain of CD4 is the receptor for IL-16 9. Multiple cell types produce IL-16 10. Neuronal IL-16 binds calcium channels and activates CD4 neural cells Functions 1. Induces migration/chemotaxis in CD4 cells 2. Primes CD4 T cells for proliferation with IL-2 or IL-15 3. Regulates chemokine receptors CCR5 and CXCR4 responsiveness 4. Regulates immune and inflammatory processes mediated through the TCR 5. Facilitates pro-B-cell differentiation 6. Suppresses HIV-1 and SIV-1 replication immunomodulatory cytokine. A better understanding of how IL-16 fits into the pathogenesis of inflammation is required as the specificity of IL-16, through CD4, is directed to those cell types that orchestrate much of the inflammatory process.

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an occluded peptide binding site. Nat. Struct. Biol. 5, 682–686. Nicoll, J., Cruikshank, W.W., Brazer, W.F. et al. (1999). Identification of domains in IL-16 critical for biological activity. J. Immunol. 163, 1827–1932. Parada, N.A., Center, D.M., Kornfeld, H. et al. (1998). Synergistic activation of CD4 T cells by IL-16 and IL-2. J. Immunol. 160, 2115–2120. Rand, T.H., Cruikshank, W.W., Center, D.M. and Weller, P.F. (1991). CD4–mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. J. Exp. Med. 173, 1521–1528. Rumsaeng, V., Cruikshank, W.W., Foster, B. et al. (1997). Human mast cells produce the CD4 T lymphocyte chemoattractant factor, IL-16. J. Immunol. 159, 2904–2910. Ryan, T.C., Cruikshank, W.W., Kornfeld, H. et al. (1995). The CD4–associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration. J. Biol. Chem. 270, 17081–17086. Sakihama, T., Smolyar, A. and Reinherz, E.L. (1995). Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120. Proc. Natl Acad. Sci. USA 92, 6444. Scala, E., D’Offizi, G., Rosso, R. et al. (1997). C-C chemokines, IL-16, and soluble antiviral factor activity are increased in cloned T cells from subjects with longterm nonprogressive HIV infection. J. Immunol. 158, 4485–4492. Sciaky, D., Brazer, W., Center, D.M. et al. (2000). Cultured human fibroblasts express constitutive IL-16 mRNA: cytokine induction of active IL-16 protein synthesis through a caspase-3-dependent mechanism. J. Immunol. 164, 3806–3814. Stoitzner, P., Ratzinger, G., Koch, F. et al. (2001). Interleukin-16 supports the migration of Langerhans cells, partly in a CD4-independent way. J. Invest. Dermatol. 116, 641–649. Szabo, P., Zhao, K., Kirman, I. et al. (1998). Maturation of B cell precursors is impaired in thymic-deprived nude and old mice. J. Immunol. 161, 2248– 2253. Theodore, A.C., Center, D.M., Nicoll, J. et al. (1996). CD4 ligand IL-16 inhibits the mixed lymphocyte reaction. J. Immunol. 157, 1958–1964. Truong, M.J., Darcissac, E.C., Hermann, E. et al. (1999). Interleukin-16 inhibits human immunodeficiency virus type 1 entry and replication in macrophages and in dendritic cells. J. Virol. 73, 7008–7013. Van Drenth, C., Jenkins, A., Ledwich, L. et al. (2000). Desensitization of CXC chemokine receptor 4, mediated by IL-16/CD4, is independent of p56lck enzymatic activity. J. Immunol. 165, 6356–6363. Wahl, S.M., Allen, J.B., Gartner, S. et al. (1989). HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes. J. Immunol. 142, 3553. Woods, D. and Bryant, P. (1995). DIgA and PSD-95/SAP90: homologous proteins in tight, septate and synaptic cell junctions. Mech. Dev. 44, 85–89. Wu, D.M., Zhang, Y., Parada, N.A. et al. (1999). Processing and release of IL-16 from CD4 but not CD8 T cells is activation dependent. J. Immunol. 162, 1287–1293.

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Xiao, X., Wu, L., Stantchev, T.S. et al. (1999). Constitutive cell surface association between CD4 and CCR5. Proc. Natl Acad. Sci. USA 96, 7496–7501. Yoshimoto, T., Wang, C.R., Yoneto, T. et al. (2000). Role of IL-16 in delayed-type hypersensitivity reaction. Blood 95, 2869–2874. Zhang, Y., Center, D.M., Wu, D.M. et al. (1998). Processing and activation of pro-interleukin-16 by caspase-3. J. Biol. Chem. 273, 1144. Zhang, Y., Kornfeld, H., Cruikshank, W.W. et al. (2000).

Nuclear translocation of the N-terminal prodomain of interleukin-16. J. Biol. Chem. 276, 1299–1303. Zhou, P., Goldstein, S., Devadas, K. et al. (1997). Human CD4 cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nat. Med. 3, 659–664. Zhou, P., Devadas, K., Tewari, D. et al. (1999). Processing, secretion, and anti-HIV-1 activity of IL-16 with or without a signal peptide in CD4 T cells. J. Immunol. 163, 906–912.

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20 Interleukin-17 [IL-17, IL-25] Mary A. Antonysamy1 and Muneo Numasaki2 1

Interferon Sciences Inc., New Brunswick, NJ, USA Department of Geriatric and Respiratory Medicine, Tohoki University School of Medicine, Tohoki, Japan 2

Pick battles big enough to matter, small enough to win Jonathan Kozol

INTRODUCTION The designation interleukin-17 (IL-17) has been assigned to a T cell-derived lymphokine that acts on a wide variety of cells. IL-17, identified originally by Rouvier et al. (1993) as murine cytotoxic T lymphocyteassociated antigen-8 (CTLA-8), was generated using subtractive hybridization approach from an ionomycin and phorbol myristate acetate (PMA) activated T-cell hybridoma in a search for molecules with immune functions. The CTLA-8 gene (GenBank Accession No. L13839) contains several AU-rich repeats in its 3 untranslated region associated with mRNA instability, as found in several cytokines, growth factors, and protooncogenes (Rouvier et al., 1993). This presumably suggests tightly controlled gene expression at the level of transcription in the molecules bearing these repeats. Conserved AU sequences in the 3 untranslated region of cytokine

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

mRNAs are reported to serve as recognition signals for an mRNA processing pathway that mediates selective mRNA degradation, resulting in transient expression of the gene (Caput et al., 1986; Shaw and Kamen, 1986). Since CTLA-8 was found to possess specific attributes that classify cytokines, the designation murine IL-17 was assigned for this factor. The CTLA-8 gene encodes a putative 150-amino-acid protein, strikingly homologous to the protein encoded by the open reading frame 13 of herpesvirus saimiri (HVS-13) (56.7% homology on 146 amino acids) (Albrecht et al., 1992). This phenomenon is analogous to the presence of the IL-6 homolog in the human herpesvirus-8 (HHV-8) or the IL-10 homolog called BamH1 C rightward fragment 1 (BCRF1) in the Epstein–Barr virus (Moore et al., 1990; Hsu et al., 1990). A number of such virus captured cellular proteins that might interfere with or modify immunologic responses have been reported (reviewed in Seow, 1998).

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CTLA-8 AND HVS-13: SEQUENCE HOMOLOGY AND EXPRESSION The close homology observed between the CTLA-8 (i.e. murine IL-17) and the simian HVS-13 sequences indicates that IL-17 and HVS-13 are probably derived from the same primordial gene. Herpesvirus saimiri, a T-lymphotropic virus, is a member of the gammaherpesvirinae subgroup (rhadinoviruses). Although squirrel monkeys (Saimiri sciureus) serve as natural hosts for HVS, the virus causes fulminant T-cell lymphomas in New World primate species, such as cottontop tamarins and common marmosets. HVS is capable of transforming non-saimiri New World primate (Albrecht et al., 1992) and human (Biesinger et al., 1992) T cells, causing their continuous growth in vitro. HVS also induces IL-2-dependent autocrine growth of human lymphocytes following crosslinking of CD2 (Mittrucker et al., 1992). These transforming and pathogenic properties of wild-type HVS were abrogated by deletion of transformation-associated genes stpC and tip. However, the replicative ability of these mutant viruses remained unaffected. Could the neighboring HVS-13 gene be a causative factor for HVS replication and pathogenesis? Knappe et al. (1998) evaluated this hypothesis using HVS-13 gene knockout mutants and reported HVS-13 gene disruption not to affect viral replication, in vitro transformation of simian and human T cells, and the pathogenicity in cottontop tamarins. Therefore, HVS13 may not be necessary for the viral pathogenicity but may be required for the apathogenic viral persistence in its natural host, the squirrel monkey (Knappe et al., 1998). However, HVS-14, another protein encoded by herpesvirus, bound to MHC class II molecules and stimulated the proliferation of peripheral blood T cells in vitro, implicating HVS-14 in the immunopathology of HVS infection (Yao et al., 1996a). The HVS genome has at least 15 genes homologous to cellular DNA or proteins, possibly acquired by retroposition (as most of the viral counterparts have no introns), suggesting that perhaps it functions as a spontaneous vector for cellular genes (Albrecht et al., 1992). HVS-13 has higher homology with human IL-17 (72%) than with murine IL-17 (56.7%), and thus may be yet another virus-captured host gene that has

provided an as yet unknown evolutionary advantage to the virus (Rouvier et al., 1993). Northern blot analysis using a murine IL-17 probe on an extended panel of cells revealed that the 1.35-kb transcript could be detected only in two T-cell clones (d10S and d18S which arose from the 10th and 18th subcloning, respectively) upon activation with PMA and ionomycin. It could not be identified in resting lymphocytes, the M12.4.1 lymphoma, lipopolysaccharide (LPS)-activated B-cell blasts, concanavalin Aactivated T cells, the KB5C20 cytotoxic T-cell line or in several T-cell hybridomas, either constitutively or following activation with PMA and ionomycin (Rouvier et al., 1993). Using in situ hybridization, the murine IL-17 gene was mapped to the IA band of the mouse genome, and the human homolog of murine IL-17 to 2q31. Southern blot studies of mouse, rat and human genomic DNA revealed the presence of a single copy IL-17 gene in all three species (Rouvier et al., 1993). Yao and colleagues (1995a) studied the expression patterns of both HVS-13 and murine IL-17. When murine IL-17 and HVS-13 were transfected into CV1/EBNA cells, supernatants and lysates from murine IL-17-expressing cells were found to contain two unique proteins with molecular masses of 17 kDa and 20 kDa, and those from HVS-13-expressing cells contained four proteins with apparent masses of 17, 20, 23 and 26 kDa. Treatment of transfectants with tunicamycin, which inhibits the addition of N-linked oligosaccharides to proteins, led to augmented expression of the 17-kDa form alone, suggesting that the other proteins contain N-linked glycans. Whereas HVS-13 possesses three potential N-glycosylation sites, murine IL-17 has only a single site.

MOUSE IL-17: ISOLATION, CHARACTERIZATION AND EXPRESSION Rouvier and colleagues (1993) isolated murine CTLA-8 from an activated rodent T-cell hybridoma of a mouse cytotoxic T-cell clone and a rat T lymphoma. Yao and colleagues (1995b) suggested that the cDNA encoding CTLA-8 could be derived from either the rat or the mouse fusion partner and subsequent studies (Yao et al., 1996b; Kennedy et al., 1996) demonstrated the sequence to be of rat origin. Polymerase chain reac-

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tion (PCR) analysis of activated rat splenocytes, using primers from the CTLA-8 sequence, revealed the rat nucleotide sequence to be identical to murine CTLA8, confirming the murine CTLA-8 to be a rat homolog of IL-17 (GenBank Accession No. Q61453). Earlier studies by Rouvier et al. (1993) and Yao et al. (1995a) reported IL-17 message in activated T cells and in a murine thymoma cell line EL4, but the exact origin of IL-17 was not determined. In an effort to identify the origin of murine IL-17 and to generate a molecular profile for abTCR CD4 CD8 (DN) cells, Kennedy et al. (1996) using subtraction techniques isolated genes specifically expressed by activated mouse abTCR DN thymocytes. Mouse IL-17 (GenBank Accession No. U43088) isolated from this cDNA library exhibited 87.3% amino-acid identity with rat IL-17 (i.e. CTLA-8), 62.5% identity with human IL-17 and 57.5% identity with HVS-13 (Kennedy et al., 1996) (Figure 20.1). Alignment of these proteins indicated conservation of six cysteines, one putative N-glycosylation site, and three consensus phosphorylation sites. The six

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conserved cysteines shared spatial features with the canonical cysteine knot motif found in nerve growth factor, transforming growth factor b (TGFb), plateletderived growth factor-BB, human chorionic gonadotropin hormone and in Toll ligand Spatzle (Lebecque et al., 2000). The abTCR DN cells, generally found in the thymus, spleen, lymph nodes and bone marrow, when activated, appeared to be the only abundant source of mIL-17 (Kennedy et al., 1996). PCR analysis of other activated T-cell subsets revealed IL-17 message, although this was not significantly high enough to be detected by Northern analysis. The abTCR DN cells were also capable of producing T helper 2 (TH2)-type cytokines (IL-4, IL-5, IL-10 and IL-13) and expressed genes commonly associated with CD8 T cells, such as interferon c (IFN c), lymphotactin, RANTES (regulated upon activation, normal T cell expressed and secreted), granzyme B and Fas ligand (FasL) (Kennedy et al., 1996). In addition, they appear to be among the first cells to be activated during an immune response.

FIGURE 20.1 Homology between mouse IL-17, HVS ORF-13, human IL-17 and murine CTLA-8 proteins. The homologous residues are within boxes. The conserved cysteines between the homologs are boxed in black. The conserved potential phosphorylation sites are within shaded boxes. The conserved potential N-linked glycosylation site is within the hatched box. (Reproduced from Kennedy et al. (1996) with permission from the publishers of the Journal of Interferon and Cytokine Research.) THE CYTOKINES AND CHEMOKINES

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HUMAN IL-17: ISOLATION, CHARACTERIZATION AND EXPRESSION A partial human IL-17 (hIL-17) sequence was obtained by amplification of fragments of human genomic DNA with degenerate primers from HVS-13 and rat IL-17 nucleotide sequences (Yao et al., 1995b). The degenerate PCR-derived partial hIL-17 sequence was used as a probe to screen a CD4 T-cell clone kgt10 library, and led to the isolation of a 155-aminoacid peptide bearing an N-terminal hydrophobic leader sequence of 19 amino acids (GenBank Accession No. U32659). The predicted mass of this protein was 17.5 kDa. Human IL-17 exhibits an overall sequence identity of 72% at the amino-acid level with HVS-13 and 63% with rat IL-17. Expression in CV1/EBNA cells generated proteins of 15 kDa and 20 kDa under reducing conditions, with the 20 kDa protein disappearing in the presence of tunicamycin. When immunoprecipitates were analyzed under nonreducing conditions, the expressed proteins had masses of 30 kDa and 38 kDa, suggestive of disulfidelinked dimers (Yao et al., 1995b). Northern analysis of various tissues indicate the constitutive expression of hIL-17 to be quite restricted. Human IL-17 mRNA was detected in PI-activated CD4 T cells, in a CD4 T-cell clone (clone 22) and in peripheral blood T cells, but not in resting PBMCs, CD4 T cells, CD8 T cells, monocytes, B cells, or in a variety of cell lines and human tissues tested (Yao et al., 1995b; Fossiez et al., 1996). The abundant expression of hIL-17 mRNA in activated human peripheral T cells is unlike that found with rat IL-17, which could not be induced by similar stimuli (Rouvier et al., 1993). Subsequently, Zhou et al., (1998a) have demonstrated IL-17 expression in a human B-cell line, AF10. Also, Shin et al. (1998) reported human CD8 T cells to express high levels of IL-17 and IFNc mRNA upon activation with ionomycin and PMA. Exposure of these activated CD8 T cells to the cAMP analogue (i.e. dibutyryl cAMP) or PGE2, caused a significant reduction in IL-17 and IFNc levels, and markedly increased IL-10 production, suggesting a means to modulate the relative role of CD8 T cells in immune responses. In a subsequent study, Shin and co-workers (1999) detected increased expression of IL-17 mRNA in memory CD845RO

T cells and CD445RO T cells following stimulation of human PBMC with ionomycin/PMA. Once again, PGE2 or dibutyryl cAMP inhibited IL-17 and IFNc expression in memory T cells, but markedly increased IL-10 levels, indicating the PKA activation pathway to play a critical role in T-cell cytokine regulation and function. In order to facilitate structural and functional characterization studies on IL-17, Murphy and colleagues (1998) developed a yeast (Pichia pastoris) expression system to generate hIL-17. The purified recombinant hIL-17 derived from the clone was found to be similar to those produced in mammalian cells. Zhou et al. (1998b) also developed a high-level expression system for hIL-17 in P. pastoris. The rhIL-17 derived from the yeast, bound the IL-17 receptor on 3T3 cells and induced their secretion of IL-6. In vivo, chronic immune reactions lead to polarized TH cytokine patterns. To classify the T-cell subset that produces IL-17 in Lyme arthritis – an aspect of Lyme disease caused by the spirochaete Borrelia burgdorferi, Infante-Duarte et al. (2000) performed single-cell cytometric analysis of TH cells for cytokine production. They found IL-17-producing Th cells to be different from the ‘classical’ TH1 or TH2 cells and to be characterized by the co-expression of the proinflammatory cytokines IL-17, tumor necrosis factor (TNFa) and granulocyte macrophage – colony stimulating factor (GM-CSF). Aarvak et al. (1999b) also attempted to categorize the TH-cell subset that produced IL-17 and developed 33 different CD4 T-cell clones from the synovial membranes and synovial fluid of rheumatoid arthritis patients. Thirteen clones were defined as TH1, four clones as TH0 and 16 clones as TH2. IL-17 was produced by some of the TH1/TH0-type cells but not by the TH2-type cells, suggesting that IL-17 could define a new subset of T cells (Aarvak et al., 1999b). Addition of TH2 cytokines inhibited the production of IL-17, while TH1-promoting cytokines increased its production. TH2 clones from rheumatoid arthritis readily switched to TH0 or TH1 phenotype, whereas the TH1 and TH0 clones were stable (Aarvak et al., 1999a). Albanesi and co-workers (1999) evaluated a panel of 80 nickel-specific CD4 T cell clones (36 TH0, 30 TH1 and 14 TH2) isolated from the peripheral blood or lesional skin of allergic contact dermatitis patients and found IL-17 to be released by about 50% of activated TH0, TH1 and TH2 clones. In another study, the antigen-induced IL-17 response in PBMCs of healthy

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controls was evaluated (Lenarczyk et al., 2000) and IL17 production was found not to correlate with either the type-1 (IFNc or TNFa) or the type-2 (IL-4 or IL-10) response at both the mRNA and protein levels. Taken together, the above studies demonstrate that IL-17 secretion does not segregate to a particular TH subset.

THE IL-17 RECEPTOR: CLONING, SEQUENCE AND EXPRESSION The mIL-17R was identified by searching for proteins that bound to an HVS-13.Fc fusion protein that was constructed by fusing a portion of the Fc region of human IgG1 with residues 19–151 of HVS-13 (Yao et al., 1995a). Screening of a cDNA expression system for binding proteins led to the isolation of two cDNA clones that specifically bound HVS-13.Fc. The open reading frame of these clones encoded an extremely long type-I transmembrane protein of 864 amino acids, which included an N-terminal signal peptide with a cleavage site after amino acid 31, followed by a 291-amino-acid extracellular domain, a 21-aminoacid transmembrane domain and a 521-amino-acid cytoplasmic tail. The IL-17R exhibits no sequence similarity with any other known cytokine receptor family (Figure 20.2). The protein has a predicted molecular mass of 97.8 kDa and an estimated isoelectric point of 4.85 (Yao et al., 1995a). The mIL-17R gene (GenBank Accession No. U31993) was localized to chromosome 6. The receptor has 12 cysteine residues in the extracellular domain with relative positions not characteristic of members belonging to the immunoglobulin superfamily or the TNF receptor family. Despite the presence of relatively large proportions of acidic and proline residues similar to those observed in many growth factor receptors, the large cytoplasmic tail has no sequence homology with any other growth factor receptor known to be a tyrosine kinase and has no recognizable motifs associated with intracellular signaling. Northern analysis with a probe to mIL-17R revealed expression in virtually all cells and tissues. This ubiquitous tissue distribution of IL-17R is in stark contrast to the restricted expression of IL-17. A soluble IL-17R.Fc fusion protein was found to significantly inhibit IL-2 production and T-cell proliferation

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induced by concanavalin A, PHA, anti-TCRab mAb and anti-CD28 mAb, suggesting a key role for endogenously produced IL-17 in T-cell proliferation (Yao et al., 1995a). Subsequently, Yao and colleagues (1997) using nucleic acid hybridization techniques, isolated a 3195-bp cDNA encoding the human homolog of the mIL-17R (i.e. hIL-17R) from a human T-cell library (GenBank accession No. U58917). The hIL-17R sequence predicted an 866-amino-acid type-I membrane glycoprotein with a 293-amino-acid extracellular domain, a 21-amino-acid carboxy-proximal transmembrane domain, and a 525-amino-acid cytoplasmic tail. Seven potential N-linked glycosylation sites were identified, with six conserved between the mouse and human receptors. The amino-acid sequence of the hIL-17R was 69% identical to the mIL-17R, and shared no homology with previously identified cytokine receptor families. The hIL-17R gene has been mapped to chromosome 22. Monoclonal antibody, m202, generated against hIL-17R, immunoprecipated the receptor and blocked IL-17induced cytokine production in human foreskin fibroblast cells. Like its murine counterpart, the hIL-17R exhibits a broad tissue distribution (Yao et al., 1997).

IL-17 SIGNALING PATHWAY Comprehension of the IL-17 signaling pathway has been particularly challenging, since the large cytoplasmic tail of the IL-17R exhibits no identifiable tyrosine kinase homology region(s) and presents no recognizable motifs associated with intracellular signaling. Binding of IL-17 to its receptor initiates the intracellular signaling cascade that eventuates in a variety of cellular responses. NFjB, a common effector of gene regulatory activities mediated by proinflammatory cytokines, is induced by IL-17 in mouse 3T3 fibroblasts (Yao et al., 1995a), in human chondrocytes (Shalom-Barak et al., 1998) and in intestinal epithelial cells (Awane et al., 1999; Andoh et al., 2001). NFjB activation is preceded by the activation of IjB kinase-a and -b(IKKs). NFjB proteins are sequestered in the cytoplasm by heterodimerization with IjB inhibitory proteins. IKKs phosphorylate two serine residues on IjB, marking them for proteolysis.

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FIGURE 20.2 Nucleotide sequence of mIL-17R cDNA. The nucleotide sequence of a cDNA encoding the IL-17R and the predicted amino acid sequence is illustrated. The signal peptide and the putative transmembrane region are underlined and doubly underlined, respectively. The potential N-linked glycosylation sites are italicized and underlined. (Reproduced from Yao et al. (1995a) with permission of the authors and publishers.)

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Phosphorylation and degradation of IjB causes the release and translocation of NFjB proteins to the nucleus where they activate gene transcription. NFjB activation by IL-17 is on a par with its ability to induce the secretion of various cytokines and chemokines (IL-1b, IL-6, IL-8, TNFa, macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1 etc.) known to be under the transcriptional control of this factor (Lebecque et al., 2000). Kehlen et al. (1999) evaluated the biologic effects of IL-17 on glial cells, and found IL-17 to up-regulate IjB-a mRNA expression in a dose- and time-dependent fashion and to stimulate the secretion of IL-6 and IL-8 from glial cells. IL-17 induced the rapid phosphorylation and degradation of IjB molecules, and in turn, caused a marked increase in NFjB DNA-binding activity in human fetal intestinal epithelial cells (Andoh et al., 2001). This was followed by an increase in nuclear factor-IL-6 and activator protein (AP)-1 DNA-binding activity. Since AP-1 is activated by a phosphorylation event mediated by mitogenactivated protein (MAP) kinases, one could speculate that IL-17 activates MAP kinases as well. In accordance with this hypothesis, in rat intestinal epithelial cells, IL-17, in addition to activating NFjB, also regulated the activities of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinases (Awane et al., 1999). IL-17-mediated activation of ERK kinases was mediated through ras and JNK activation was dependent on functional TRAF-6 (Awane et al., 1999). IL-17 is also known to regulate claudin expression in intestinal epithelial cells through the ERK–MAP kinase pathway (Kinugasa et al., 2000), and IL-6 and IL-8 secretion in primary bronchial epithelial cells via activation of ERK1 and ERK2, but not p38 or JNK kinases (Kawaguchi et al., 2001b). A selective MAP kinase kinase inhibitor blocked IL-17-induced IL-6 and IL-8 production in the latter cell population (Kawaguchi et al., 2001b). Laan et al. (2001) also evaluated the role of MAP kinases in IL-17-induced release of IL-6 and IL-8 from bronchial epithelial cells and found p38 MAP kinase and ERK kinase pathway inhibitors to block the release of these cytokines in a concentration-dependent manner. These studies further support the involvement of MAP kinases in IL-17-mediated signaling. In chondrocytes, IL-17 in addition to inducing NFjB DNA-binding activity, also induced MAP kinases, p38, ERK1, ERK2 and JNK (Shalom-Barak

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et al., 1998). Evaluation of the effects of dexamethasone (an antagonist of NFjB DNA-binding activity) and SB203580 (a p38 kinase inhibitor) on IL-17induced nitric oxide (NO) production and gene expression, revealed the p38 kinase inhibitor to be moderately efficient and dexamethasone to be highly efficient in inhibiting IL-17-mediated NO release, iNOS, COX-2 and IL-6 protein expression. In addition, dexamethasone inhibited IL-17-induced activation of MAP kinases, especially activation of JNK, and reduced IL-17-induced NF-jB activity. These findings suggest that p38 may not be essential, but dexamethasone-sensitive signal transduction events may be important in mediating IL-17-induced activation of chondrocytes. Glucocorticoids are known to regulate NFjB activity by enhancing IjB expression. In this study, however, dexamethasone reduced, but did not abolish IL-17-induced NFjB activity, an effect attributed to lack of IjB induction in dexamethasonetreated chondrocytes (Shalom-Barak et al., 1998). It is therefore concluded that NFjB may not be the key transcription factor in mediating IL-17 signal transduction. Martel-Pelletier et al. (1999) reported NFjB and MAP kinases together to regulate stimulation of NO production by IL-17 in human osteoarthritic chondrocytes. NFjB appeared to be necessary for MAP kinase activation, with downstream activation of MAP kinase-activated protein kinase (MAPKAPK) acting perhaps as a transactivating factor, to induce iNOS expression (Martel-Pelletier et al., 1999). Receptor activator of the NFjB ligand (RANKL), expressed on osteoblast/stromal lineage cells binds to its receptor RANK, expressed on osteoclasts, and induces their differentiation. IL-17 is known to increase the expression of RANKL and decrease the expression of its receptor RANK, a mechanism by which it is believed to promote and regulate osteoclastic bone resorption (Van Bezooijen et al., 1999; Martel-Pelletier et al., 1999; Nakashima et al., 2000). Although it is now well documented that IL-17 activates NFjB and MAP kinases, the upstream signaling events remain to be elucidated. Schwander et al. (2000) report the requirement for TNF receptor-associated factor (TRAF)-6 for IL-17-induced NFjB and JNK activation. IL-17 failed to activate IKK and JNK in embryonic fibroblasts derived from TRAF-6 knockout mice, and as a result inhibited IL-17-induced IL-6 and

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intercellular adhesion molecule (ICAM)-1 expression in these cells. This defective IL-17 response was not observed in embryonic fibroblasts derived from TRAF-2 knockout mice, indicating that TRAF-6 and not TRAF-2 may be essential in the IL-17-signaling cascade (Schwander et al., 2000). Upon IL-17 stimulation signaling may be initiated at the cell surface by the direct activation of receptor protein tyrosine kinases, a rapid event linked to several downstream signaling elements. IL-17 is known to induce phosphorylation of several cellular proteins, including activation of raf-1 serine/threonine kinase (Subramaniam et al., 1999a). Shin et al. (1998) propose the protein kinase-A (PKA) activation pathway to play a prominent role in regulating IL-17 expression in CD8 T cells. A significant reduction in IL-17 and IFNc expression was observed in human CD8 T cells following their exposure to cAMP analog, dibutyryl cAMP or PGE2. These results are supported by the work of Jovanovic and colleagues (1998), who found IL-17-induced production of TNFa in macrophages to be either completely or partially inhibited by PKA-specific or non-specific tyrosine kinase inhibitors and by MAP kinase kinase inhibitors, respectively. In human macrophages, IL-17 stimulated a transient Ca2 influx and a reduction in intracellular cAMP and exposure of these cells to Calphositin C, an agent that inhibits Ca2-regulated protein kinase C, inhibited their response to IL-17, including a decrease in IL-17-induced IL-1b and TNFa production (Jovanovic et al., 1998). Early signaling events triggered by IL-17 in human U937 monocytic leukemic cells include a time-dependent stimulation of tyrosine phosphorylation of JAK1, 2 and 3, Tyk 2 and STAT 1, 2, 3 and 4. Findings suggest that the JAK/STAT signaling pathway plays a major role in transducing IL-17 signals from the receptor to the nucleus in human cells (Subramaniam et al., 1999b).

NEW MEMBERS OF THE IL-17 FAMILY AND THEIR IMMUNOBIOLOGY IL-17B and IL-17C Progress in biotechnology and the recent advances in human genome sequencing have facilitated the discovery of new genes. Li et al. (2000) identified two new

proteins that are related to IL-17 (approximately 27% overall amino-acid identity including five conserved cysteine residues), IL-17B and IL-17C (GenBank Accession Nos AF152098 and AF152099), establishing that there exists a family of IL-17-related molecules. Like IL-17, the two new members are 150–200 amino acids in length and are secreted from cells via a hydrophobic leader sequence. However, the genes for the new family members are dispersed and not localized to the IL-17 gene 2q31. IL-17B has been localized to 5q32–34 and IL-17C to 16q24. Unlike IL-17, IL-17B and IL-17C mRNA transcripts could not be detected in activated CD4 T cells, with IL-17B expression limited to normal human adult pancreas, small intestine and stomach, and IL-17C expression to adult prostrate and fetal kidney libraries (Li et al., 2000). The new members bound to the monocytic cell line, THP-1 and stimulated the release of TNFa and IL-1b, while IL-17 only had a weak effect in this system. Likewise, IL-17B and IL-17C were not active in an IL-17 assay and were unable to induce IL-6 release from human fibroblasts. Evidently, these new members do not bind the IL-17R, alluding to the existence of other receptor molecules (Li et al., 2000). Concurrently, Shi et al. (2000) isolated and characterized the ligand–receptor pair, IL-17B:IL-17BR (GenBank Accession Nos AF212311 and AF212365). The IL-17B amino acid sequence was identical to the IL-17B sequence characterized by Li et al. (2000). The ligand was highly transcribed in human spinal cord, testis and small intestines, with low levels of expression in many other organs. The AU-rich repeats indicative of transient expression found in IL-17 and other cytokines was absent in the 3-untranslated region of the IL-17B transcript. Unlike the IL-17R, IL-17BR exhibited a more restricted tissue expression pattern, largely limited to the kidney, liver, pancreas and intestines. The IL-17BR also had a short cytoplasmic tail, which may account for possible differences from the IL-17R in downstream signaling mechanisms. The IL-17BR protein sequence is 19.2% and 18.2% identical to the human and murine IL-17R sequences, respectively, and the map location of IL-17BR was determined at 3p21.1 (Shi et al., 2000). Treatment of mice with recombinant IL-17B protein induced a dose-dependent influx of neutrophils into the peritoneal cavity, suggesting IL-17B to be involved in inflammatory processes.

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IL-17E / IL-25 Characterization of the newly identified proteins described above suggested proinflammatory activity to be a key attribute for this family of cytokines. Hence, identification of other IL-17 homologs would be of clinical significance. Renewed interest in the search for other IL-17 members and their receptors led to the identification of IL-17E and its receptor. Lee et al. (2001) identified IL-17E, a 177-amino-acid protein that was 16–20% identical to IL-17, IL-17B and IL-17C, and was expressed at very low levels in several peripheral tissues (GenBank Accession No. AF305200). IL-17E was found to bind IL-17BR, the IL-17B receptor earlier reported by Shi et al. (2000). Owing to its ability to bind more than one ligand, the IL-17BR was designated IL-17 receptor homolog 1 (IL-17Rh1). IL-17E induced NFjB activation and stimulated the proinflammatory chemokine IL-8 in human TK-10 kidney-derived cells (Lee et al. 2001). In a subsequent study, the same research group demonstrated transgenic mice overexpressing IL-17E to have up-regulated several cytokines, namely IL-4, IL-10, IL-13, IL-5, IFNc and TNFa (GenBank Accession No. for mIL-17E is AY034088). In addition, these animals expressed pathologic changes in multiple organs characterized by mixed inflammatory infiltration, epithelial hyperplasia and hypertrophy, were jaundiced and growth retarded, indicating unique pleiotropic proinflammatory activities for IL-17E (Pan et al., 2001). Furthermore, IL-17E was found to induce catabolism of the human articular cartilage (Cai et al., 2001). More recently, Fort et al. (2001) have identified a sequence that bears homology to the IL-17E sequence reported by Lee et al. (2001). Produced by TH2 cells, this new member was named IL-25, a designation approved by the IUIS subcommittee on interleukin nomenclature (Fort et al., 2001). Although IL-25 is structurally related to IL-17, it displays unique biologic functions distinct from those described for IL-17 and its other newly identified members, hence its classification as another interleukin (Fort et al., 2001). However, this nomenclature leaves the reader uncertain as to whether IL-17E be referred to as IL-25 or vice versa. In mice, in vivo treatment with IL-25 (GenBank Accession Nos for IL-25; NM_080729, AF458060) induced a significant increase in the cytokines IL-4, IL-5 and IL-13, augmented serum

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IgG1, IgE and IgA, caused eosinophilia and splenomegaly; effects attributed to mobilization of hematopoietic progenitors. Furthermore, treated mice also exhibited striking histologic changes in the lungs and GI tract that included eosinophilic and mononuclear infiltrates, increased mucus production, epithelial cell hyperplasia and hypertrophy (Fort et al., 2001). Most of these observations, like those reported by Pan et al. (2001), suggest a distinct role for this cytokine, possibly in TH2-mediated inflammatory responses.

IL-17F and ML-1 The next member to be included in the IL-17 family of proteins was IL-17F, identified independently by Starnes et al. (2001) and Hymowitz et al. (2001). Starnes et al. (2001) reported IL-17F to be selectively expressed in monocytes and activated CD4 T cells. The gene encoding IL-17F was located adjacent to the IL-17 gene at chromosome 6p12 (Accession no. AL355513). The recombinant form of human IL-17F regulated angiogenesis and cytokine production (IL-2, TGFb and MCP-1) in human endothelial cells, functions distinctive from those reported for other IL-17 members. Hymowitz and colleagues (2001) also identified IL-17F, at the same time as Starnes et al. (2001), and found it to be expressed in activated T cells, and to stimulate IL-6, IL-8 and G-CSF production in primary human fibroblasts. The chromosomal location as well as the amino-acid sequence of this homolog was identical to the one described by Starnes et al. (2001). The mature protein exhibited 50% amino-acid identity to IL-17 and is encoded by 163 amino acids, which include a signal sequence of 30 residues. IL-17 and IL-17F share a lesser 16–30% amino-acid identity with IL-17B, C and E, suggesting that the two may form a distinct subgroup within the IL-17 family (Hymowitz et al., 2001). Interestingly, IL-17F does not bind either of the two reported receptors, IL-17R or IL-17Rh1. Since, IL-17F shared several features with IL-17, and IL-17 has been implicated in the pathogenesis of rheumatoid arthritis, the authors evaluated the role of IL-17F on cartilage matrix metabolism. Although less potent than IL-17 in inducing IL-6 production in human and porcine cartilage, IL-17F was as potent in

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FIGURE 20.3 Structure of IL-17F. (A) The ribbon trace of IL-17F monomer. The strands are numbered and the disulfides depicted as a ball-and-stick with the sulfur atoms shaded gray. Inset denotes a cartoon representation of the cysteine knot fold. Open circles represent the cysteine residues present in IL-17 members and the two filled circles represent the missing residues. (B) The ribbon trace of IL-17F dimer. (C) The structure of NGF from NGFTrkA complex. (Reproduced from Hymowitz et al. (2001) with permission of the authors and publishers.) regulating cartilage matrix turnover (Hymowitz et al., 2001). In RA pathogenesis, activated T cells may stimulate other cells such as macrophages and fibroblasts to release cytokines, which then amplify the local immune response and promote synovitis. Since IL-17F is a T-cell cytokine that activates IL-6 production and cartilage matrix turnover, like IL-17 it could also be a key player in the RA disease process. Surprisingly, the crystal structure of IL-17F reveals IL-17 family members to adopt a monomer fold typical of the cysteine knot family of proteins, named so for its unusual but distinctive cysteine linkages (Hymowitz et al., 2001). The cysteine knot fold is characterized by two pairs of b strands (strands 1 and 2 and 3 and 4) that are connected by two disulfide linkages between strands 2 and 4 (Figure 20.3) and a third disulfide bridge passes through this macrocycle to connect strands 1 and 3. In IL-17F, the two disulfide linkages, Cys72/Cys122 and Cys77/cys124, form the macrocycle of the typical cysteine knot, and residues 50 and 90, located in the same three-dimensional space as the third disulfide in cysteine knot proteins are made up of serines in IL-17F (Figure 20.4). Notably, the serines are conserved in these positions in all IL-17 members (Figure 20.4). Despite lacking the third disulfide responsible for defining the ‘knot’ structure, IL-17F dimerizes in a parallel fashion simi-

lar to nerve growth factor (NGF), and features an unusually large cavity (two per dimer) on its surface. The cavity is located on the same position where NGF binds its high-affinity receptor, TrkA, suggesting similarities between IL-17s and neurotrophins with regard to receptor recognition. The cysteine knot fold including the location of b sheets and the macrocycle disulfide linkages are supposedly preserved among all the IL-17 members. Nevertheless, IL-17B, C and E, each have long N-terminal sequences and additional cysteine residues making them significantly different to IL-17 and IL-17F (Hymowitz et al., 2001). Additional structural information on these IL-17 members will be required to predict accurately molecular and structural homology. As part of ongoing molecular genetic studies of complex diseases, Kawaguchi et al. (2001a), identified a novel gene from a human genomic DNA clone and from human T cell cDNA sequences, and called it ML-1. The expressed sequence, with a centromerictelomeric orientation is made up of two exons, of 221bp and 238 bp, respectively. Homology searches revealed an overall amino-acid sequence homology of 70% between ML-1 and IL-17, and only 20% between ML-1 and IL-17B and IL-17C. Alignment of the amino-acid sequences for ML-1, IL-17, IL-17B and IL-17C revealed several conserved cysteine residues.

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FIGURE 20.4 Sequence alignment of IL-17F with other IL-17 members. Residue numbering is the start of mature sequences. Regions similar between IL-17F and IL-17 are highlighted in gray and other members that have similar residues in the same position are also in gray. All cysteine residues are indicated with white letters on black. Conserved serines that replace the canonical knot cysteines are highlighted with white letters on gray. The bonded cysteines representing the disulfide bonds, which are likely to be conserved between IL-17 members, are connected with black lines. The two cysteines that form the inter-chain disulfide in IL-17F are marked with asterisks. The secondary structural elements in IL-17F are depicted above the sequences as arrows (b strands) or a cylinder (a helix). (Reproduced from Hymowitz et al. (2001) with permission of the authors and publishers.)

These residues are identical to the two disulfide linkages, Cys72/Cys122 and Cys77/cys124, that form the macrocycle of the typical cysteine knot described by Hymowitz et al. (2001), expected to be conserved in all IL-17 members. Interestingly, the ML-1 sequence (GenBank Accession No. AF332389) appears to be a

truncated form of the IL-17F sequence identified by Starnes et al. (2001) and Hymowitz et al. (2001). The ML-1-coding region starts with an internal Met at residue 25 of the mature protein, and lacks a significant portion of the N-terminus and the signal sequence. The tissue expression patterns of IL-17F

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and ML-1 are also different. ML-1 has a wider expression pattern compared with IL-17, with gene expression in activated PBMCs, CD4 T cells, TH0, TH1 and TH2 clones, and cell types involved in allergic responses, namely activated basophils and mast cells. In addition to stimulating primary bronchial epithelial cells to express high levels of IL-6, ML-1 also induces high levels of IL-8 and ICAM-1; molecules involved in facilitating leukocyte recruitment and activation in the airway epithelium (Nocker et al., 1996 and Manolitsas et al., 1994). Seemingly, this IL-17 member and not IL-17, is up-regulated following allergen challenge in asthmatic patients, suggesting its involvement in allergic inflammatory disorders (Kawaguchi et al., 2001b). As is evident from the above studies, the functional parameters each of the research teams evaluated for IL-17F and ML-1 are different, making it difficult to resolve the functional identity of this new member at the present time.

BIOLOGIC ACTIVITIES OF IL-17 Production of cytokines, chemokines and other immune effectors IL-17 is characterized as a cytokine-inducing cytokine that exhibits pleiotropic biologic activities on various cell types (Table 20.1). The predominantly proinflammatory and hematopoietic activities of IL-17 are due to its ability to stimulate the release of secondary cytokines and chemokines (Table 20.1). IL-17 stimulated stromal cells derived from various tissues to secrete IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and PGE2, an effect that can be specifically blocked by an anti-IL-17 mAb (Fossiez et al., 1996). In human macrophages, IL-17 augmented the production of proinflammatory cytokines TNFa, IL-b, PGE2 and IL-6, as well as IL-10, IL-12, IL-1R antagonist and stromelysin (Jovanovic et al., 1998). Thiele and co-workers (2000) found cell–cell contact of human T cells with fibroblasts, to augment IL-17 and IL-17R expression in lymphocytes, and to enhance IL-6 and IL-8 production by fibroblasts. The enhanced secretion of these proinflammatory cytokines was mediated by IL-17, as antibodies to IL-17 abrogated the effect. IL-17 also induced a plethora of chemokines in

various cell subsets; namely, IL-8, IP-2, MIP-2, growth related oncogene (GRO)-a and alpha chemokines in bronchial epithelial cells (Laan et al., 1999; Molet et al., 2001; Kawaguchi et al., 2001b), IL-8, cytokineinduced neutrophil chemoattractant (CINC) – a rat homologue of human GRO-a, and MCP-1 in intestinal epithelial cells (Andoh et al., 2001; Awane et al., 1999), IL-8, MCP-1 and RANTES in tubular epithelial cells (Van Kooten et al., 1998; Woltmann et al., 2000) and IL-8, MIP-3a in synoviocytes and keratinocytes (Chaubaud et al., 2001d; Homey et al., 2000). Because of the ability of hIL-17 to induce IL-8 in various cell types and the capture of these two genes by HVS, the possibility of a functional relationship between IL-8R and IL-17 has been hinted at. In addition, both the IL-8R gene cluster and the hIL-17 gene map to human chromosome 2q31–q35 (Fossiez et al., 1996). HVS-13 (vIL-17) secreted by HVS-infected cells may bind to adjacent uninfected cells expressing IL-17R, enhancing differentiation and/or proliferation, thereby making them susceptible targets for viral infection and replication. Therefore, abnormalities in IL-17 secretory levels may indicate disease progression. In addition to inducing cytokines and chemokines, IL-17 presents itself with certain other unique functions. IL-17 enhanced surface expression of ICAM-1 in human fibroblasts (Fossiez et al., 1996) and keratinocytes (Teunissen et al., 1998; Albanesi et al., 1999), and was found to promote modestly maturation of dendritic cell (DC) progenitors as evidenced by increased surface expression of CD11c, CD40, CD80, CD86 and MHC class II antigens (Antonysamy et al., 1999b). IL-17 also regulated complement factor B and C3 gene expression and protein synthesis in human skin fibroblasts (Katz et al., 2000) and induced NO production in chondrocytes, osteoblasts and astrocytes (Attur et al., 1997, Shalom-Barak et al., 1998; Martel-Pelletier et al., 1999; Rifas and Avioli, 1999; Van Bezooijen et al., 2001; Trajkovic et al., 2001). Like most cytokines, IL-17 is known for its ability to synergize with other cytokines. For example, neither IL-17 nor TNFa alone had any effect on GM-CSF secretion, but together were capable of inducing GM-CSF production by synovial fibroblasts (Fossiez et al., 1996; 1998). IL-17 enhanced TNFa-induced synthesis of complement factor B, IL-1, IL-6 and IL-8 by skin and synovial fibroblasts (Katz et al., 2000, 2001). Besides,

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TABLE 20.1 An overview on the in vitro biologic effects of IL-17 Cell type

Cytokines, chemokines and other immune effector functions induced

References

Macrophages

TNFa, IL-1b, IL-1R antagonist, IL-6, IL-10, IL-12, PGE2, MMP9, tissue inhibitor of metalloproteinase 1 (TIMP-1) and stromelysin

Jovanovic et al., 1998

Dendritic cell progenitors

Induced maturation

Antonysamy et al., 1999b

T lymphocytes

Increased proliferation

Yao et al., 1995b

Bone marrow (BM) cells

IL-6, IL-1a, EPO, CFU-GM and BFU-E progenitors

Jovcic et al., 2001

BM stromal cells

G-CSF and SCF

Schwarzenberger et al., 2000

Fibroblasts

IL-6, IL-8

Yao et al., 1995a, 1995b; Fossiez et al., 1996; Kennedy et al., 1996; Zhou et al., 1998b; Thiele et al., 2000; Schwander et al., 2000; Molet et al., 2001 Katz et al., 2001 Fossiez et al., 1996; Cai et al., 1998 Fossiez et al., 1996 Fossiez et al., 1996; Schwander et al., 2000 Molet et al., 2001; Chaubaud et al., 1998 Molet et al., 2001 Katz et al., 2000 Chaubaud et al., 1998, 2001a

Synergizes with TNFa to induce IL-6, IL-1b and IL-8 G-CSF PGE2, synergizes with TNFa to induce GM-CSF ICAM-1 IL-11 a chemokines Complement components, C3 and factor B Leukemia inhibitory factor (LIF), IL-6 Chondrocytes

IL-6, NO

Attur et al., 1997; Shalom-Barak et al., 1998; Martel-Pelletier et al., 1999

Synoviocytes

IL-6, IL-8, G-CSF and PGE2 Synergizes with T cells to induce IL-6, IL-8 and PGE2 Synergizes with IL-1b to induce IL-6 and LIF Synergizes with IL-1b and TNFa to induce MIP-3a

Chaubaud et al., 1998 Yamamura et al., 2001 Chaubaud et al., 1998; 1999 Chaubaud et al., 2001d

Osteoblasts

Synergizes with TNFa to induce IL-6 and NO IL-6 PGE2 and ODF/RANK expression

Van Bezooijen et al., 2001 Rifas and Avioli, 1999 Kotake et al., 1999

Bone

Increased TNFa-induced IL-1a, IL-1b, IL-6 IL-6

Van Bezooijen et al., 1999 Chaubaud et al., 2001b

Cartilage

NO

Attur et al., 1997

Bronchial epithelial cells

IL-8

IP-2, MIP-2 Growth-related oncogene (GRO)-a, a chemokines

Laan et al., 1999; Molet et al., 2001, Kawaguchi et al., 2001b Kawaguchi et al., 2001b; Laan et al., 2001 Laan et al., 1999 Molet et al., 2001

Intestinal epithelial cells

IL-8 and MCP-1 CINC and MCP-1

Andoh et al., 2001 Awane et al., 1999

Gastric epithelial cells

IL-8

Luzza et al., 2000

IL-6

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TABLE 20.1 Continued Cell type

Cytokines, chemokines and other immune effector functions induced

References

Tubular epithelial cells

Complement component C3, IL-6, IL-8, and MCP-1 Synergized with CD-40L to induce IL-6, IL-8, MCP-1 and RANTES

Van Kooten et al., 1998; Woltman et al., 2000

Endothelial cells

IL-6, IL-8 IL-8

Fossiez et al., 1996 Laan et al., 1999

Mesothelial cells

GRO-a

Witowski et al., 2000

Keratinocytes

IL-6 IL-8, synergized with IFNc to induce ICAM-1 Synergized with IFNc to induce HLA-DR MIP-3a/CCL20 and its receptor CCR6

Teunissen et al., 1998 Teunissen et al., 1998; Albanesi et al., 1999 Teunissen et al., 1998 Homey et al., 2000

IL-1b, IL-6 and NO

Trajkovic et al., 2001

Astrocytes Glioblastoma cells

IL-6, IL-8

Kehlen et al., 1999

Melanoma cells

IL-6, IL-8

Tartour et al., 1999

Cervical carcinoma cells

IL-6, IL-8

Tartour et al., 1999

TNFa, IL-1b and IFNc synergized with IL-17 and augmented IL-17-induced secretion of IL-6 by rheumatoid synoviocytes (Fossiez et al., 1996) and up-regulated NO and PGE2 production by human osteoarthritic knee menisci (LeGrand et al., 2001). IL-17 was also found to have an enhancing effect on IL-1-induced IL-6 and leukemia inhibitory factor (LIF) production by rheumatoid synoviocytes (Chabaud et al., 1998). In human keratinocytes, IL-17 specifically and dose-dependently augmented IFNc-induced ICAM-1 expression and the induction of IL-8 ( Teunissen et al., 1998; Albanesi et al., 1999), IL-6 and HLA-DR (Teunissen et al., 1998). In fetal mouse long bones, IL-17 increased TNFa-induced IL-1a, IL-1b, and IL-6 and together with TNFa caused a dose-dependent increase in bone resorption (Van Bezooijen et al., 1999). Similarly, TNFa potentiated IL-17-induced release of the CXC chemokine IL-8, from human bronchial and venous endothelial cells (Laan et al., 1999). Chaubaud et al. (2001d) evaluated the effect of IL-17 on MIP-3a production in RA synoviocytes and found IL-17, IL-1 and TNFa to have a synergistic effect on MIP-3a production. The proinflammatory and hematopoietic properties of IL-17 together with its synergistic potential identifies IL-17 to be a potential target for

therapeutic intervention and supports combination therapy to be an effective therapeutic modality.

IL-17 and hematopoiesis The ability of IL-17 to induce key hematopoietic regulators such as erythropoietin (EPO), stem cell factor (SCF), G-CSF, IL-6, IL-8 and IL-11, supports the hypothesis that IL-17 is the communicative link by which T lymphocytes interact with the hematopoietic system. The studies discussed herein tend to support this hypothesis. Notably, hIL-17 when added to fibroblast cells enhanced their ability to sustain the growth of CD34 umbilical cord-derived hematopoietic progenitors and directed their preferential maturation into neutrophils. These effects were associated with the increased secretion of hematopoietic cytokines (IL-1, IL-6, IL-8, G-CSF and IFNc) by the fibroblast feeder cells (Fossiez et al., 1996). Fine and co-workers (1997) examined the mechanism of IL-17enhanced G-CSF expression in fibroblasts. Treatment of 3T3 fibroblasts with IL-17 resulted in an increased steady state of mRNA expression and augmented production of G-CSF protein. Addition of LPS and IL-17 to fibroblasts further enhanced the level of G-CSF mRNA and protein. Stability studies have

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revealed IL-17 to stabilize LPS-induced G-CSF mRNA expression, with a t / of 4 hours compared with less than 2 hours in cells treated with LPS alone (Fine et al., 1997; Cai et al., 1998). Schwarzenberger and colleagues (1998) in an attempt to evaluate the in vivo hematopoietic effects of IL-17 overexpressed the cytokine in mice using recombinant adenovirus expression system. Adenovirus-mediated cytokine delivery of mIL-17 cDNA targeted to mice liver, resulted in leukocytosis; fivefold increase in the peripheral WBC count, that included a 10-fold increase in absolute neutrophil count, with the neutrophil count returning to baseline by day 21 and the WBC count remaining elevated. This was associated with splenomegaly and stimulation of splenic hemopoiesis as demonstrated by a 50% increase in cellularity of the spleen over 7–14 days following gene transfer (Schwarzenberger et al., 1998). These studies demonstrated IL-17 to stimulate hematopoiesis, particularly granulopoiesis in vivo. The authors hypothesized that IL-17 mediated granulopoiesis by its ability to induce the secretion of SCF and G-CSF – factors known to synergize strongly in mediating optimal granulopoiesis, from BM stromal cells. Subsequently, Schwarzenberger et al. (2000) demonstrated that IL-17 stimulated the release of G-CSF and induced enhanced expression of the transmembrane form of SCF in BM-derived stromal cell lines. To test the hypothesis that IL-17-augmented SCF was required for granulopoiesis in vivo, they evaluated IL-17-mediated granulopoiesis in Steel–Dickie (Sl/Sld ) mice which lacked functional transmembrane SCF. The synergistic requirement for G-CSF was also studied by neutralizing G-CSF in the Sl/Sld mice. They demonstrated the in vivo effects of IL-17-mediated hematopoiesis and secondary granulopoiesis required both G-CSF and functional SCF for optimal activity. In addition, IL-17 independently induced neutrophil maturation in Sl/Sld mice treated with anti-G-CSF neutralizing antibodies, protecting these mice from G-CSF neutralization-induced neutropenia (Schwarzenberger et al., 2001). The in vivo ability of IL-17 to mobilize peripheral blood stem cells was also studied (Schwarzenberger et al., 2001). Overexpression of mIL-17 using recombinant adenovirus led to effective mobilization of hematopoietic precursor cells (CFU granulocyte– 1

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erythrocyte–macrophage–monocyte, CFU-high proliferative potential) and primitive hematopoietic stem cells (Lin/lowc-kitSca1) with both short- and longterm repopulating ability (Schwarzenberger et al., 2001). This study supports clinical potential for IL-17mobilized peripheral blood stem cells. The ability of IL-17 to regulate hematopoiesis is reported to be dependent on the physiological status of the individual (Jovcic et al., 2001). In normal mouse bone marrow IL-17 induced IL-6 and EPO secretion, and increased granulocyte–macrophage (CFU-GM) and erythroid (BFU-E) progenitors, but reduced CFU-Ederived colony numbers. On the other hand, in irradiated mouse bone marrow, although IL-17 augmented IL-6, EPO and IL-1a secretion, its effect on hematopoietic progenitors varied depending on their lineage, stage of differentiation and time after irradiation (Jovcic et al., 2001).

IL-17 IN IMMUNOPATHOLOGIC CONDITIONS IL-17 in transplantation Based on the observations that IL-17 is a proinflammatory cytokine one could postulate that blocking the effects of this cytokine could modify alloimmune responses. Indeed, administration of soluble IL-17R.Fc fusion protein for varying periods post transplant to MHC-mismatched cardiac allograft recipients led to a significant increase in cardiac allograft median survival time (Antonysamy et al., 1999a, 1999b). IL-17 mediated allogeneic immunity in part via a maturation-inducing effect on DC, as evidenced by increased cell surface expression of CD40, CD11c, CD80, CD86 and MHC class II antigen and by promoting their allostimulatory capacity in vitro (Antonysamy et al., 1999b). The study supports IL-17 antagonism to therefore have potential for therapy in allograft rejection, possibly in conjunction with immunosuppressive agents that possess complimentary modes of action. Tang et al. (2001) evaluated the influence of IL-17 in the pathogenesis of acute and chronic aortic allograft rejection, using a MHC-mismatched murine model that bears both morphological and developmental similarities to

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post-transplant vasculopathy in humans. Although IL-17 antagonism suppressed immunopathogenesis of acute vascular rejection, it did not prevent ensuing chronic graft vascular disease, in particular neointimal formation, suggesting that IL-17 may not play an essential role in the pathogenesis of chronic rejection (Tang et al., 2001). Local production of cytokines could be a determining factor in transplant outcome. Immunofluorescence studies have identified the presence of IL-17 in kidney biopsies from patients suffering graft rejection, whereas pre-transplant biopsies and normal kidneys were negative. Analysis of whole kidney isolates and graft-infiltrating T lymphocytes by RT-PCR confirmed the presence of IL-17 message (Van Kooten et al., 1998). The presence of IL17 transcripts in renal biopsies was found to be both sensitive and specific (Strehlau et al., 1997) and the use of IL-17 detection as an early marker for kidney rejection was suggested (Strehlau et al., 1997; Van Kooten et al., 1998). More recently, Hsieh et al. (2001) evaluated the role of IL-17 in subclinical renal allograft rejection. Reportedly, IL-17 was detected in human biopsy samples of rejected renal allografts as well as in mononuclear cells isolated from urinary sediment of patients suffering borderline subclinical rejection (Hsieh et al., 2001). Using a rat renal allograft model Hsieh et al. demonstrated that on day 2 post transplant when kidney allografts underwent borderline changes based on Banff classification scale, IL-17 mRNA was expressed, but blood urea nitrogen and serum creatinine levels remain unchanged. IL-17 levels peaked at day 5, becoming undetectable by day 9, when most animals died. Once again these observations support the use of IL-17 expression as a predictive parameter for detecting borderline subclinical renal allograft rejection (Loong et al., 2000; Hsieh et al., 2001). CD40L, a product of activated T cells and IL17 are both expressed in kidneys undergoing rejection. CD40 on the other hand, is expressed on most graft-infiltrating cells as well as on resident tubular epithelial cells. Woltman et al. (2000) investigated the influence of IL-17 and CD-40L on human primary tubular epithelial cells, and found them to have a synergistic effect on IL-6, IL-8 and RANTES production and an additive effect on MCP-1 secretion by these cells. Van Kooten et al. (1998) have pre-

viously demonstrated IL-17 to augment IL-6, IL-8 and MCP-1 secretion in primary human proximal tubular epithelial cells, cells known to regulate local interstitial inflammatory responses. These studies indicate that T-cell interaction with parenchymal cells both by direct cell contact and via release of soluble mediators could be a determining factor in renal allograft rejection.

IL-17 in cancer Tartour et al. investigated the influence of IL-17 in human cervical tumors, given its ability to up-regulate IL-6 and IL-8, cytokines implicated in the pathogenesis of cervical cancer. IL-17 transfection of two human cervical cell lines caused a significant increase in tumor size compared with the parent tumor when transplanted in nude mice, with increased IL-6 production and macrophage recruitment to the tumor site (Tartour et al., 1999). IL-17 mRNA was also detected in a significant proportion of ovarian cancers, with increased number of blood vessels in the IL-17-positive tumors, indicating a role for IL-17 in promoting tumor angiogenesis (Kato et al., 2001). These studies support a paradoxical tumor-promoting role for IL-17. Hirahara and colleagues (2000), however, found hIL-17-gene-transfected Chinese hamster ovary (CHO) cells injected into the tail vein of nude mice to cause significantly smaller metastatic nodules in the lungs compared with CHO- or CHO/neo-injected mice. In this model IL-17 significantly lowered the metastatic potential of CHO cells by directly modulating the invasiveness and metastasis of CHO cells as well as by enhancing NK cell activity (Hirahara et al., 2000). Subsequently, Hirahara and coworkers (2001) reported the possibility for development of a tumor vaccine that will incorporate IL-17transfected tumor cells. Murine Meth-A fibrosarcoma cells were transfected with hIL-17 gene by lipofectin method. When the various tumor cells (i.e. parent Meth-A, Meth-A transfected with vector alone and Meth-A/IL-17 cells) were transplanted subcutaneously into BALB/c nude mice, there was no difference in the in vivo growth rates of these cells. However, conventional BALB/c mice when challenged with the tumor cells, rejected the Meth-A/ IL-17 cells, while the other two tumor cells grew.

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When these mice were rechallenged with parent Meth-A or syngeneic plasmacytoma cells, they were able to specifically reject the parent Meth-A cells alone. Meth-A/IL-17 cells appear to have induced T cell-dependent tumor-specific immunity in these animals, as injection of anti-thy 1, 2 (CD-90), antiCD4 or anti-CD8 monoclonal antibodies resumed the growth of Meth-A/IL-17 cells (Hirahara et al., 2001). These results indicate a therapeutic role for IL-17 in tumor. Bruserud et al. (2000) evaluated the effects of IL-16 and IL-17 on T-cell responses in patients with acute leukemia and chemotherapy-induced leukopenia. They report that although IL-17 and IL-16 functioned as growth factors for a large subset of pre-activated monoclonal T cell populations prepared from long-term in vitro cultures, the cytokines had little effect on polyclonal, non-expanded T-cell responses for cytopenic patients (Bruserud et al., 2000).

IL-17 in autoimmune disorders Rheumatoid arthritis Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by heavy lymphocytic infiltration into the synovial cavity, with complex cell–cell interactions and secretion of various cytokines and chemokines that ultimately lead to the destruction of joint tissue. A decade ago, monocyte-derived cytokines, IL-1b and TNFa were identified to be the major inducers of chronic destructive arthritis (reviewed in van den Berg et al., 2001). In contrast to the abundance of monocyte-derived proinflammatory cytokines like TNFa and IL-1, T cell-derived cytokines have been less pronounced in the rheumatoid synovium, leaving the contribution of T cells in RA pathogenicity a matter of debate. More recently, however, several clinical studies have indicated a pivotal role for T cell-derived IL-17 in RA. One of the earliest observations indicating a role for IL-17 in RA was by Fossiez et al. (1996). IL-17 induced secretion of IL-6 by rheumatoid synoviocytes and both TNFa and IFNc synergized with IL-17 and augmented this activity. IL-17 together with TNFa also induced GM-CSF production by synovial fibroblasts (Fossiez et al.1996). Subsequently, Lotz et al. (1996) evaluated the role of IL-17 in the regulation of synoviocytes and chondro-

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cytes and found IL-17 to be effective in promoting synovial inflammation and cartilage degradation. IL-17 inhibited chondrocyte proliferation, induced NO production and stimulated the expression of various genes (i.e. inducible cyclo-oxygenase and inducible NO synthase) in normal human chondrocytes. In addition, several other genes associated with inflammation and cartilage destruction, namely osteoclastogenic factors IL-6 and IL-1b, and stromelysin, were all augmented in IL-17-treated chondrocytes (Shalom-Barak et al., 1998; Lotz et al., 1996). Consequently, several other investigators reported high levels of biologically active IL-17 in the synovial fluid of patients with RA, suggesting a role for this cytokine in inflammation and skeletal destruction, characteristic of the disease (Chaubaud et al., 1998, 1999; Kotake et al., 1999; Ziolkowska et al., 2000; Jovanovic et al., 2000; Lubberts et al., 2001). Neutralization of IL-17 in these synovial cultures from RA patients caused a substantial reduction in the biologic activity of the diseased synovium (Chaubaud et al., 1998, 1999; Kotake et al., 1999; Lubberts et al., 2001). Immunohistochemical analysis of synovial and cartilage biopsies identified IL-17 positive cells exclusively in RA. On the other hand, synovial endothelial cells and chondrocytes from different types of arthritis were found to express the IL-17R, suggesting perhaps a second ligand for the receptor (Honarati et al., 2001). In an attempt to characterize the cellular and cytokine interactions governing tissue outgrowth in RA patients, Wakisaka and co-workers (2000) cultured single cell suspensions of dissociated synovial tissues of RA patients in vitro for prolonged periods to permit tissue outgrowth. T cells from the tissue outgrowth expressed lymphocyte function-associated antigen-1 (LFA-1) and CD2, and the synovial cells expressed ICAM-1 and LFA-3, suggesting interactions via LFA-1/ ICAM-1 and CD2/LFA-3. T cell-derived IL-17 and IFNc, synovial-cell derived fibroblast growth factor-1 and IL-15, were all present in the tissue outgrowth (Wakisaka et al., 2000). IL-15 is newly identified to be a candidate of the proinflammatory cytokine cascade. Fibroblast-like synoviocytes isolated from the joints of RA patients were found to spontaneously secrete large amounts of IL-15 (Ziolkowska et al., 2000). Interestingly, synovial IL-15 induced IL-17 secretion by

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PBMCs isolated from RA patients, a process that was effectively blocked by CsA and methylprednisolone, indicating a role for CsA in controlling the cytokine cascade (Ziolkowska et al., 2000). It has been suggested that interactions between T cells and synovial cells could trigger hyperactivity in RA by inducing proinflammatory cytokines and matrix metalloproteinase (MMP)-1, and by promoting the proliferation of lymphocytes and synovial cells. Chaubaud and co-workers (2001a) evaluated the contribution of cell–cell interactions on cytokine induction and type I collagen synthesis by co-culturing fixed synovium T-cell clones on synoviocytes. IL-17-producing TH1 cells were identified as the highest inducers of IL-6 and LIF, and inhibitors of collagen synthesis, indicating potential contribution of IL-17-producing TH1 cells to defective repair activity in joint inflammation. TH1-mediated effects were partially corrected with the TH2 cytokine, IL-4. Yamamura et al. (2001) reported synergy between the effects of T cell- derived IL-17 and resting T cells in the absence of exogenous antigen, to induce expression of IL-6, IL-8, stromelysin and PGE2, molecules relevant to joint inflammation and destruction, by cultured synovial fibroblasts. Recent models for understanding the pathogenesis of RA highlight interactions between T cells and professional APC in initiating responses to autoantigens and other antigenic stimuli found in the synovial tissue. Chabaud et al. (1998) observed an enhancing effect of IL-17 on IL-1-induced IL-6 and LIF production by RA synoviocytes. When low concentrations of IL-17 and IL-1 were used, a synergistic effect on the production of IL-6 and an additive effect on LIF production was observed, demonstrating that low levels of cytokines produced by monocytes and T cells can act together on synoviocytes (Chabaud et al., 1998). Lubberts et al. (2001) report an IL-1-independent role for IL-17 in the pathogenesis of arthritis. IL-17 accelerated bone erosion in collagen-induced mouse arthritis and blocked endogenous IL-17-suppressed joint damage. Although local IL-17 enhanced IL-1 levels in the synovium, blocking IL-1 with neutralizing antibodies had no influence on IL-17-induced joint inflammation and damage, suggesting IL-17 to be more destructive by amplifying T cell-driven arthritis (Lubberts et al., 2001). However, several studies have demonstrated synergy between IL-17 and TNFa in

the induction of proinflammatory cytokines (Fossiez et al., 1996; Laan et al., 1999; Van Bezooijen et al., 1999; Katz et al., 2001). IL-17 and TNFa synergistically induced osteoclastic bone resorption in vitro. IL-17 also augmented TNFa-induced IL-1a, IL-1b and IL-6 expression in fetal mouse bones (Van Bezooijen et al., 1999), and enhanced TNFa-induced IL-1b, IL-6 and IL-8 expression in synovial fibroblasts (Katz et al., 2001). IL-17-induced TNFa expression in human macrophages and synovial fibroblasts can be suppressed by PGE2 by its induction of early growth response protein-1 expression (Di Battista et al., 1999). While evaluating a cellular mechanism of action for IL-17, Jovanovic and co-workers (2000) proposed IL-17 to contribute to RA pathology by interacting with macrophages in the rheumatoid synovium and by inducing their unbalanced production of proinflammatory cytokines and MMP-9. Upon evaluating the regulation of tissue inhibitor of metalloproteinase (TIMP)-1 in IL-17-stimulated human monocytes and macrophages, an increased level of MMP-9 production relative to TIMP-1 production was observed. This excess of MMP-9 over TIMP-1 production, as well as the decreased inhibition of MMP-9 activity in chronic rheumatoid diseases, could exacerbate cartilage degradation and joint destruction (Jovanovic et al., 2001). IL-17 can thus influence bone metabolism in pathological conditions like RA, characterized by the presence of activated T cells and TNFa production. MIP-3a is a chemokine involved in the migration of T cells and immature dendritic cells. IL-17, IL-1b and TNFa all induce MIP-3a production from rheumatoid synoviocytes in a dose-dependent manner (Chabaud et al., 2001d). IL-1b was more potent than IL-17 or TNFa when used at optimal concentrations. At low concentrations, however, a synergistic effect was observed. A 95% inhibition in MIP-3a production was achieved only when all three soluble receptors were used in combination (Chabaud et al., 2001d). This study further supports an interactive role for monocyte- and T cell-derived cytokines in RA, in their ability to recruit T cells and dendritic cells by enhancing MIP-3a production by synoviocytes. Chabaud et al. (2001c) using ex vivo models of RA inflammation and bone destruction, have established combination therapy involving TNFa, IL-1 and IL-17 blockade to be more effective in inhibiting IL-6 production and colla-

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gen degradation, than any one blockade considered alone. Given the interactive role of these cytokines in several models of RA, combination cytokine therapy may increase the numbers of responding RA patients and the degree of individual patient response (Chabaud et al., 2001c, 2001d; Miossec, 2001). Dudler and colleagues (2000) demonstrated a direct catabolic effect for IL-17 on cartilage tissue in addition to its stimulatory effects on macrophages and synoviocytes. Direct repeated intra-articular injections of murine IL-17 into the knee joints of normal mice induced joint inflammation and cartilage proteoglycan depletion as evaluated by histological scoring, reiterating IL-17 involvement in the pathogenesis of arthritis. Chondrocyte activation is known to contribute to cartilage matrix destruction and IL-17 induces direct chondrocyte-mediated cartilage damage. Local overexpression of IL-4, suppressed synovial IL-17 and osteoclast differentiation factor (ODF) expression, impeding joint damage and bone erosion in the knees of mouse with type II collagen induced arthritis (Lubberts et al., 2000a). IL-4 suppressed IL-17’s ability to inhibit chondrocyte proteoglycan synthesis in cartilage explants, by enhancing type I procollagen synthesis thereby aiding in tissue repair (Lubberts et al., 2000b). Cai et al. (2001) also reported IL-17 to be a direct and potent inducer of matrix breakdown and inhibitor of matrix synthesis in articular cartilage explants. Although IL-17 up-regulated expression of MMPs in chondrocytes, the mechanism by which IL-17 mediated matrix breakdown, was by the stimulation of aggrecanase(s) and LIF. The newly identified member, IL-17E, also induced catabolism of human articular cartilage (Cai et al., 2001). Chabaud et al. (2000) reported IL-17 to induce MMP-1 production by synoviocytes spontaneously. However, IL-1b was more potent causing a nine-fold increase in MMP-1 production compared with the five-fold increase caused by IL-17. Addition of antiinflammatory cytokines like IL-4, IL-13 and IL-10 to the synoviocyte cultures reduced IL-17-induced and/or IL-1b-induced MMP-1 production and augmented production of TIMP-1. Antibodies to IL-17 reduced MMP-1 production and collagenase activity in the synovium by 50% (Chabaud et al., 2000). All of the above studies support an indirect as well as a direct role for IL-17 in the RA inflammatory process. Adjuvant arthritis (AA) is an experimental model of

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RA in rats. Bush et al. (2001) investigated the immune processes controlling the initiation and spontaneous resolution of AA. IL-17 was up-regulated in the early stages of the disease along with TNF and IFNc, proposing an important role for these cytokines in disease initiation. An increase in IL-4 levels in the later stages of AA implies that IL-4 mediates spontaneous resolution of the disease (Bush et al., 2001). Osteoarthritis (OA) is a chronic joint disease characterized by progressive degenerative changes in the articular cartilage and menisci-intra-articular fibrocartilagenous structures in the knee joint. Bioactive IL-17 has been reported in a few OA synovial biopsies, although not as prevalent as in RA synovial biopsies (Chabaud et al., 1999). NO and PGE2 have been implicated as mediators of inflammation and cartilage destruction in OA (Attur et al., 1997; Martel-Pelletier et al., 1999; LeGrand et al., 2001). IL-17 could be a critical candidate in provoking cartilage destruction in OA given its ability to augment inducible NO synthase (iNOS) expression and NO production in chondrocytes (Attur et al., 1997; Shalom-Barak et al., 1998; Martel-Pelletier et al., 1999; LeGrand et al., 2001) and PGE2 synthesis in synoviocytes and osteoblasts (Fossiez et al., 1996; Kotake et al., 1999). In fact, IL-17 synergized with IL-1 and TNFa and upregulated the spontaneous secretion of NO and PGE2 from explants of human OA knee menisci (LeGrand et al., 2001). Studies by Attur et al. (1997) demonstrated IL-17 to augment the spontaneous production of NO in OA cartilage via NFjB activation, a pathway independent from IL-1b and sensitive to cyclohexamide and pyrrolidine dithiocarbamate. IL17-induced NFjB and MAP kinases, in turn regulate NO production by IL-17 in human OA chondrocytes (Shalom-Barak et al., 1998; Martel-Pelletier et al., 1999). Osteoblasts express ODF (also referred to as TRANCE/RANKL/OPGL), a membrane bound factor that promotes the differentiation of osteoclast progenitors into osteoclasts in response to various bone resorbing factors through a mechanism requiring cell–cell contact (Kotake et al., 1999). Adding IL-17 to co-cultures of bone marrow cells and osteoblasts stimulated both COX-2-dependent PGE2 synthesis and ODF gene expression in osteoblasts, which in turn induced the differentiation of osteoclast progenitors into mature osteoclasts. Anti-IL-17 antibody

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significantly inhibited osteoclast formation induced by culture media of RA synovial tissues, further suggesting IL-17 to be a crucial cytokine for osteoclastic bone resorption in RA patients (Kotake et al., 1999). Osteoclasts are potent bone-resorbing cells, and RANKL has been shown to be a key regulator of osteoclastogenesis. T cell-derived IL-17 is implicated in osteoclastic bone resorption, due to its ability to induce RANKL expression (Van Bezooijen et al., 1999; Martel-Pelletier et al., 1999). Co-culturing human PBMC with murine ST-2 stromal cells induced the differentiation of osteoclast precursors from PBMC. Upon co-culture ST-2 cells specifically expressed the message for ODF, while PBMC specifically expressed RANK. Together, they expressed IL-17, IL-1/IL-1R, IL11R, IL-6/IL-6R, IL-18/IL-18R, M-CSF and TGFb and the osteoclastogenesis inhibition factor, OPG (Atkins et al., 2000). Hence, osteoclast differentiation is regulated by coordinated cytokine responses from both stromal and hematopoietic cells and the RANKL/RANK balance appears to be of critical importance in osteoclastogenesis and the bone erosion process. Recent investigations, however, suggest the existence of other pathways that may mediate osteoclastic bone resorption. IL-17 when combined with TNFa, induced NO production in osteoblasts and fetal mouse bones by a NFjB-dependent mechanism that was associated with elevated mRNA levels of NFjB isoforms RelA and p50 (Van Bezooijen et al., 2001). Using inhibitors to NO synthesis, NFjB activation and the RANK/RANKL pathway, the authors have demonstrated both NO production and NFjB signaling not to be involved, and the RANK/RANKL pathway to be partly involved in the IL-17TNFa-induced stimulation of osteoclastic bone resorption. These observations suggest the involvement of additional pathways in mediating osteoclastic bone resorption (Van Bezooijen et al., 2001). Yano et al. (2001) investigated the involvement of OPG in the pathogenesis of bone destruction in RA. Inflammatory cytokines like IL-17, IL-1b and TNFa that are elevated in the synovial fluid of RA patients, up-regulated the production of OPG via stimulation of PGE2, high enough to inhibit osteoclastogenesis in vitro. While basic fibroblast growth factor inhibited OPG production by inflammatory cytokines, it stimulated PGE2-mediated OPG production and inflammatory cytokine secretion.

Thus, a reciprocal regulation of OPG production by inflammatory cytokines and basic fibroblast growth factor was identified. Lyme disease is caused by a tick-borne spirochaete, Borrelia burgdorferi, and is a multisystem disease primarily affecting the skin, nervous system, heart and joints. The long-term outcome of the disease is Lyme arthritis. Infante-Duarte and co-workers (2000) have found a high percentage of IL-17-producing synovial fluid T cells in patients with Lyme arthritis and in vitro have demonstrated microbial stimuli, such as B. burgdorferi and Mycobacterium bovis lysates, and synthetic lipopeptides derived from B. burgdorferi outer surface lipoproteins, to induce IL-17 expression in both murine and human T cells. The authors propose chronic IL-17 production by microbes to be an important mediator in infection-induced immunopathology. Manipulation of IL-17 could therefore be of importance in chronic inflammatory conditions, such as antibiotic-resistant arthritis, that is sometimes induced by B. burgdorferi infection. Hypoestoxide, a natural diterpene, inhibits NO production in IL-17- or IL-1b-stimulated chondrocytes. Hypoestoxide inhibits NFjB activation by directly inhibiting IjB kinase activity and thus can be useful as an anti-inflammatory agent (Ojo-Amaize et al., 2001). Major pathologic manifestations of RA and OA include joint inflammation and articular cartilage resorption by proinflammatory cytokine-stimulated MMPs and aggrecanases. The Chinese herbal remedy Tripterygium wilfordii Hook F (TWHF) is effective in the treatment of various types of arthritis, and its antiinflammatory activity is attributed to the inhibition of cyclooxygenase-2 and PGE2 synthesis. Sylvester and co-workers (2001) have now demonstrated TWHF to either completely or partially inhibit mRNA and protein expression of TNFa, IL-1 and IL-17-inducible MMP-3 and MMP-13, in articular chondrocytes. This suppression was mediated by TWHF’s ability to partially inhibit the DNA binding capacity of cytokinestimulated transcription factors, AP-1 and NFjB.

Systemic lupus erythematosus IL-17 has also been implicated in systemic lupus erythematosus (SLE), an autoimmune phenomenon induced by an imbalance in T helper-cell cytokine secretion. Wong et al., (2000) evaluated plasma levels

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of the TH2 cytokine IL-4, and proinflammatory cytokines IL-17, IL-18 and IL-12, in SLE patients, and found elevated levels of both TH1 and TH2 cytokines. This imbalance in the cytokine profile portrays a flaw in the cytokine immunoregulatory pathway, a predictive factor in SLE pathogenesis.

Multiple sclerosis Multiple sclerosis (MS) is a CNS inflammatory disease characterized by myelin-directed autoimmunity. Elevated IL-17 mRNA expression was found in mononuclear cells of the cerebrospinal fluid and blood of MS patients, with the highest number of cells expressing the message in blood during clinical exacerbation of the disease (Matusevicius et al., 1999). IL-17 synergized with exogenous IL-1b and TNFa, and induced a dose-dependent enhancement of IFNc-triggered NO synthesis from rodent astrocytes (Trajkovic et al., 2001). Given the neurotoxic activity of NO this study further supports a potential role for IL-17 in inflammatory diseases of the CNS. Activation of NFjB, an important transcription factor in the regulation of neuronal and glial cell function, involves degradation of its cytoplasmic inhibitor, IjB-a, which allows the nuclear translocation of NFjB and ensures transcriptional activation of genes. Kehlen et al. (1999) evaluated the biological effects of IL-17 on glial cells, and found IL-17 to up-regulate IjB-a mRNA expression in a dose- and time-dependent fashion and to stimulate the secretion of IL-6 and IL-8 from glial cells, supporting a role for IL-17 in neurological diseases.

Experimental autoimmune neuritis Experimental autoimmune neuritis (EAN) is a CD4 T cell-mediated demyelinating disease of the peripheral nervous system. Intranasal administration of IL-17 to rats with EAN enhanced the acute phase and inhibited the chronic phase of the disease suggesting an immunoregulatory capacity for IL-17 in this disease model (Pelidou et al., 2000).

IL-17 in gastric disorders IL-17, in addition to inducing NF-jB and AP-1 DNAbinding activities, also regulates the activities of MAP kinases in intestinal epithelial cells, and therefore

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could be involved in enteric disorders (Awane et al., 1999; Andoh et al., 2001). Heliobacter pylori (Hp)associated gastritis is characterized by massive mucosal infiltration and cytokine production by polymorphonuclear leukocytes, T cells, macrophages and plasma cells, all eventually contributing to the maintenance and progression of gastric inflammation. It has been recently established that IL-8, a major chemoattractant of polymorphonuclear leukocytes in humans, plays a key role in Hp-associated acute inflammatory responses. Since T cell-derived IL-17 is a potent stimulator of IL-8, Luzza et al. (2000) evaluated the possibility of IL-17’s involvement in Hpassociated gastritis. High levels of IL-17 mRNA and protein were found in the whole gastric mucosal and lamina propria mononuclear cells isolated from Hpinfected patients, with eradication of the pathogen leading to a marked down-regulation of IL-17 expression. Addition of an anti-IL-17 neutralizing antibody to gastric lamina propria mononuclear cell cultures caused a significant inhibition in IL-8 production by these cells. The data provided herein support an important role for IL-17 in the inflammatory response to Hp-colonization (Luzza et al., 2000).

IL-17 in airway diseases Activated CD4 T cells are known to play a central role in airway inflammation, as antibodies to CD4 inhibit the influx of eosinophils and neutrophils into the murine airways. Although IL-5, the eosinophil regulatory cytokine, is believed to link the activation of CD4 T cells to the influx of eosinophils into the airways, the agent(s) responsible for recruiting neutrophils has not been identified. The receptor for IL-17 is expressed in the airways (Yao et al., 1995a) and IL-17 stimulates the expression of neutrophilpreferring adhesion molecule, ICAM-I in fibroblasts (Yao et al., 1995b). In fact, ICAM-I expression has been detected in patients with allergic asthma (Wong et al., 2001). Furthermore, IL-17 has the potential to stimulate IL-6, IL-8, IL-1b, TNFa and GM-CSF – cytokines implicated in airway remodeling within the lungs of asthmatic subjects. Could T cell-derived IL-17 be the link between T-cell activation and neutrophil recruitment in the airways? This was the basis for the investigations by Laan et al. (1999) and Hoshino et al. (1999). Intratracheal instillation of

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IL-17 selectively recruited neutrophils into the rat airways via the release of MIP-2, a rat correlate of human IL-8, from bronchial epithelial cells (Laan et al. 1999), an effect that was modulated by endogenous tachykinins acting via NK-1 receptors (Hoshino et al., 1999). Tachykinins like substance P and neurokinin A (NKA) are co-localized in unmyelinated sensory nerves of the airways and are believed to modulate neutrophil recruitment by binding their receptors neurokinin (NK)-1 and NK-2, respectively. In addition, IL-17 selectively recruits neutrophils into the peritoneal cavity by inducing the release of the neutrophil-specific chemokine, GRO-a from the peritoneal mesothelial cells (Witowski et al., 2000). Does IL-17 activate neutrophils in addition to recruiting them into the airways? Hoshino and colleagues (2000) characterized the bronchoalveolar fluid following intratracheal administration of IL-17 in terms of neutrophil count, myeloperoxidase activity and elastase activity. IL-17 increased myeloperoxidase and elastase activity, as well as the neutrophil count in the BAL fluid of IL-17-treated rats. Direct stimulation of neutrophils with IL-17 in vitro did not augment myeloperoxidase activity. In vivo activation of neutrophils by IL-17 is probably achieved indirectly by stimulation of other airway cells. Mechanisms linking T-cell activation to recruitment and activation of neutrophils in airway inflammation has thus been demonstrated (Laan et al., 1999; Hoshino et al., 1999, 2000; reviewed in Linden et al., 2000). Interestingly, eosinophils and not just T cells are identified as being significant sources of IL17 in the asthmatic airways (Molet et al., 2001). It is therefore tempting to speculate that T cells and eosinophils may both regulate IL-17-mediated neutrophil influx into the airways and this needs to be investigated. IL-17 is also capable of amplifying the inflammatory responses in asthma by its ability to enhance the secretion of profibrotic cytokines, IL-6 and IL-11 and inflammatory mediators such as IL-8, alpha chemokines and GRO-a from human bronchial fibroblasts (Molet et al., 2001). IL-17 may augment host defense against bacterial pneumonia. Ye et al. (2001a, 2001b) tested this hypothesis using murine Klebsiella pneumoniae lung infection models that either overexpressed IL-17 or were genetically deficient in IL-17R expression. Overexpression of IL-17 in the pulmonary compartment

using a recombinant adenovirus expression system caused the local induction of TNFa, IL-1b, MIP-2 and G-CSF, and increased infiltration by polymorphonuclear leukocytes, leading to enhanced bacterial clearance and survival following K. pneumoniae infection (Ye et al., 2001a). In contrast, mice deficient in IL-17R exhibited significantly delayed neutrophil recruitment to the alveoli, causing the bacteria to disseminate resulting in 100% mortality compared with only 40% mortality in controls. The delay in neutrophil recruitment was associated with a reduction in steady-state levels of G-CSF and MIP-2 in the lung following intranasal challenge with K. pneumoniae (Ye et al., 2001b). IL-17R signaling may therefore be critical in dictating host defenses against K. pneumoniae lung infection. Inhalation of high doses of LPS in mice caused IL-17 expression in the lung (Larsson et al., 2000). Alcohol consumption suppresses release of IL-17 into lung tissue, decreases neutrophil recruitment and increases mortality in experimental K. pneumonia. In vivo administration of IL-17 to these recipients normalized neutrophil recruitment and mortality after bacterial challenge, indicating susceptibility of the IL-17 pathway to immunosuppression following alcohol abuse (Shellito et al., 2001).

IL-17 in inflammatory skin disorders Psoriasis is a common chronic inflammatory skin disease characterized by a heavy inflammatory infiltrate that includes activated T cells, neutrophils, macrophages and dendritic cells, with hyperproliferation of keratinocytes. None the less, the factors responsible for the recruitment of pathogenic cells and mediators to skin lesions remain to be established. Homey et al. (2000) found the expression of CC chemokine, MIP3a, recently renamed CCL20, and its receptor CCR6 to be significantly augmented in psoriasis and report the proinflammatory mediators IL-17, IFNc, TNFa, IL-1b and CD40L, to induce CCL20 expression within the psoriatic lesions. The major sources of CCL20 were identified to be cultured primary keratinocytes, dermal fibroblasts and dermal microvascular endothelial and dendritic cells (Homey et al., 2000). Within the lesions CCL20-expressing keratinocytes co-localized with skin-infiltrating T lymphocytes, strongly suggesting a major role for IL-17 in the indirect recruitment and establishment of chronic skin disease. In addi-

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tion, T cell-derived-IL-17 induced the secretion of proinflammatory cytokines, IL-6 and IL-8 by human keratinocytes in vitro. This combined with the detection of IL-17 mRNA in CD4 and CD8 T-cell clones derived from psoriatic skin lesions and lesional psoriatic skin biopsies, suggests IL-17 to be a key player in the pathology of dermatosis (Teunissen et al., 1998). Albanesi et al. (1999) evaluated whether haptenspecific T cells isolated from patients with allergic contact dermatitis to nickel produced IL-17, and if so, did the released IL-17 influence immune activation of keratinocytes. Skin biopsies and skin-derived nickel specific CD4 T cells were found to express the message and protein for IL-17, IFNc and TNFa. IL-17 specifically and dose-dependently augmented IFNcinduced ICAM-1 expression on keratinocytes. In addition, IL-17 both directly and in synergy with IFNc and/or TNFa induced IL-8 production by keratinocytes. Furthermore, IL-17 inhibited IFNc- and TNFa-induced RANTES production, but did not alter MCP-1 production. Thus, IL-17 either synergized or antagonized the effects of IFNc and TNFa, and regulated ICAM-1 expression and chemokine production in human keratinocytes (Albanesi et al., 1999). IL-17 in addition to regulating keratinocyte expression of adhesion molecules and chemokines also potentiated both IFNc- and IL-4-induced activation of keratinocytes (Albanesi et al., 2000a). IL-4 exerts its proinflammatory influence on keratinocytes by potentiating IFNc- and TNFa-mediated induction of CXC chemokines (i.e. IFN-induced protein of 10 kDa (IP-10), monokine induced by IFNc (Mig) and IFN-inducible T-cell a-chemoattractant (I-TAC)), leading to the significant recruitment of T cells expressing the receptor CXCR3 into skin lesions (Albanesi et al., 2000b). From these observations one could infer that IL-17 has the potential to participate in the immunopathogenesis of skin disorders.

IL-17 in other inflammatory disorders Systemic sclerosis is a connective tissue disorder of unknown etiology characterized by fibrosis and microvascular abnormalities of the skin and visceral organs. IL-17 is overproduced in the peripheral blood and fibrotic lesions of the skin and lung of systemic sclerosis patients (Kurasawa et al., 2000). In vitro, IL-17 induced IL-1 secretion in endothelial cells and

augmented their expression of adhesion molecules ICAM-1 and VCAM-1 in a dose-dependent manner. IL-17 also enhanced the proliferation of fibroblasts. These observations indicate that overproduction of IL-17 in the early stages of systemic sclerosis could play an important role in the disease pathology (Kurasawa et al., 2000). Ischemic brain injury secondary to arterial occlusion is characterized by acute local inflammation, and infiltration by PMN. Kostulas et al. (1999) evaluated mRNA levels of IL-1b, IL-8, IL-17 and MIP-1a in blood mononuclear cells from patients with ischemic stroke in an effort to assess the factors involved in the selective recruitment and accumulation of inflammatory cells into the ischemic brain tissue. They found increased expression of all cytokines tested, but the chemokine MIP-1a remained unaffected. Systemic up-regulation of IL-17 and the other proinflammatory cytokines could contribute to the pathogenesis associated with ischemic stroke. In a rat model, IL-17 and IFNc mRNA expression were elevated in the ischemic hemispheres of the brain and in peripheral blood mononuclear cells as early as 1 hour following permanent middle cerebral artery occlusion. This study further supports the notion that alterations in proinflammatory cytokines levels may influence the outcome of brain ischemia (Li et al., 2001).

CONCLUSIONS Significant progress in interleukin-17 research over the last 5 years has established this cytokine as a molecule of tremendous functional potential and complexity. At least four new members structurally related to IL-17 have been identified, thus establishing a family of IL-17-related molecules. The IL-17 family of proteins, produced predominantly by activated T cells, exhibits a broad range of activities and is represented in a spectrum of pathophysiologic conditions. There is now ample evidence for IL-17 as a regulator of hematopoiesis, and as a mediator of inflammation in several T cell-mediated disorders. These observations necessitate further research and development of this cytokine for potential clinical applications for conditions associated with compromised BM status, as well as in transplantation, cancer, autoimmunity and other T cell-mediated inflammatory disorders.

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ACKNOWLEDGEMENTS The authors thank Dr Melissa A. Starovasnik, Genentech, Inc., and Dr Shau-Ku Huang, Johns Hopkins University School of Medicine for the helpful discussions and clarifications on the IL-17 family members.

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21 Granulocyte–macrophage colony-stimulating factor Thomas Enzler and Glenn Dranoff Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

Granulocyte–macrophage colony-stimulating factor: If I didn’t define myself for myself, I would be crunched into other people’s fantasies for me and eaten alive. Andre Lorde

INTRODUCTION Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a 22-kDa glycoprotein encoded by a gene on the long arm of human chromosome 5 (Gasson et al., 1984; Le Beau et al., 1986). GM-CSF alone or in combination with other cytokines stimulates the growth of CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-DC, CFU-Eo, CFU-Mk and BFU-E (Sieff et al., 1985; Fleischmann et al., 1986; Vadhan-Raj et al., 1987; Migliaccio et al., 1988; Segal et al., 1988; Bot et al., 1989; Bussolino et al., 1989; Ferrero et al., 1989; Straneva et al., 1989; Caux et al., 1992). These striking properties have led to the clinical application of GM-CSF for ameliorating neutropenia and enhancing hematopoietic recovery after cytotoxic chemotherapy or bone marrow transplantation. Since GM-CSF also augments the functional activities of phagocytes, including neutrophils (Gasson

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

et al., 1984), macrophages (Cannistra et al., 1988; Kleinerman et al., 1988; Morrissey et al., 1989; Wing et al., 1989), dendritic cells (Romani et al., 1994; Caux et al., 1996), and eosinophils (Vadas et al., 1983; Lopez et al., 1986; Owen et al., 1987), additional clinical uses of GM-CSF for immunomodulation are currently under active study. While mice rendered deficient in GM-CSF by gene targeting techniques surprisingly maintain normal steady-state hematopoiesis, they develop a lung disease resembling human pulmonary alveolar proteinosis (PAP) (Dranoff et al., 1994; Stanley et al., 1994). In a cohort of patients with idiopathic PAP, high titers of neutralizing IgG antibodies against GM-CSF could be found (Kitamura et al., 1999). Remarkably, some of these patients underwent a symptomatic, physiologic and radiographic improvement following administration of recombinant GM-CSF (Seymour et al., 1996; Barraclough and Gillies, 2001; Kavuru et al., 2000).

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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PROTEIN STRUCTURE The primary structures of murine (Gough et al., 1984, 1985; Sparrow et al., 1985), human (Lee et al., 1985; Wong et al., 1985b), gibbon (Wong et al., 1985a), and bovine (Maliszewski et al., 1988) GM-CSF were determined by either partial amino acid sequence analysis or isolation of cDNA clones. In each species, the amino terminus of the protein encodes a 25 amino acid hydrophobic leader sequence. Mature murine GM-CSF is comprised of 124 amino acids (molecular mass 14 kDa), whereas human, gibbon and bovine GM-CSF are slightly longer, reflecting the insertion of three amino acids between the counterparts of murine residues 27 and 28. Interestingly, these additional amino acids reside within the first of four a helices, a region that contributes to binding the bc subunit (‘b chain’) of the heterodimeric GM-CSF receptor. Murine and human GM-CSF display only 56% sequence identity and are not cross-reactive in receptor binding or biologic activity. Murine and human GM-CSF contain two potential N-linked glycosylation sites. The glycosylated, higher Mr forms are more acidic as determined by twodimensional gel electrophoresis, reflecting increased sialation (Cebon et al., 1990). Recombinant GM-CSF produced in yeast or baculovirus shows differences in glycosylation compared with the native protein, but nonetheless is biologically active (Price et al., 1987; Chiou and Wu, 1990). Moreover, recombinant GMCSF generated in Escherichia coli also has high biological activity (Libby et al., 1987; Schrimsher et al., 1987; Wingfield et al., 1988). Taken together, these findings demonstrate that glycosylation is not essential for GM-CSF function, although it does influence the affinity of receptor binding (Kaushansky et al., 1987). The tertiary structure of GM-CSF comprises, analogous to several other hematopoietic growth factors, two pairs of anti-parallel a helices (Diederichs et al., 1991; Rozwarski et al., 1996). The pattern of disulfide bonding has been determined, with pairing of the first and third, and second and fourth residues, respectively (Schrimsher et al., 1987). Amino acids 18–22, 34–41, 52–61 and 94–115 are involved in receptor binding (Gough et al., 1987; Kaushansky et al., 1987; Clark-Lewis et al., 1988; Shanafelt and Kastelein, 1989; LaBranche et al., 1990; Shanafelt et al., 1991a, 1991b).

Residues interacting with the bc subunit of the receptor are located within the first a helix (residues 18–22). Mutating residue 21 from glutamine to arginine abolishes bc subunit binding, but preserves a subunit interactions (Lopez et al., 1992). Further, systematic mutagenesis of 11 residues on the helix A/helix C face confirmed the importance of Gln-21 and revealed that modifying Gly-75 and Gln-86, located on helix C, resulted in a four-fold drop in biologic activity. Interestingly, Gln-21 and Gly-75, but not Gln-86, are structurally equivalent to residues involved in the binding of growth hormone to its receptor (Rozwarski et al., 1996).

GENE EXPRESSION AND REGULATION GM-CSF was first purified from lung-conditioned medium of endotoxin-primed mice (Sparrow et al., 1985). Subsequent studies identified a number of cell types with the capacity to synthesize GM-CSF, including T lymphocytes, macrophages, NK cells, NKT cells, endothelial cells, stromal cells, fibroblasts, respiratory epithelial cells and others. In many cases, GM-CSF secretion requires stimulation of the producer cell, for example by other cytokines, antigens, or inflammatory agents. Injection of mice with endotoxin results in a rapid release of GM-CSF into the circulation, with macrophages and/or endothelial cells likely being the major sources. Nonetheless, almost all tissues and organs from endotoxin-primed mice release GM-CSF after culture, and this appears to reflect de novo synthesis, rather than release of preformed protein (Nicola et al., 1979). In T cells, important synergies between TCR and CD28 signaling result in GM-CSF production (Kruger et al., 1996). The fact that GM-CSF is produced mainly in response to cellular activation agrees with its role in immune and inflammatory responses. The GM-CSF gene comprises two distinct transcriptional control regions. These include the promoter, spanning 120 bp beginning with the transcription start site, and an enhancer approximately 3 kb upstream (Nimer et al., 1988, 1990; Shannon et al., 1988; Cockerill et al., 1993; Naora et al., 1994; Jenkins et al., 1995). Different regions within the promoter respond to a wide array of signals,

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including phorbol myristate acetate (PMA) and Ca2 ionophores, tumor necrosis factor (TNF), and interleukin-1 (IL-1) (Koeffler et al., 1988; Nimer et al., 1988, 1990; Kaushansky, 1989; Slack et al., 1990; Falkenburg et al., 1991; Coles et al., 2000). The transcription factors which mediate these responses belong to the NF-jB/Rel and AP-1 transcription factor families (Schreck and Baeuerle, 1990; Miyatake et al., 1991; Koyano-Nakagawa et al., 1993; Naora et al., 1994; Jenkins et al., 1995). The NF-jB/Sp1 binding sites in the promoter seem to be crucial for chromatin remodeling and efficient transcription (Cakouros et al., 2001). The enhancer is also responsive to PMA/Ca2 ionophores and can be inhibited by cyclosporin A (Cockerill et al., 1993). NF-AT/AP-1 protein complexes are essential for enhancer function (Cockerill et al., 1995). Although increased transcription of GM-CSF is evident after inductive stimulation, a more important regulatory mechanism may involve control of mRNA degradation (Thorens et al., 1987; Ernst et al., 1989; Kaushansky et al., 1989; Bickel et al., 1990; Akahane et al., 1991; Hahn et al., 1991). The mRNAs of many cytokines and transcription factors that display short cytoplasmic half-lives have repetitive 5-AUUUA-3 motifs in their 3 untranslated regions (Shaw and Kamen, 1986). The GM-CSF mRNA contains eight copies of this sequence, which is required for both stabilization and degradation of the transcript (Shaw and Kamen, 1986; Iwai et al., 1991; Akashi et al., 1991, 1994; Buzby et al., 1996; Lai and Blackshear, 2001).

1989), and the bc subunit alone has no detectable binding activity (Hayashida et al., 1990). Nonetheless, the two subunits together generate a ligand-specific, high-affinity receptor capable of effective signal transduction (Hayashida et al., 1990; Muto et al., 1996). A single tyrosine residue in the membraneproximal domain of the bc subunit (when associated with the specific a subunit) is necessary and sufficient for high affinity binding and signaling by the ligand (Woodcock et al., 1996). The cytoplasmic domain of the bc subunit is larger (approximately 430 amino acid residues) than the a subunit intracellular domain (54 amino acid residues), consistent with its dominant role in signal transduction. GM-CSF receptors are found on myeloid cells (but not B or T lymphocytes) and many non-hematopoietic cells, including endothelial cells, respiratory epithelia, placental cells and various malignant tumors (Morrissey et al., 1987). Soluble forms of the GMCSF receptor have been described (Fukunaga et al., 1990). Although the roles of these proteins are incompletely understood, they may bind GM-CSF and serve a regulatory function. The human GM-CSF receptor a subunit exists in at least eight isoforms, resulting from alternative splicing (Lilly et al., 2001), and six of them have no intracytoplasmic sequences (Raines et al., 1991; Hu et al., 1994; Chopra et al., 1996; Hu and Zuckerman, 1998). The a subunit expression is inhibited by the B cell-specific transcription factor pax5a (Chiang and Monroe, 2001).

SIGNAL TRANSDUCTION

RECEPTOR The GM-CSF receptor is composed of two distinct subunits: a ligand-specific a subunit (Gearing et al., 1989) and a bc subunit (‘b chain’) (Hayashida et al., 1990). The receptor belongs to the class I cytokine receptor superfamily. Members of this family show common features in their extracellular domains, including conserved cysteine residues, the ‘WSXWS’ motif, and fibronectin type III-like domains (Bazan, 1990). Moreover, the GM-CSF receptor shares the bc subunit with the IL-3 and IL-5 receptors, whereas each receptor has a unique a subunit (Kitamura et al., 1991; Tavernier et al., 1991). The ligand-specific a subunit binds GM-CSF with low affinity (Gearing et al.,

Like other members of the class I cytokine receptor superfamily, the GM-CSF receptor does not possess intrinsic kinase activity, but rather serves as a docking site for adaptor molecules with kinase activity. Signaling events in response to GM-CSF, IL-3 or IL-5 include activation of JAK2 tyrosine kinase, the Ras activation pathway including Vav, Shc, Raf and MAP kinase, the Src family kinases Fyn and Lyn, Fps/Fes, phosphatidylinositol-3 kinase p85, protein kinase C, calcium flux and inositol phosphate mobilization (Mui et al., 1995). There appear to be at least two main signaling pathways linked to the GM-CSF receptor, each involving a distinct region of the cytoplasmic part of the

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bc subunit (Sato et al., 1993). The first pathway, which leads to the induction of c-myc and activation of DNA replication, is associated with Box1, a conserved membrane proximal region within the bc subunit (Quelle et al., 1994). Box1 is required for JAK2 binding with subsequent activation of its intrinsic kinase activity (Brizzi et al., 1994; Quelle et al., 1994; Itoh et al., 1996; Watanabe et al., 1996). JAK2 phosphorylates the bc subunit, creating a docking site for STAT proteins (signal transducers and activators of transcription) (Mui et al., 1995; Wang et al., 1995). STATs exist in the cytoplasm as latent, inactive forms until they become phosphorylated on tyrosine residues in the context of an activated receptor, whereupon they translocate to the nucleus, bind to specific DNA elements, and stimulate transcription. bc signaling involves STAT5 and, to a lesser extent, STAT1 and STAT3 (Azam et al., 1995; Mui et al., 1995; Brizzi et al., 1996). Interestingly, carboxyl truncations of the a subunit impair JAK2 activation (Doyle and Gasson, 1998; Quelle et al., 1994), indicating an important role for the a subunit as well (Doyle and Gasson, 1998). The second signaling pathway, involving activation of the Ras/Raf-1/MAP kinase cascade, is initiated through the C-terminal, membrane distal region of the bc subunit (Okuda et al., 1992). This pathway leads to the induction of c-fos, c-jun and other genes crucial for hematopoietic differentiation (Sato et al., 1993). Lastly, the protein tyrosine phosphatase SHP-1, the SH2 containing 5-inositol phosphatase SHIP, and the recently described suppressor of cytokine signaling proteins (SOCS) play important roles in modulating GM-CSF signaling (Yasukawa et al., 2000).

GENETICS The genes for GM-CSF and IL-3 are closely linked on human chromosome 5 and mouse chromosome 11 (Yang et al., 1988). They are positioned within a cytokine cluster that encompasses IL-4, IL-5, IL-9 and IL-13. The GM-CSF gene in both species consists of four exons and three introns (Miyatake et al., 1985). The genes encoding the a subunits of the GM-CSF and IL-3 receptors are closely linked in humans (but not mice) within the pseudoautosomal region of the

 and Y chromosomes (Rappold et al., 1992; Kremer et al., 1993). The a subunit of the GM-CSF receptor spans approximately 45 kb and consists of 13 exons, similar to other members of the cytokine receptor superfamily (Nakagawa et al., 1994). The human bc subunit of the GM-CSF receptor is encoded at 22q12–q13 (Shen et al., 1992), whereas the murine bc and the related bcIL-3 subunit (that exclusively mediates IL-3 signaling in the mouse) are closely linked on mouse chromosome 15 (Gorman et al., 1992).

IN VITRO ACTIVITIES The multiple activities of GM-CSF detected in vitro include stimulation of hematopoietic cell proliferation, differentiation, function and survival. Together, the in vitro studies reveal a prominent role for GM-CSF in hematopoiesis and immunity.

Hematopoietic progenitors GM-CSF alone or in combination with other cytokines stimulates the growth of CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-DC, CFU-Eo, CFU-Mk, and BFU-E (Sieff et al., 1985; Fleischmann et al., 1986; Vadhan-Raj et al., 1987; Migliaccio et al., 1988; Segal et al., 1988; Bot et al., 1989; Bussolino et al., 1989; Ferrero et al., 1989; Straneva et al., 1989; Caux et al., 1992).

Neutrophils GM-CSF stimulates neutrophil antimicrobial activity through a combination of augmenting phagocytosis and intra-cellular killing mechanisms. GM-CSF increases the expression of surface molecules important for the uptake of opsonized pathogens, including FccRI (CD64), the IgA Fc receptor, CR-1 (CD35) and CR-3 (CD11b) (reviewed in Weisbart et al., 1988; Ruef and Coleman, 1990; Jones, 1994). GM-CSF primes neutrophils for increased production of several inflammatory mediators such as the lipid leukotriene LTB4 (Stankova et al., 1995). The cytokine also stimulates neutrophil trans-endothelial migration, in part through induction of CD11b (Socinski et al., 1988b; Yong and Linch, 1993).

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GM-CSF exerts pleiotropic effects on monocyte/ macrophages (Jones, 1996). The cytokine markedly increases phagocytosis, in part by up-regulating FccRI, FccRII and complement receptors (Grabstein et al., 1986). GM-CSF and TNF-a synergize in promoting phagocytosis of Cryptococcus neoformans (Collins and Bancroft, 1992). Alveolar macrophages from GMCSF-deficient mice show impaired phagocytosis of Pnemocystis carinii (Paine et al., 2000). GM-CSF enhances microbiocidal killing through both reactive oxygen and reactive nitrogen species (Reed et al., 1987). The cytokine augments the antigen presenting cell function of macrophages through increased expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules (Morrissey et al., 1987; Fischer et al., 1988). GM-CSF also stimulates the production of multiple pro-inflammatory cytokines including TNF-a, IL-1a, IL-1b, IL-6, IL-8, IL-12, macrophage colony-stimulating factor (M-CSF) and IL-1Ra (Bergamini et al., 2000). Lastly, GM-CSFinduced expression of critical adhesion molecules, such as CD11b and ICAM, promotes emigration into inflammatory foci (Cannistra et al., 1988; Finbloom et al., 1993; Bernasconi et al., 1995).

GM-CSF promotes eosinophil survival, cytotoxicity, leukotriene production, and adhesion (Silberstein et al., 1986; Sung et al., 1997; Gasson, 1991). The cytokine also induces histamine release from basophilic granulocytes (Haak-Frendscho et al., 1988; Hirai et al., 1988). Although GM-CSF receptors have not been detected on T lymphocytes, some evidence suggests that GM-CSF may enhance the ability of IL-2 to stimulate T cell growth in vitro (Santoli et al., 1988). NKT cell precursors can differentiate in vitro into mature NKT cells through the actions of GM-CSF (Sato et al., 1999). Lastly, GM-CSF is active on a variety of non-hematopoietic cells, including fibroblasts, endothelial cells, and smooth muscle cells (Dedhar et al., 1988; Bussolino et al., 1989).

Dendritic cells

GM-CSF-transgenic mice

Dendritic cells are specialized for antigen capture, migration and T cell stimulation (Banchereau and Steinman, 1998). They constitute a rare population in most tissues and are difficult to isolate. The recognition that GM-CSF elicits striking effects on Langerhans cells in vitro suggested that this cytokine might influence dendritic cells generally (WitmerPack et al., 1987). Indeed, GM-CSF (alone or with IL-4 or TNF-a) was subsequently shown to promote the development of dendritic cells from murine and human hematopoietic precursors (Inaba et al., 1992; Romani et al., 1994; Sallusto and Lanzavecchia, 1994; Caux et al., 1996; Zhou and Tedder, 1996). Despite this ability, dendritic cell numbers are not altered in GMCSF-deficient mice, suggesting that other factors such as flt3-ligand are required for steady-state production (Dranoff et al., 1994; Saunders et al., 1996; McKenna et al., 2000).

Transgenic mice expressing GM-CSF develop accumulations of macrophages in multiple tissues, resulting in blindness and lethal tissue damage (Lang et al., 1987). Substantial increases in macrophage reactive oxygen species production contribute to this toxicity (Elliott et al., 1991). Wild-type mice transplanted with hematopoietic cells engineered to secrete GM-CSF develop striking increases in myeloid cell populations. Extensive neutrophil, eosinophil and macrophage infiltrates in the spleen, lung, liver, peritoneal cavity, heart and skeletal muscle rapidly culminate in death (Johnson et al., 1989). The intra-tracheal administration of adenoviral vectors expressing GMCSF induces a marked pulmonary accumulation of eosinophils, neutrophils, macrophages and dendritic cells (Xing et al., 1996; Wang et al., 2000).

IN VIVO ACTIVITIES IN EXPERIMENTAL MODELS The functions of GM-CSF in vivo have been explored through genetic manipulations in murine models and the pharmacologic administration of recombinant protein.

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GM-CSF-deficient mice Despite the ability of GM-CSF to stimulate hematopoiesis, homozygous inactivation of the gene in mice does not alter steady-state blood cell production (Dranoff et al., 1994; Stanley et al., 1994). Normal numbers of peripheral blood cells, bone marrow progenitors, and tissue hematopoietic populations, including splenic dendritic cells, are maintained throughout the lifespan of these animals (Dranoff and Mulligan, 1994). Lethally irradiated mutant recipients can also be efficiently reconstituted with mutant bone marrow (Dranoff and Mulligan, 1994). In contrast to these findings, GM-CSF-deficient mice display profound pulmonary abnormalities. Mutant animals develop progressive accumulation of surfactant proteins and lipids in the alveoli, a condition that closely resembles the human disease pulmonary alveolar proteinosis (PAP) (Dranoff et al., 1994; Stanley et al., 1994). Analysis of surfactant protein mRNA transcripts in these mice revealed no alterations, suggesting that surfactant production was not increased. Rather, metabolic labeling studies showed that surfactant clearance and catabolism were markedly impaired (Ikegami et al., 1996, 2001). These findings suggest that GM-CSF functions in the lung to render alveolar macrophages competent to metabolize oxidatively damaged surfactant. Consistent with this idea, the transgenic expression of GM-CSF in the lung epithelium of GM-CSF-deficient mice corrected the surfactant accumulation (Huffman et al., 1996). GM-CSF-deficient mice further manifest compromised antigen-specific IgG and cytotoxic T cell responses, IFN-c production and phagocyte function (Wada et al., 1997; Noguchi et al., 1998; Scott et al., 1998; Zhan et al., 1999). These immune defects confer an increased susceptibility to infection with Listeria monocytogenes, group B Streptococcus and Pneumocystis carinii, but partial protection against endotoxin challenge and collagen-induced arthritis (Basu et al., 1997; Campbell et al., 1998; Zhan et al., 1998; LeVine et al., 1999; Paine et al., 2000). An additional important role for GM-CSF in the female reproductive tract was revealed by a partial impairment in fertility (Robertson et al., 1999). The mean number of resorbing and malformed fetuses was increased in pregnant GM-CSF-deficient females, and the mean litter sizes of homozygous GM-CSF-

deficient breeding pairs was slightly reduced compared with controls (Robertson et al., 1999). GM-CSF-deficient mice have been interbred with other cytokine knockouts to explore potential redundancies of function. Mice deficient for GM-CSF and granulocyte colony-stimulating factor (G-CSF) show impaired neonatal granulopoiesis, an increased incidence of pneumonia, compromised reproductive capacities, and amyloidosis (Seymour et al., 1997). Mice deficient for GM-CSF and M-CSF display PAP and osteopetrosis, but still possess circulating monocytes and lung macrophages (Lieschke et al., 1994). Mice deficient for GM-CSF and IL-3 develop modest increases in circulating eosinophils and markedly reduced contact hypersensitivity responses (Gillessen et al., 2001).

bc subunit-deficient mice Mice deficient for the bc subunit of the GM-CSF receptor develop PAP resembling that found in GM-CSF-deficient mice and generate reduced numbers of eosinophils, similar to IL-5-deficient mice (Nishinakamura et al., 1995; Foster et al., 1996 Reed et al., 2000). Transplantation of wild-type bone marrow into bc-deficient mice corrects the PAP, supporting the idea that abnormal alveolar macrophage function contributes to the surfactant accumulation (Nishinakamura et al., 1996b; Cooke et al., 1997). The lymph nodes of bc-deficient mice show a minimal decrease in dendritic cell numbers compared with controls (Vremec et al., 1997). Mice deficient for bc and IL-3 develop PAP and mount reduced eosinophil responses, but show no additional abnormalities (Nishinakamura et al., 1996a).

Pharmacologic administration Rodents The intraperitoneal injection of GM-CSF increases granulocyte and macrophage numbers, but reduces bone marrow cellularity in normal mice (Metcalf et al., 1987). Peritoneal macrophages show increased proliferation and phagocytic capacity. Similarly, GM-CSF administration induces a peripheral neutrophilia and monocytosis in rats that peaks between 4 and 8 h and returns to baseline by 12 h (Ulich et al., 1990).

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Vaccination

GM-CSF administration by continuous infusion or daily subcutaneous or intravenous injection elicits a marked leukocytosis in healthy monkeys, with fourfold increases in granulocytes, monocytes and lymphocytes, but no effects on platelet or erythrocyte counts (Donahue et al., 1986; Mayer et al., 1987). Granulocytes from treated animals show enhanced oxidative metabolism and bactericidal capacity (Mayer et al., 1987). The sequential administration of IL-3 followed by GM-CSF dramatically increases leukocyte and platelet responses (Donahue et al., 1988; Stahl et al., 1992).

Dendritic cells (DCs) are specialized to initiate immunity because of their abilities to process antigens efficiently into both MHC class I and II pathways and their high level expression of costimulatory molecules (Banchereau and Steinman, 1998). These cells acquire antigens in peripheral tissues and migrate to organized lymphoid structures to stimulate antigenspecific CD4 and CD8 positive T lymphocytes and B cells. GM-CSF induces the development of DCs in vitro (Caux et al., 1992; Inaba et al., 1992; Reid et al., 1992; Scheicher et al., 1992) and in vivo (Mach et al., 2000). Vaccination with irradiated tumor cells engineered to secrete GM-CSF stimulates the expansion and maturation of DCs and results in a potent, specific and long-lasting anti-tumor immunity in mice (Dranoff et al., 1993; Mach et al., 2000). The administration of recombinant GM-CSF protein or plasmid DNA encoding GM-CSF similarly enhances anti-tumor immunity in numerous model systems (Dranoff, 1998).

Disease models Myelosuppression GM-CSF enhances neutrophil and platelet recovery following sublethal irradiation of rhesus monkeys (Clark and Kamen, 1987; Monroy et al., 1987; Nienhuis et al., 1987).

GM-CSF DEFICIENCY OR EXCESS STATES IN HUMANS

Infections GM-CSF improves host responses to a variety of infectious challenges. Mice infected with Candida albicans show enhanced survival and clearance of the organism following GM-CSF administration (Liehl et al., 1994). The cytokine augments alveolar macrophage function and improves survival after challenge with Pneumococcus (Hebert and O’Reilly, 1996). GM-CSF also reduces the intensity of Pneumocystis carinii infection, particularly in immunocompromised mice (Paine et al., 2000).

Wound healing GM-CSF stimulates an increase in wound healing in normal rodents and diabetic rats (Vyalov et al., 1993; Canturk et al., 1999). The cytokine improves wound tensile strength and cellularity in several models, leading to faster resolution of injury (Jyung et al., 1994; Molloy et al., 1995).

GM-CSF deficiency Kitamura et al. found that 11 individuals with idiopathic PAP, but not healthy controls or patients with other lung disease, showed high titers of neutralizing IgG antibodies against GM-CSF (Kitamura et al., 1999). This suggests that some cases of PAP may involve an autoimmune response to GM-CSF. Mutations in the bc subunit have been observed in a subset of pediatric patients with PAP (Dirksen et al., 1997). A third mechanism underlying PAP may involve aberrant IL-10 expression, leading to reduced GM-CSF production (Tchou-Wong et al., 1997; Thomassen et al., 2000). Interestingly, the administration of GMCSF has resulted in symptomatic, physiologic and radiographic improvement in some patients with PAP (Seymour et al., 1996; Kavuru et al., 2000; Barraclough and Gillies, 2001).

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Enhanced GM-CSF production Increased levels of GM-CSF are associated with some forms of asthma and other eosinophil-mediated inflammatory lung diseases (Robinson et al., 1993; Walker et al., 1994).

CLINICAL APPLICATIONS OF GM-CSF The ability of GM-CSF to enhance the proliferation and function of neutrophils, eosinophils, monocytes and dendritic cells has led to its application in diverse clinical settings (Ruef and Coleman, 1990; Gasson, 1991; Kanz et al., 1993; Romani et al., 1994; Caux et al., 1996).

After therapy-related myelosuppression or bone marrow transplantation GM-CSF produced in either yeast or bacteria has been registered worldwide for use in reducing the duration of neutropenia and thrombocytopenia following cytotoxic chemotherapy or bone marrow transplantation. Parenteral administration of GM-CSF induces a dose-dependent increase in peripheral blood neutrophil counts by altering the kinetics of myeloid progenitor cell development within the bone marrow (Kaplan et al., 1989). The cytokine induces rapid entry of neutrophil progenitor cells into the cell-cycle and shortens cell-cycle time (Broxmeyer et al., 1989). GMCSF allows chemotherapy dose-intensification, as the rapid amelioration of neutropenia permits reduction in treatment intervals (Yau et al., 1996; Pfreundschuh et al., 2001). This advantage is primarily during the early courses of chemotherapy, since late-cycle thrombocytopenia is not prevented (Neidhart et al., 1992; Grem et al., 1994; Lachance et al., 1995; Moghrabi et al., 1995; Yau et al., 1996). Additional benefits conferred by the cytokine include shorter periods of hospitalization and reduced platelet-transfusion requirements (Gulati and Bennett, 1992; Bennett et al., 1999). GM-CSF improves survival following bone marrow graft failure after transplantation (Nemunaitis et al., 1990).

Mobilization of peripheral blood stem cells The use of autologous or allogeneic peripheral blood stem cells (PBSC) to repopulate the bone marrow after cytotoxic chemotherapy or radiation damage has become an increasingly important tool in the management of cancer. Different approaches have been used in evaluating the role of GM-CSF in mobilization of PBSC. Since GM-CSF-induced mobilization of PBSC is more efficient with prior ablative chemotherapy, various combinations of chemotherapeutics have been tested in mobilization of autologous PBSC (Socinski et al., 1988b; Gianni et al., 1989, 1990; Brugger et al., 1991; Lane et al., 1995). Cell collections from patients whose bone marrow has not been previously damaged by extensive chemotherapy or radiation therapy are more efficient than those from patients whose bone marrow has been impaired (Campos et al., 1993). In the last few years, interest has increased in improving mobilization of PBSC with cytokines alone without having to administer chemotherapy. This is especially true in the allogeneic transplant setting, where a nontoxic mobilization regimen that follows for collection of a sufficient number of cells to promote engraftment in a minimum number of leukaphereses is most critical. In this context, GM-CSF has been tested alone, and together with other cytokines. In one study of normal donors, the stem cell yield with combination G-CSF and GM-CSF treatment was significantly greater than with either cytokine alone (Lane et al., 1995).

Drug-induced agranulocytosis Patients with drug-induced agranulocytosis show a faster normalization of the peripheral blood granulocyte count and a reduced incidence of fatal complications with the application of GM-CSF (Sprikkelman et al., 1994).

Neutropenia and sepsis Patients suffering from sepsis complicating prolonged neutropenia may benefit from GM-CSF administration. In this setting, GM-CSF may reduce

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the number of lengthy hospitalizations by up to 50% (Riikonen et al., 1994). On the other hand, GM-CSF does not reduce the incidence of sepsis associated with brief periods of neutropenia (Steward et al., 1998).

Infections Although GM-CSF enhances the antimicrobial activities of phagocytes, controlled clinical trials are necessary to define specific indications during infection. Functional studies of neutrophils and monocytes isolated from cancer patients treated with GM-CSF show enhanced phagocytosis and killing of Staphylococcus aureus (Verhoef and Boogaerts, 1991). GM-CSF also augments the fungicidal activity of macrophages and neutrophils (Ruef and Coleman, 1990). In this context, the addition of GM-CSF to amphotericin B therapy in cancer patients suffering disseminated Candida infections may be of clinical benefit (Bodey et al., 1993). Administration of GM-CSF to leukemia patients and bone marrow transplant recipients significantly reduces the incidence and severity of fungal infections (Nemunaitis et al., 1991; Rowe et al., 1996).

Human immunodeficiency virus (HIV) infection GM-CSF is effective in ameliorating chemotherapyinduced neutropenia in HIV patients with Kaposi’s sarcoma (Scadden et al., 1991, 1996; Gill et al., 1992). Although initial studies indicated that GM-CSF enhanced HIV replication in monocyte/macrophages (Folks et al., 1987; Koyanagi et al., 1988; Hammer et al., 1990; Kitano et al., 1991), there is now convincing evidence that GM-CSF in combination with antiretroviral therapy suppresses HIV replication (Matsuda et al., 1995; Skowron et al., 1999). In part, this may reflect the ability of GM-CSF to increase the intracellular phosphorylation of some dideoxynucelosides, such as zidovudine and stavudine (Hammer and Gillis, 1987; Perno et al., 1992). In a phase III study of patients with advanced HIV disease, the addition of GM-CSF to anti-retroviral therapy resulted in a significant increase in CD4 cell counts and a decrease in overall infections and development of drug-resistance as well (Angel et al., 2000). GM-CSF augments the phagocytosis of Mycobacterium avium-intracellulare (MAC)

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by HIV-infected monocytes/macrophages and may prove beneficial against this important pathogen (Kedzierska et al., 2000).

Neonates The dysregulation of cytokine and hematopoietic growth factor synthesis and action may be an important contributory part to the complex deficiency of immunologic and hematologic function, especially in the preterm neonate. In one randomized, controlled study, the administration of prophylactic GM-CSF at birth to premature newborns ameliorated neutropenia (Carr et al., 1999). GM-CSF also increases neutrophil, eosinophil, monocyte, lymphocyte and platelet counts in septic neonates and can reduce overall mortality (Bilgin et al., 2001).

Myelodysplasia and aplastic anemia Several studies have examined the use of GM-CSF in myelodysplasia (MDS) (Ganser and Hoelzer, 1996). Although pre-clinical experiments demonstrated a potential synergy between GM-CSF and erythropoietin in vitro for the production of erythrocytes from MDS precursors, clinical testing of the combination failed to reveal a reduction in transfusion requirements (Stasi et al., 1999; Thompson et al., 2000). Long-term treatment with GM-CSF can reduce the frequency of infections, but may be associated with allergic reactions (Yoshida et al., 1995). The use of GM-CSF for the treatment of aplastic anemia remains investigational. Therapy of severe disease with anti-lymphocyte globulin (ALG), GMCSF and erythropoietin induced a complete remission rate of 91% in one study (Shao et al., 1998).

Acute myeloid leukemia The malignant blasts in acute myeloid leukemia (AML) express GM-CSF receptors and respond to cytokine addition (Vellenga et al., 1987; Aglietta et al., 1991; Gasson, 1991). This finding motivated clinical trials aimed at stimulating leukemic cell entry into S phase prior to the administration of cytotoxic chemotherapy (Bettelheim et al., 1991; Buchner et al., 1993; Frenette et al., 1995; Thomas et al., 1999). Although GM-CSF infusion did promote leukemic cell

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DNA synthesis in vivo, it failed to increase complete remission rates or prolong disease-free survival compared with control groups (Estey, 1994; Heil et al., 1995; Thomas et al., 1999). Moreover, although GMCSF administration following chemotherapy may promote normal myeloid cell recovery in some patients, no significant survival differences were observed compared with controls (Buchner et al., 1993; Stone et al., 2001).

Vaccines for infectious diseases The important contributions of GM-CSF to the maturation and function of antigen-presenting cells, such as dendritic cells and macrophages, and its ability to promote T and B cell immunity, provides the basis for its potential role as a vaccine adjuvant. Results of several preliminary studies using GM-CSF in conjunction with hepatitis B (Hess et al., 1996; Tarr et al., 1996) and influenza vaccines (Operschall et al., 1999; Pauksen et al., 2000) support the ability of the cytokine to enhance antigen specific immune responses.

Tumor immunity Studies evaluating the vaccination activity of tumor cells engineered to secrete GM-CSF suggest that the cytokine may prove useful for cancer immunotherapy. In multiple murine tumor models, the expression of GM-CSF at tumor sites dramatically stimulated the development of anti-tumor immunity (Dranoff et al., 1993; Armstrong et al., 1996; Mach et al., 2000). A phase I clinical trial of vaccination with irradiated autologous melanoma cells engineered to secrete GM-CSF in patients with metastatic melanoma also revealed the induction of significant anti-tumor immunity. In particular, metastases resected after immunization exhibited dense infiltration with T lymphocytes and plasma cells associated with extensive tumor necrosis, fibrosis and edema in 11 of 16 patients studied (Soiffer et al., 1998). Further, adjuvant systemic GM-CSF therapy of resected stage III and IV melanoma patients led to significantly prolonged disease-free survival compared with matched historical controls (Spitler et al., 2000). GM-CSF has also been studied as a vaccine adjuvant with defined tumor antigens, including the melanosomal differentiation proteins and HER-2/neu (Jager et al., 1996;

Samanci et al., 1998; Bendandi et al., 1999; Disis et al., 1999; Leong et al., 1999). Several investigations have explored the effects of GM-CSF on activated killer cell (AKC) activity in patients with AML undergoing BMT (Bendall et al., 1995; Richard et al., 1995). In one study, the actuarial risk of relapse was 37.4% in GM-CSF-treated patients compared with 49.5% in controls after a median follow-up of 24 months. Interestingly, none of the seven investigated patients with an AKC activity  20% in the first 2–5 weeks after autologous BMT relapsed compared with six of nine patients with an AKC activity 20% (Richard et al., 1995).

Wound healing and mucositis GM-CSF enhances the migration and proliferation of endothelial cells and promotes keratinocyte growth, factors crucial to wound repair (Hancock et al., 1988; Bussolino et al., 1989). A number of case reports and small series have explored the use of GM-CSF as a treatment for poorly healing wounds with encouraging results (da Costa et al., 1994; Raderer et al., 1997; Groves and Schmidt-Lucke, 2000). A study of 38 elderly patients with chronic venous insufficiency revealed that topical GM-CSF followed by application of a compression dressing resulted in complete healing in 90% of the cases (Jaschke et al., 1999). Moreover, chronic, non-healing leg ulcers of Necrobiosis lipoidica diabeticorum were successfully treated with topical GM-CSF (Remes and Ronnemaa, 1999). Similarly, GM-CSF has also been found to be beneficial in patients with severe mucositis after chemotherapy or radiation (Hejna et al., 1999; Karthaus et al., 1999).

Primary pulmonary alveolar proteinosis Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of lipoproteinaceous material within the alveoli. As discussed above, abnormalities in GM-CSF function have been associated with some form of this disease. Preliminary studies show that some patients with idiopathic PAP benefit by the administration of GM-CSF (Seymour et al., 1996; Barraclough and Gillies, 2001; Kavuru et al., 2000).

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TABLE 21.1 Summary of possible therapeutic applications of GM-CSF Therapeutic use

Effects

References

Neutropenia and thrombocytopenia after chemotherapy or BMT

Shortens the period of neutropenia and thrombocytopenia by inducing the proliferation and egress of bone marrow-derived neutrophils, eosinophils, monocytes and platelets

(Gasson, 1991) (Straneva et al., 1989)

Increasing chemotherapy dose intensity

Allows chemotherapy dose intensification by interval reduction

(Pfreundschuh et al., 2001)

Peripheral blood stem cell mobilization (PBSC) for BMT

Mobilizes PBSC after chemotherapy or in combination with G-CSF

(Socinski et al., 1988a) (Gianni et al., 1989) (Lane et al., 1995)

Serious bacterial or fungal infections

Enhances chemotaxis, up-regulates surface antigens and increases phagocytic activity of neutrophils, macrophages and monocytes

(Rossman et al., 1993) (Williams et al., 1995)

HIV infections and its complications

Suppresses HIV replication Augments antiretroviral activity of zidovudine and stavudine Promotes killing of Mycobacterium aviumintracellulare (MAC)

(Perno et al., 1992) (Matsuda et al., 1995) (Hammer et al., 1990) (Onyeji et al., 1995)

Preterm neonate

Abolishes postnatal neutropenia Significantly decreases mortality in septic neonate

(Carr et al., 1999) (Bilgin et al., 2001)

Myelodysplasia (MDS) and aplastic anemia (AA)

MDS: Abrogates neutropenia and relieves transfusion requirements in combination with erythropoietin (Epo) AA: Enhances complete remission rates in combination with anti-lymphocyte globulin and Epo

(Stasi et al., 1999) (Shao et al., 1998)

Prior to cytotoxic chemotherapy in AML

Stimulates leukemic cells to initiate DNA synthesis. Possible enhancement of cell cytotoxicity

(Vellenga et al., 1987) (Thomas et al., 1999)

Vaccine adjuvant

Increases antigen presenting cell function and stimulates T and B cell responses

(Operschall et al., 1999) (Dranoff et al., 1993)

Antitumor therapy

Enhances tumoricidal functions Facilitates tumor antigen presentation

(Richard et al., 1995)

Wound healing

Stimulates migration and proliferation of endothelial cells Promotes keratinocyte growth Increases formation of granulation tissue Increases breaking strength of incisional wounds.

(Jyung et al., 1994)

Pulmonary alveolar Proteinosis (PAP)

Improves symptoms, physiology, and radiographic findings in some patients with PAP

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(Braunstein et al., 1994) (Kaplan et al., 1992) (Bussolino et al., 1989) (Kavuru et al., 2000)

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Pharmacokinetics

ACKNOWLEGEMENTS

The pharmacokinetics of GM-CSF are determined by the route of administration. Peak serum concentrations are higher after intravenous than subcutaneous injection, although the bioavailability is comparable. Serum concentrations are more prolonged after subcutaneous administration (Stute et al., 1995). The elimination of GM-CSF occurs principally by nonrenal pathways (Schwinghammer et al., 1991). The increase in absolute neutrophil count with a specific dose of GM-CSF is greater after subcutaneous injection than after a 2-h intravenous infusion (Schwinghammer et al., 1991). Lastly, the pharmacokinetics of GM-CSF are similar among healthy individuals and patients (Schwinghammer et al., 1991).

Toxicities The common side effects of systemic GM-CSF administration are fever, myalgia, malaise and arthralgia (Stern and Jones, 1992). Infrequent adverse events include irritation at injection sites, fluid retention and dyspnea. Cardio-pulmonary toxicities including supraventricular tachycardia and capillary leak syndrome are rare complications.

SUMMARY GM-CSF stimulates the proliferation, differentiation and function of multiple myeloid cells including neutrophils, monocytes/macrophages, eosinophils and dendritic cells. These properties may prove beneficial for a broad array of diseases (Table 21.1). The cytokine ameliorates myelosuppression following cytotoxic chemotherapy and bone marrow transplantation. GM-CSF may be useful as prophylaxis or adjunctive treatment for serious bacterial and fungal infections, especially in immunocompromised hosts. Its potent immunomodulatory activities might be further exploited in vaccine development against tumors and pathogens. The ability of GM-CSF to enhance endothelial cell and keratinocyte growth may be advantageous for wound healing and mucositis. Lastly, deficiency of GM-CSF underlies the pathogenesis of at least some forms of PAP, rendering this previously idiopathic disease amenable to replacement therapy.

This study was supported by the Hanne Liebermann Foundation, Zurich, Switzerland (T.E.), the Cancer Research Institute/Partridge Foundation, CA74886 and CA39542 (G.D.). G.D. is a Clinical Scholar of the Leukemia and Lymphoma Society.

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Yang, Y.C., Kovacic, S., Kriz, R. et al. (1988). The human genes for GM-CSF and IL 3 are closely linked in tandem on chromosome 5. Blood 71, 958–961. Yasukawa, H., Sasaki, A. and Yoshimura, A. (2000). Negative regulation of cytokine signaling pathways. Annu. Rev. Immunol. 18, 143–164. Yau, J.C., Neidhart, J.A., Triozzi, P. et al. (1996). Randomized placebo-controlled trial of granulocyte–macrophage colony-stimulating factor support for dose-intensive cyclophosphamide, etoposide, and cisplatin. Am. J. Hematol. 51, 289–295. Yong, K.L. and Linch, D.C. (1993). Granulocyte–macrophage colony-stimulating factor differentially regulates neutrophil migration across IL-1-activated and nonactivated human endothelium. J. Immunol. 150, 2449–2456. Yoshida, Y., Nakahata, T., Shibata, A. et al. (1995). Effects of long-term treatment with recombinant human granulocyte–macrophage colony-stimulating factor in patients with myelodysplastic syndrome. Leuk. Lymphoma 18, 457–463. Zhan, Y., Lieschke, G.J., Grail, D. et al. (1998). Essential roles for granulocyte–macrophage colony-stimulating factor (GM-CSF) and G-CSF in the sustained hematopoietic response of Listeria monocytogenes-infected mice. Blood 91, 863–869. Zhan, Y., Basu, S., Lieschke, G.J. et al. (1999). Functional deficiencies of peritoneal cells from gene-targeted mice lacking G-CSF or GM-CSF. J. Leuk. Biol. 65, 256–264. Zhou, L.J. and Tedder, T.F. (1996). CD14 blood monocytes can differentiate into functionally mature CD83 dendritic cells. Proc. Natl Acad. Sci. USA 93, 2588–2592.

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22 Granulocyte colony-stimulating factor Scott M. White and David J. Tweardy Baylor College of Medicine, Houston, TX, USA

At the bottom there is only one treatment for all diseases and that is to stimulate the phagocytes. Stimulate the phagocytes, drugs are a delusion. George Bernard Shaw

INTRODUCTION Neutrophilic granulocytes (neutrophils) serve as the critical circulating cell for nonspecific host defense against microbial infection. The short-lived circulating population of neutrophils is continuously renewing, being replenished via newly developed cells originating from the bone marrow hematopoietic stem cell population. Granulocyte colony-stimulating factor (G-CSF) is a 20 000 Mr glycoprotein that plays a critical role in this process, acting to stimulate the basal and stress-induced production of neutrophils and enhancing some of their anti-microbial processes. G-CSF is produced by a variety of cell types, including bone marrow stromal cells, fibroblasts, endothelial cells and activated macrophages. Production of G-CSF in large quantities as a recombinant protein in Escherichia coli has allowed its clinical use as a drug to treat neutropenia due to a variety of causes. The enhancement of myeloid cell proliferation, The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

maturation and function that is triggered by G-CSF stimulation occurs due to signals transduced via its cell surface receptor (G-CSFR). The G-CSFR is a type 1 membrane protein of the hematopoietic growth factor receptor family. Its expression is limited and has been demonstrated on myeloid progenitors and circulating neutrophils, as well as non-hematopoietic cell types such as placental cytotrophoblasts. Signal transduction classically occurs following the binding of one G-CSF molecule to one G-CSFR molecule with subsequent receptor dimerization producing activation of signal transduction molecules. While the G-CSFR does not contain any intrinsic protein kinase activity, tyrosine phosphorylation of signaling molecules and the receptor itself occurs due to the activation of members of the Janus kinase (JAK) family. Following is a summary of the molecular and biochemical properties of G-CSF and its receptor, their role in normal and pathologic physiology, a detailed description of G-CSFR signal transduction and areas of ongoing research.

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GRANULOCYTE COLONY-STIMULATING FACTOR AND ITS RECEPTOR Biochemical properties of G-CSF Murine G-CSF was initially purified from culture medium conditioned by lung tissue obtained from mice injected with bacterial endotoxin (Nicola et al., 1983). The identification of G-CSF was based upon its ability to drive differentiation of the murine myelomonocytic leukemia cell line WEHI-3BD cells into granulocytes and monocytes. With an Mr of 24 000–25 000 and a pI of 4.5–5.8, this acidic glycoprotein is reasonably stable at extremes of pH (2–10), temperature (loss of 50% activity following 30 min at 70 C) and under strong denaturing conditions (8 M urea, 0.1% sodium lauryl sulfate, or 6 M guanidine hydrochloride). Human G-CSF was subsequently isolated from the conditioned tissue culture medium of various tumor cell lines and found to have an Mr of 18 000–19 000 and a pI of 5.5–6.1 depending upon the extent of sialylation (Welte et al., 1985; Nomura et al., 1986). The sequencing of purified human G-CSF protein allowed the cloning of its cDNA by three separate groups (Nagata et al., 1986a; Souza et al., 1986; Tweardy et al., 1987a). The 204 amino acid peptide sequence includes 30 amino acids of signal sequence. Its molecular weight is calculated to be 18 671 Da. While Souza et al. and Tweardy et al. isolated only single clones, Nagata et al. isolated a second cDNA clone, the result of alternative splicing, containing an additional nine base pairs that insert three amino acids at positions 36–38. This longer isoform of G-CSF has less biologic activity than the shorter isoform and is less abundant. Endogenously produced human G-CSF is Oglycosylated at Thr-133, with N-acetyl-neuraminic acid a(2–6){galactose, b(1–3)} N-acetylgalactosamine (Souza et al., 1986; Oheda et al., 1988). This glycosylation inhibits aggregation and thereby stabilizes the molecule, but it is not required for biologic activity. Recombinant human G-CSF produced in Escherichia coli is not glycosylated, but has activity similar to endogenous G-CSF. Four of the five cysteine residues of the G-CSF molecule connect via disulfide bonds (Cys-36–Cys-42 and Cys-64–Cys-74) and are required

to maintain proper folding and function (Lu et al., 1989). These disulfide bridges are conserved between mouse and human G-CSF and those in interleukin 6 (IL-6) (Kishimoto et al., 1994). The amino acid sequence homology between human G-CSF and that of other species exceeds 70% and allowed the cloning of G-CSF cDNAs of murine, canine, bovine and rat origins (Tsuchiya et al., 1986; Lovejoy et al., 1993; Han et al., 1996). The G-CSFs from these different species fully cross-react with regards to their activity. The cloning of G-CSF from various species has allowed X-ray diffraction study of human, canine and bovine species and NMR (nuclear magnetic resonance) analysis of human G-CSF. These studies have revealed a bundled structure, including two antiparallel pairs of a-helices (Plate 22.1) (see Plate section) similar to that seen in other cytokines such as interleukin 2 (IL-2), interferons (IFNs), granulocyte– macrophage colony-stimulating factor (GM-CSF) or growth hormone (Hill et al., 1993; Lovejoy et al., 1993; Zink et al., 1994). The mutational analysis of the Nterminal and C-terminal regions of human G-CSF demonstrate that both portions are critical to the interaction between G-CSF and its receptor (ReidhaarOlson et al., 1996). The details of this interaction will be discussed below.

G-CSF gene structure and expression The single gene for human G-CSF is found on chromosome 17q21 proximal to the breakpoint characteristic of acute promyelocytic leukemia (APL) (Simmers et al., 1987; Tweardy et al., 1987a; Le Beau et al., 1987; Kanda et al., 1987). The murine G-CSF gene is found on chromosome 11 (Buchberg et al., 1988). Both genes have the structure of five exons separated by four introns and are approximately 2.5 kb in length. The localization of the human G-CSF gene separates it from other hematopoietic growth factors, including IL-3, IL-4, IL-5 and GM-CSF that are located on the long arm of chromosome 5 (Nicola, 1989). This is mirrored in the murine system where G-CSF is on a region of murine chromosome 11 that is homologous to human chromosome 17, whereas GM-CSF and IL-3 are in a region of murine chromosome 11 that is homologous to the human chromosome 5 (Nicola, 1989). In the human gene at the 5 terminus of the second

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intron there are two potential splice donor sites separated by only nine bases (Nagata et al., 1986b). When the more proximal donor site is used, the 204 amino acid isoform of G-CSF is produced. In CHU-2 cells, the more distal splice donor site was also used, producing a less active isoform of G-CSF that is 207 amino acids in length (Nagata et al., 1986b). The biologic relevance of the longer isoform and the mechanisms involved in the recognition of the distal splice donor site are not known. The murine G-CSF gene does not demonstrate this tandem splice donor site structure (Tsuchiya et al., 1987a). The G-CSF gene is transcribed in an assortment of cell types such as macrophage/monocytes, astroglial cells, bone marrow stromal cells and cells of mesodermal origin including vascular endothelium, fibroblasts and mesothelial cells (Koeffler et al., 1987; Fibbe et al., 1988; Zsebo et al., 1988; Demetri et al., 1989; Tweardy et al., 1990). G-CSF production in these cells is increased by a variety of stimuli. Tumor necrosis factor-a (TNF-a) and IL-1 stimulate production of GCSF by endothelial cells, astroglial cells, fibroblasts and macrophages, with bacterial lipopolysaccharide (LPS) also stimulating G-CSF production by macrophages (Metcalf and Nicola, 1985; Broudy et al., 1987; Koeffler et al., 1987; Seelentag et al., 1987; Kaushansky et al., 1988; Lu et al., 1988; Vellenga et al., 1988; Nishizawa and Nagata, 1990; Tweardy et al., 1990). Therefore LPS-stimulated macrophages that also produce TNF-a and IL-1 will drive production of G-CSF by fibroblasts and endothelial cells. This is a means to explain the increased serum levels of G-CSF and associated granulocytosis observed in humans during the acute stage of bacterial infection (Kawakami et al., 1990). G-CSF is also produced constitutively by a number of different neoplasms, including squamous cell carcinoma, melanoma, glioblastoma, transitional cell carcinoma, hepatoma, ovarian carcinoma, epithelial skin tumors chronic B-cell leukemia and human T-lymphotrophic virus-1 (HTLV-1)-transformed endothelial cells (Tweardy et al., 1987b; Nagata, 1990; Takashima et al., 1996; Wang et al., 1996; Savarese et al., 2001; Brandstetter et al., 2001; Hirai et al., 2001). The clinical relevance of this aberrant G-CSF production remains to be determined. The transcriptional regulation of G-CSF production appears to be controlled through three cis-regulatory promoter elements (G-CSF promoter elements GPE1,

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GPE2 and GPE3) (Nishizawa and Nagata, 1990; Nishizawa et al., 1990) within the 300 or so nucleotides immediately upstream of the ATG initiation codon. This region is highly homologous between both the human and murine G-CSF gene. These sites as studied in murine monocyte/macrophage cell lines and human carcinoma cell lines include GPE1, a region approximately 180 bases upstream of the major transcription start site with binding sites for nuclear factor-jB (NF-jB) {PuGAGPuTTCCACPu} and NFIL-6 {TT/GNNGNAAT/G}. The NF-jB binding site is observed upstream of many cytokine genes including GM-CSF and IL-3 (Stanley et al., 1985; Shannon et al., 1988; Miyatake et al., 1988). GPE2 is an octamer sequence {ATTTGCAT} 110 bases proximal to the TATA box that interacts with an octamer transcription factor (OTF). GPE3 is a region of approximately 40 bases in length that is suspected to regulate G-CSF transcription and bears no homology to promoter elements observed in other genes (Asano and Nagata, 1992). In a model using LPS to stimulate macrophage production of G-CSF all three of these promoter elements appear to interact with their respective transcription factors in a collaborative manner (Asano et al., 1991). The role of the NF-IL-6 site in LPS and TNF-a-mediated G-CSF production has been confirmed in studies using NF-IL-6 null mice (Tanaka et al., 1995) and antisense oligonucleotides targeting NF-IL-6 (Kiehntopf et al., 1995). In addition to transcriptional regulation of G-CSF production, there is also a mechanism of posttranscriptional regulation that involves mRNA stability. The 3 untranslated region of the G-CSF transcript contains numerous AUUUA sequences (Shaw and Kamen, 1986) allowing an IL-10-mediated mechanism to immediately destabilize the transcript and thus increase its rate of degradation (Brown et al., 1996). Such poly-AUUUA sequences have been noted in a growing list of growth factor and growth regulatory molecule genes (Akashi et al., 1994). Conversely, TNF-a-exposed fibroblasts demonstrate increased G-CSF mRNA stability (Koeffler et al., 1988). The details of the mechanism through which G-CSF mRNA stability is controlled remain unknown. Of interest outside of the role of G-CSF in granulopoiesis and neutrophil functional regulation are reports of G-CSF/G-CSFR expression on cells of the maternal/fetal interface in both mice and humans

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(Shorter et al., 1992; Saito et al., 1994; Vandermolen and Gu, 1996; Miyama et al., 1998). While G-CSF has been shown to have a proliferative effect on trophoblasts (Miyama et al., 1998) its complete role during gestation is not yet understood. G-CSF is also present in human milk (Gilmore et al., 1994; Calhoun et al., 2000) with the G-CSFR correspondingly expressed by human fetal intestine (Calhoun et al., 2000). Again, the functional role of G-CSF/G-CSFR in this setting remains to be delineated. Mice deficient in either G-CSF or G-CSFR expression develop normally (Lieschke et al., 1994; Liu et al., 1996), which suggests that their role in the maternal/fetal interface or in intestinal epithelial cell function is redundant or able to be compensated by other cytokine/receptor systems.

Biochemical properties of G-CSFR G-CSFR expression is critical for the normal development of cells within the granulocytic lineage as is evident by the marked neutropenia seen in mice lacking G-CSFR expression (Liu et al., 1996). The receptor number per cell is in the range of 50–500 (Nicola and Metcalf, 1985), with cells expressing increasing receptor number with granulocytic maturation (Nicola et al., 1986). The dissociation constant (Kd) for G-CSF with its receptor has been measured at approximately 90–900 pM (Nicola and Metcalf, 1985; Nicola et al., 1986), whereas the biologic response is at halfmaximal at a concentration of 3 pM (Nicola and Metcalf, 1985) suggesting that only a low percentage of receptors need to be occupied for relevant signal transduction to occur. Cross-linking studies of the murine G-CSFR on WEHI-3B D cells suggested an Mr of 150 000 (Nicola and Peterson, 1986), while unpublished reports of solubilized G-CSFR from murine NFS-60 cells with an Mr of 100 000–130 000 are discussed by Fukunaga et al. (1990a). The murine G-CSFR cDNA was first isolated using expression cloning (Fukunaga et al., 1990a) and was found to encode 837 amino acids of which the N-terminal 25 amino acids served as a signal peptide. The structure of the 812 amino acid mature protein is that of a single transmembrane domain of 24 amino acids placing the 601 amino acid N-terminal region in the extracellular position and the 187 amino acid C-terminal region in the cytosol (Plate 22.2) (see Plate

section). The calculated Mr of the G-CSFR is 90 814, much lower than the measured Mr from earlier studies and is likely due to glycosylation at any of the putative 11 N-linked glycosylation sites (Asn-X-Ser/Thr) within the extracellular domain. Sub-domains within the extracellular portion of the receptor include an Iglike domain of approximately 100 amino acids at the N-terminus. The next 200 amino acids make up the cytokine-related homology domain (CRH domain), so named because of the shared homology between a large number of cytokine and hematopoietic growth factor receptors including growth hormone, prolactin, leptin, leukemia inhibitory factor, gp130 (or IL-6 receptor b-chain) and ciliary neurotrophic factor (Davis et al., 1991; Nagata and Fukunaga, 1993). Within the CRH domain are four conserved cysteine residues that form disulfide bonds, and the downstream Trp-Ser-X-Trp-Ser motif. The relevance of this domain to ligand binding is discussed along with the signal transduction activity of the G-CSFR below. The CRH domain is followed by three repeated domains similar to fibronectin type III in sequence. The murine G-CSFR has a predicted 24 amino acid transmembrane domain and ends with a 187 amino acid cytosolic domain. The details of the amino acid sequence of the cytosolic domain are discussed below as they relate to the signal transduction activities of the G-CSFR. The human G-CSFR was cloned by Fukunaga et al. (1990b) and Larsen et al. (1990) and has a structure that is highly homologous to the murine G-CSFR with a sequence similarity of 62.5%. While multiple cDNA isoforms were isolated, as is discussed below, the most homologous isoform is a mature peptide of 813 amino acids with a signal peptide of 24 amino acids removed from the N-terminus. The human G-CSFR extracellular domain retains the subdomain structures mentioned above for the murine G-CSFR. The cytosolic domain consists of 183 amino acids and has areas of sequence that are highly conserved between mouse and human versions.

G-CSFR gene structure and expression The gene for the murine G-CSFR has been localized to chromosome 4 downstream of a non-functional G-CSFR pseudogene (Ito et al., 1994). The human G-CSFR gene occurs as a single copy and has been

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mapped to the long arm of 1p32–35 (Inazawa et al., 1991; Tweardy et al., 1992). Both the human and murine G-CSFR genes consist of 17 exons (Seto et al., 1992; Ito et al., 1994) with peptide coding sequence occurring on exons 3–17 with receptor subdomains being encoded by clusters of exons.

Alternative splicing of human G-CSFR transcript While the murine G-CSFR does not appear to undergo alternative splicing that involves the cytosolic domain protein sequence, such alternative splicing has been identified for the human G-CSFR. There are to date seven known alternative splice isoforms of the human G-CSFR transcript that involve the transmembrane and or cytosolic domain (Figure 22.3) (Fukunaga et al., 1990b; Larsen et al., 1990; Seto et al., 1992; Tweardy et al., 1992; Dong et al., 1995b; Bernard et al.,

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1996). These isoforms have been designated class I–VII and their known functional and signaling activities are discussed in detail below. The class I isoform is the homologue to the murine G-CSFR, is the primary isoform detected in myeloid cells and was originally isolated from placental tissue, the histiocytic cell line U937 and the myeloid leukemia cell line HL60 (Fukunaga et al., 1990b; Larsen et al., 1990; Tweardy et al., 1992). The class II and III isoforms were also isolated from the cell line U937 (Fukunaga et al., 1990b) with the class II isoform appearing to be a soluble receptor isoform lacking the transmembrane domain due to the deletion of the majority of exon 15 via use of an alternative 3 splice acceptor site for intron 14 that is near the 3 end of exon 15. The class III isoform is derived through the use of an alternative proximal 3 splice acceptor site within intron 15 and adds 81 bases in-frame within the cytosolic domain to the class I isoform. Isoforms class IV–VII are derived

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FIGURE 22.3 Human G-CSFR alternative splice isoforms. The genomic structure and the known alternative splicing patterns involving exons 14–17 of the human G-CSFR (Fukunaga et al., 1990; Larsen et al., 1990; Dong et al., 1995; Bernard et al., 1996). THE CYTOKINES AND CHEMOKINES

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through the recognition of a cryptic intron within the terminal exon 17. Isoform class VI deletes exon 1b. Isoform class VII makes use of a more proximal splice acceptor site in exon 15. The class IV isoform, originally cloned from placental tissue, is present in normal and leukemic myeloid cells (White et al., 1998). Isoforms class V–VII were detected as rare transcripts in myeloid cells and are of no apparent physiologic significance (Dong et al., 1995b; Bernard et al., 1996).

Regulation of G-CSFR transcription Initial observations with the IL-3-dependent murine myeloblastic cell line 32Dcl 3 determined that G-CSF stimulation leads to increased expression of the G-CSFR mRNA transcript (Steinman and Tweardy, 1994) suggesting that either G-CSFR signal transduction directly or indirectly influences the G-CSFR promoter region. The transcription factor of primary importance for promoting G-CSFR transcription in early myeloid progenitors appears to be C/EBP-a binding to its consensus sequence {GCAAT} 49 bases proximal to the transcription initiation start site (Scott et al., 1992; Smith et al., 1996). In mice carrying null mutation of the C/EBP-a gene are incapable of expressing the G-CSFR (Zhang et al., 1997). Fetal liver cells from these mice transduced with a retroviral vector containing a dominant-negative retinoic acid receptor are able to up-regulate G-CSFR mRNA expression when stimulated with GM-CSF and further enhancement was seen with the addition of all-trans retinoic acid (Collins et al., 2001). This indicates that there are transcription factors other than C/EBPa that are able to increase G-CSFR mRNA transcription. As granulocytic maturation occurs, there is a switch from C/EBPa to C/EBPe expression with C/EBPe also up-regulating G-CSFR expression (Nakajima and Ihle, 2001). This suggests that other C/EBP family members may be able to compensate for the lack of C/EBPa in the C/EBPa/ knock-out mouse model. Within the 5 untranslated region of the G-CSFR are two binding sites for the transcription factor PU.1 from the ets family of transcription factors. While mice carrying null mutation of the PU.1 gene are still capable of expressing the G-CSFR (Olson et al., 1995), mutation of the PU.1 binding sites diminishes G-CSFR transcription by 75% (Smith et al., 1996).

PHYSIOLOGIC ROLES OF G-CSF AND ITS RECEPTOR Biologic activities of G-CSF Effect of G-CSF on cells within the marrow G-CSF and its receptor are critical to the normal production of fully functional circulating neutrophils. The effect of G-CSF on basal and stress-induced production of neutrophils begins with its effect on the myeloid progenitor cells within the marrow (Metcalf and Nicola, 1983). While the CD34/CD33 cell fraction of the marrow does not respond to G-CSF stimulation, the myeloid committed CD34/CD33 fraction does respond to G-CSF with granulocytic colony formation in semisolid culture medium (Ema et al., 1990a, 1990b). G-CSF also accelerates the proliferation and maturation of granulocytic progenitors and precursors in the marrow, as well as promoting release of neutrophils from the marrow into the peripheral circulation (Welte et al., 1987; Bronchud et al., 1988). A study in mice details these observations demonstrating that G-CSF decreases the marrow transit time of stem cell to circulating neutrophil from 116 h to 45 h (Uchida and Yamagiwa, 1992). It also revealed a 20-fold increase in the rate of neutrophil turnover in the peripheral circulation. No other cytokine or growth factor has such a profound effect upon myeloid cell physiology. At the molecular level, G-CSF has been shown to alter the expression of a growing list of molecules during myeloid development. The list of genes include myeloperoxidase, neutrophil elastase, c-myb, lactoferrin, chloroacetate esterase and p27kip (Morishita et al., 1987; Fukunaga et al., 1993; Yoshikawa et al., 1995; Bellon et al., 1997; de Koning et al., 2000). There is also an increase G-CSFR expression with prolonged G-CSF stimulation as has been shown in a murine cell line model of G-CSF-mediated myelopoiesis (Steinman and Tweardy, 1994).

Effects of G-CSF or G-CSFR deletion Mice with null mutations of both alleles of the G-CSF or G-CSFR genes have demonstrated the importance of G-CSF stimulation to the baseline and stressinduced production of circulating neutrophils

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(Lieschke et al., 1994; Liu et al., 1996). The first of these mice to be developed and examined was the G-CSF knock-out mouse (Lieschke et al., 1994). These animals demonstrate normal fertility and viability, but have a circulating neutrophil count only 20–30% of that in the wild-type mice. The marrow of G-CSF/ mice has only half the normal numbers of the granulocytic precursors and reduced numbers of progenitor cells, particularly granulocyte, granulocyte/ macrophage and macrophage colony-forming units. All of these defects are corrected with the administration of recombinant human G-CSF for 4 days. When G-CSF null mutation mice are infected with Listeria monocytogenes, they exhibit a reduced and delayed production of circulating neutrophils (Lieschke et al., 1994). Interestingly, when G-CSF/ mice are infected with Candida albicans they have a normal neutrophilic response along with an increase in the number of neutrophilic progenitors in the marrow (Basu et al., 2000). When G-CSF null mice are subjected to a chronic infection with Mycobacterium avium, the most pronounced effect observed was a decrease in macrophage production of nitric oxide and an increase in T-cell production of c-interferon (Mannering et al., 2000). There was no depletion of myeloid precursor cells in the marrow space in this study and the infection itself was not exacerbated. Mice carrying a homozygous null mutation of the G-CSFR gene also demonstrate severe effects on myelopoiesis (Liu et al., 1996). While these mice develop normally and are fertile and indistinguishable from their normal counterparts, they display a resting neutropenia with neutrophil counts at 12% of those seen in wild-type mice. The G-CSFR null mice also demonstrate a moderate decrease in the number of hematopoietic progenitors in their marrow. Interestingly, the numbers of myeloid precursors was similar to wild-type mice, whereas the G-CSF/ mice display a decrease in their myeloid precursors counts. This suggests an additional mode of action by G-CSF in driving myeloid precursor expansion that is independent of G-CSFR or that mouse strain-specific differences exist in the mechanism of compensation for G-CSF/G-CSFR-independent neutrophil production. The recruitment of neutrophils to the peritoneal cavity following intraperitoneal thioglycollate injection appeared normal in the G-CSFR null mice as did their myeloperoxidase production. The neutrophils from

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G-CSFR/ did, however, demonstrate an increased susceptibility to apoptosis. The studies of mice carrying knock-out mutations of G-CSF or its receptor provide insight into the critical requirement of the G-CSF/G-CSFR system for normal basal and stress-induced granulopoiesis. The fact that these knock-out mice can still produce limited numbers of morphologically normal neutrophils does not establish a stochastic model of myeloid differentiation. Rather quite the opposite, it demonstrates the need for growth factor instruction to coordinate signal transduction to produce normal numbers of physiologically competent cells for host defense against infection. This interpretation is supported by studies done with dogs to treat canine cyclic neutropenia. During the course of this work, normal dogs received recombinant human G-CSF that induced neutralizing anti-G-CSF antibodies that cross-reacted with endogenously produced canine G-CSF (Hammond et al., 1991). The result was the reduction of circulating neutrophil counts to 2–10% of that observed in untreated dogs. The marrow from these animals during this period of circulating neutropenia displayed minimal decreases in the numbers of neutrophil precursors suggesting either a prolongation of the myeloid maturation process or apoptosis of postmitotic neutrophils prior to release from the marrow.

Effects on circulating neutrophils Pharmacodynamic study of human recombinant GCSF given subcutaneously reveals a bi-exponential mode of clearance with a 30-min tissue distribution half-life and a 3.8 h elimination half-life (Cohen et al., 1987). When G-CSF is administered in vivo to rats, mice, hamsters, primates and humans there is an acute margination of circulating neutrophils that occurs within 10 min (Cohen et al., 1987; Tsuchiya et al., 1987b; Welte et al., 1987; Okada et al., 1990; Itoh et al., 1991; Katoh et al., 1992; Shank and Balducci, 1992). No effect is seen on the number of circulating erythrocytes, monocytes, lymphocytes or platelets. The abrupt circulating neutropenia resolves within 30–150 min and a rise in the circulating neutrophil count is then observed over the following 4–6 h. This rise in neutrophil number is due to release of mature neutrophils from the bone marrow and is followed by an expansion of the myeloid cell population within

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the marrow. When the surface molecules and functional aspects of the neutrophils are examined before and after the G-CSF-induced margination, it appears that the G-CSF-marginated neutrophils exit the circulation and are replaced with neutrophils released from the marrow space (Katoh et al., 1992). Recent studies reveal that this G-CSF-induced margination may be due to activation of surface CD11a and CD11b (LFA-1 and Mac-1, respectively) on neutrophils allowing them to interact with ICAM-1 on vascular endothelial cells (Chakraborty et al., 2002). G-CSF also alters the survival and functional capacity of circulating neutrophils. The prolonged survival of circulating neutrophils treated with G-CSF has been demonstrated both in vitro and in vivo (Colotta et al., 1992; Adachi et al., 1994; Leavey et al., 1998). There is also enhanced bacteriocidal activity, chemokinesis, phagocytosis and superoxide anion production by neutrophils stimulated with G-CSF (Bober et al., 1995; Carulli, 1997). The difficulty with performing such evaluations of neutrophil function is that both the administration of G-CSF to a host and/or the isolation of neutrophils from a host will change to characteristics of the isolated neutrophils (Carulli, 1997). A murine in vivo model of G-CSF administration demonstrates a clear improvement in outcome of disseminated Candida albicans infection when G-CSF is administered in the acute phase of infection (Kullberg et al., 1999). Giving G-CSF during chronic candidiasis had less of an impact on outcome.

Clinical uses of G-CSF The approval of recombinant human G-CSF as a pharmacological agent by the FDA in 1991 was based on the ability of G-CSF to increase circulating neutrophil numbers and significantly reduce the neutropenia and fever following myelosuppressive chemotherapeutic regimens (Clark and Kamen, 1987; Duhrsen et al., 1988; Morstyn et al., 1988). Since that approval, G-CSF has been given to patients for chemotherapy-induced neutropenia and studied in a broad variety of clinical settings including its use as an adjuvant to chemotherapeutic regimens (Rubenstein, 2000). While such uses are becoming more common, the degree of benefit from the addition of G-CSF to various treatment regimens remains to be fully elucidated (Rubenstein, 2000).

Neutropenias of other etiologies have also responded to therapy with G-CSF. These include severe congenital neutropenia (SCN), congenital cyclic neutropenia, chronic idiopathic neutropenia, chronic autoimmune neutropenia, non-cytotoxic drug-induced neutropenia and HIV-associated neutropenia (Hammond et al., 1989; Boxer et al., 1992; Willfort et al., 1993; Ganser and Karthaus, 1996; Welte and Dale, 1996; Welte et al., 1996; Bauduer, 1998; Armstrong and Kazanjian, 2001). The side-effects from such uses of G-CSF remain minimal, with mild to moderate bone pain that is typically easily managed. Rare cases of acute febrile neutrophilic dermatosis (Sweet’s syndrome) have been associated with G-CSF administration (Park et al., 1992; Paydas et al., 1993), but typically resolve upon withdrawal of G-CSF therapy. Other rare side-effects include the acute exacerbation of rheumatoid arthritis during G-CSF treatment of neutropenia in Felty’s syndrome (Yasuda et al., 1994; Farhey and Herman, 1995; Hayat et al., 1995; McMullin and Finch, 1995; Vidarsson et al., 1995). Anaphylactic reactions to the recombinant human G-CSF preparation have also been reported and can be related to E. coli-derived antigens in the preparation (Jaiyesimi et al., 1991; Adkins, 1998; Stone et al., 1998; Keung et al., 1999; Khoury et al., 2000). Such reactions have even been reported in patients with their first dose of G-CSF (Batel-Copel et al., 1995). G-CSF treatment increases the numbers of CD34 cells mobilized from the bone marrow into the peripheral circulation, with this effect enhanced by the addition of other cytokines such as GM-CSF, stem cell factor (SCF), or flt3 ligand (Yan et al., 1994; Lane et al., 1995; Varas et al., 1996; Brasel et al., 1997). This has allowed the collection of hematopoietic stem cells suitable for autologous or allogeneic transplantation to be performed by leukapheresis rather than via bone marrow harvesting under general anesthesia. What is interesting is that the most primitive hematopoietic stem cells, those that retain their pluripotent nature, do not express detectable G-CSFR yet are mobilized in subjects treated with G-CSF alone, suggesting an indirect effect of G-CSF that leads to their mobilization (Ebihara et al., 2000). One such mechanism involves the G-CSF-mediated release of neutrophil elastase and cathepsin G by neutrophils in the marrow that cleaves VCAM-1 expressed

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on marrow stromal cells thus releasing hematopoietic stem cells into the circulation (Levesque et al., 2001). This evidence is supported by work done in mice that reveals the need for a functional G-CSFR on hematopoietic cells, but not on stromal cells or the mobilized hematopoietic stem cells (Liu et al., 2000). The biology of the hematopoietic stems cells mobilized by various cytokine cocktails continues to be explored to determine which cytokine treatment will yield the preferred population of transplanted cells (Lemoli et al., 1997; Ebihara et al., 2000; Gyger et al., 2000). Based on its ability to mobilize neutrophils and enhance their functional capacities, G-CSF has been examined as an adjunct to treatment of infections in non-neutropenic patients. In a trial using G-CSF in conjunction with antibiotics for severe diabetic foot infections there was a significant improvement in clinical outcome with an associated increase in circulating neutrophil superoxide anion production (Gough et al., 1997) There may also be a role for G-CSF in the treatment of neonatal sepsis in some cases as preliminary studies have demonstrated a clear benefit of G-CSF administration (Kocherlakota and La Gamma, 1998). In contrast, the role of G-CSF in the treatment of adult bacterial pneumonia is not as clear, with some studies demonstrating a benefit while others show no significant difference in outcome (Nelson, 2001; Wunderink et al., 2001). The ability of G-CSF to mobilize neutrophils into the circulation has stimulated a return to investigations into neutrophil transfusion therapy for infections in patients lacking normal numbers of functional neutrophils, however, currently the utility of such interventions remains to be determined (Hubel et al., 2001). Because G-CSF was initially identified based on its capacity to promote differentiation of murine leukemic cell lines, its ability to differentiate human leukemic myeloid cells was an early topic of investigation. Initial work suggested a possible maturational effect on fresh leukemic myeloblasts in vitro (Souza et al., 1986) however, subsequent studies on larger numbers of samples demonstrated a lack of significant differentiation (Vellenga et al., 1987a, 1987b). In fact, most studies involving G-CSF stimulation of fresh human leukemic myeloid cells in vitro exhibit a strictly proliferative response (Kelleher et al., 1987; Vellenga et al., 1990). However, in vivo work in mice

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has demonstrated that administration of G-CSF does not promote or accelerate the development of leukemia in an experimental model and furthermore delayed the detection of leukemic cells in the peripheral circulation and improved survival (Tamura et al., 1989). More recent evidence in humans suggests that the administration of G-CSF to subgroups of patients with acute myeloid leukemia (AML) is safe and potentially beneficial both medically and financially (Takahashi et al., 1997; Saito et al., 2000; Bradstock et al., 2001; Clavio et al., 2001). Since the introduction of recombinant human GCSF produced from E. coli, there have been additional means of administering G-CSF to patients. It is now possible to give patients G-CSF via the rectal route (Watanabe et al., 1996). There have also been animal trials with G-CSF produced by transduced marrow stromal cells (Chang et al., 1989) or by implanted transfected myoblasts or fibroblasts (Tani et al., 1989; Bonham et al., 1996; Lejnieks et al., 1996). A glycosylated version of human G-CSF (lenograstim) produced in Chinese hamster ovary (CHO) cells is available for use in some countries. It does appear to be more potent that filgrastim on a weight-by-weight basis for CD34 cell mobilization in some, but not all, studies. Therefore the biologic and clinical advantages of glycosylated G-CSF are still being determined (Hoglund, 1998).

Pathology associated with altered G-CSFR expression Aberrant G-CSFR expression has been demonstrated in disorders of myelopoiesis, such as myeloid leukemias and neutropenia. One area of investigation concerns mutations within an allele of the G-CSFR gene in patients with SCN that progress to AML. Typically the mutation that is observed in the G-CSFR allele is a non-sense mutation that truncates the receptor’s cytosolic domain by 80–100 amino acids (Dong et al., 1994, 1995a; Tidow et al., 1997). Such truncated mutant receptor isoforms lack peptide sequence critical to the maturation signal transduction activity and ligand-mediated receptor internalization, yet retain portions of the receptor capable of driving proliferation (Dong et al., 1994, 1995a, 1995b; Hunter and Avalos, 1999; Ward et al., 1999c). Given the pro-proliferative potential of these

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truncated mutant G-CSFR forms, such non-sense mutations have been knocked-in to the murine GCSFR gene by two laboratories to determine their in vivo effect upon myeloid proliferation and maturation (McLemore et al., 1998; Hermans et al., 1999). Both groups found the myeloid precursors to be hyper-proliferative in response to G-CSF stimulation. The mice produced by McLemore et al. had normal baseline circulating neutrophil counts in both the heterozygous and homozygous knock-in mutants, whereas Hermans et al. observed a reduction of the circulating neutrophil count of 60% in their homozygous mutant animals and 30–40% in their heterozygous mutant animals. The reason for this disparity remains unclear. McLemore et al. also observed an increase in the number of G-CSF-responsive progenitor cells in the marrow of their heterozygous and homozygous mutant mice. Neither group observed a block in myeloid maturation as is seen in patients with SCN. Since such non-sense mutations are only found in the subset of SCN patients that have progressed to AML while receiving G-CSF therapy and are not observed in other forms of AML, it is likely that these G-CSFR mutations contribute to AML in conjunction with other unidentified mutations in SCN patients. A defect that appears in a majority of AML patients is aberrant regulation of the alternative splicing of the G-CSFR transcript (White et al., 1998). Normal immature myeloid cells from adult bone marrow express between 5% and 8% of their G-CSFR mRNA as the class IV isoform, with the remainder as the class I isoform. In contrast, blast cells from approximately 60% of AML patients express 8–15% of their G-CSFR as the class IV G-CSFR mRNA isoform (White et al., 1998; and unpublished data). This relative level of class IV G-CSFR mRNA expression was reproduced in the murine myeloid cell line 32Dcl3, a cell line that expresses the murine G-CSFR and differentiates into phenotypic neutrophils with G-CSF stimulation. It was observed that by expressing the human class IV G-CSFR along with the murine G-CSFR at relative levels similar to those in many AML patients, the normal G-CSF-induced pattern of myeloid maturation was delayed or completely blocked (White et al., 2000).

G-CSF RECEPTOR SIGNAL TRANSDUCTION Interaction between ligand and receptor Based on work from other cytokine–cytokine receptor interactions, it was anticipated that the G-CSFR would interact via its CRH domain to bind its ligand and induce signal transduction. Similar to the prolactin, growth hormone and erythropoietin receptors, the G-CSFR appears to function as a homodimer (Ishizaka-Ikeda et al., 1993). G-CSFR retains its signal transduction capacity even as a homotrimer or homo-oligomer as was determined using a chimeric receptor that placed the Fas receptor extracellular domain above the transmembrane and cytosolic portion of the G-CSFR (Takahashi et al., 1996). When cells expressing this chimeric receptor were exposed to Fas ligand or anti-Fas antibodies, thus inducing trimerization or oligomerization, proliferation was observed. This suggests that signal transduction via the G-CSFR requires the co-association or two or more receptor molecules without strict limitations on the number of receptor molecules forming the complex. Using high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy, it was specifically determined that the stoichiometry of G-CSF:G-CSFR interaction was 1:2 (Anaguchi et al., 1995; Hiraoka et al., 1995). This project involved the use of recombinant proteins including the Ig-like domain and CRH domain (consisting of BN {amino-terminal} and BC {carboxy-terminal} subdomains) produced from a baculovirus expression system and non-glycosylated G-CSF. It was also determined that the Try-Ser-X-Try-Ser motif within the BC subdomain is not directly involved in receptor–ligand contact, but was critical to maintaining proper protein folding in the ligand-binding domain of the receptor, an observation that may be relevant to all receptors with this motif. More recent work examining similar recombinant proteins by the use of X-ray crystallography has determined that two 1:1 complexes of G-CSF:G-CSFR molecules interact in a slightly asymmetric conformation (Plate 22.4) (see Plate section) (Aritomi et al., 1999).

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Growth and differentiation mediated by distinct G-CSFR domains Once the G-CSFR has been dimerized through ligand binding, signal transduction occurs which involves the tyrosine phosphorylation of multiple proteins including the G-CSFR itself (Plate 22.5) (see Plate section) (Pan et al., 1993). The detailed molecular events of signal transduction will be discussed later. G-CSFR signal transduction leads to both proliferative and maturational responses in myeloid cells. These distinct signal responses have been examined using a variety of deletion, truncation and point mutations. Such studies have revealed that the membrane proximal 57 amino acids of the human class I G-CSFR cytosolic domain are all that are required to provide signals for a proliferative response (Ziegler et al., 1993) in the murine IL-3 dependent pro-B cell line BAF/BO3. The addition of the subsequent 39 amino acids, including one of four tyrosine phosphorylation sites, enhances the proliferative response in this model, with the remainder of the G-CSFR providing no additional proliferative impetus. A study using similar truncated versions of the human G-CSFR in comparison to the class I and class IV G-CSFR in the BAF/BO3 cell line, as well as the murine myeloblastic cell lines 32D and L-GM (Dong et al., 1993) provided nearly identical results. Of interest is that the cell lines 32D and L-GM are capable of some degree of myeloid maturation and both display a blunted proliferative response to activation of the class I G-CSFR, but a markedly enhanced proliferative response to G-CSF when working via the class IV G-CSFR. This region of the G-CSFR cytosolic domain involved in proliferative signal transduction contains two regions of sequence homology shared with the gp-130 signal transduction chain of the IL-6 receptor that have been named Box 1 and Box 2 (Plate 22.5) (Murakami et al., 1991; Fukunaga et al., 1993; Ziegler et al., 1993). The Cterminal 87 amino acid portion of the human class I or murine G-CSFR cytosolic domain appears to contain peptide sequence that, in combination with the membrane proximal domain, is critical for signal transduction for myeloid maturation and gene induction (Dong et al., 1993; Fukunaga et al., 1993; Ziegler et al., 1993). The human class IV G-CSFR replaces the C-terminal peptide sequence in the full-length G-CSFR critical to driving myeloid maturation and

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was found to delay or block G-CSF-mediated myeloid maturation when co-expressed with the full-length G-CSFR (White et al., 2000). This inhibition of murine G-CSFR-driven myeloid maturation occurred with only 10–25% of the total G-CSFR mRNA being the human class IV isoform.

Molecules involved in G-CSFR signaling The identification of distinct cytosolic functional domains has lead to the investigation of the specific signal transduction molecules activated by these portions of the G-CSFR. As mentioned above, the first step in G-CSFR signal transduction that occurs following ligand binding is the tyrosine phosphorylation of multiple proteins including four tyrosines on the G-CSFR itself (Pan et al., 1993). Given the lack of any intrinsic tyrosine kinase activity within the G-CSFR, it appears that one or more of the Janus kinase (JAK) family members perform this initial signal transduction step. Jak 1 has been shown to constitutively interact with the G-CSFR and become activated with G-CSF binding (Nicholson et al., 1994). Other JAK kinase family members, particularly Jak2 and Tyk2, are also activated by the G-CSFR with sequence within the membrane proximal portion of the cytosolic domain required for their activation (Shimoda et al., 1994; Nicholson et al., 1995; Tian et al., 1996; Avalos et al., 1997). Whether the activation of the different JAK family members is merely a redundancy or a coordinated mechanism is not yet understood. Cytokine receptor-activated JAK kinases typically tyrosine phosphorylate STAT (signal transducer and activator of transcription) molecules leading to their dimerization and delivery of the STAT dimer to the nucleus where it acts as a transcription factor. The G-CSFR has been shown to strongly activate Stat3 and to a lesser extent activate Stat5 and Stat1 (Tian et al., 1994; Nicholson et al., 1995; Tweardy et al., 1995). These STAT molecules form homodimers once tyrosine phosphorylated, with G-CSFR-initiated heterodimers of Stat1:Stat3 and Stat3:Stat5 also reported (Chakraborty et al., 2000). The recruitment of specific STATs to the G-CSFR appears to occur via different portions of the receptor. Stat3 has been shown to interact with phosphorylated

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Y704 and phosphorylated Y744 of the human G-CSFR (Chakraborty and Tweardy, 1998; Chakraborty et al., 1999; Ward et al., 1999a). At high levels of G-CSF, there has been demonstrated an independent mechanism of Stat3 tyrosine phosphorylation though the physiologic relevance of this mechanism is unclear (Ward et al., 1999a). G-CSFR-mediated activation of Stat3 has been associated with both proliferative and maturational responses (Nicholson et al., 1995; Tweardy et al., 1995). More recent observations support the concept that the role of Stat3 in G-CSFR signaling is to promote cell survival and myeloid maturation (Shimozaki et al., 1997; Chakraborty and Tweardy, 1998; de Koning et al., 2000). An added layer of complexity to the role of Stat3 in G-CSFR signaling is the alteration in Stat3 isoform expression during G-CSFmediated myeloid maturation, with Stat3a being predominant in human CD34 cells and Stat3b becoming the prominent isoform as cells progress to the postmitotic phase of myeloid maturation (White et al., 1998; Biethahn et al., 1999). This change in Stat3 isoform expression during G-CSF-mediated myelopoiesis presumably alters Stat3-responsive gene regulation since the two isoforms possess different peptide sequence in their transactivation domains, however, the details of these effects remain to be explored. While Stat3 is the most prominent STAT activated by the G-CSFR, Stat5 and Stat1 are also activated by the G-CSFR. Activation of Stat5 by the G-CSFR has been associated with the proliferative response to G-CSF (Dong et al., 1998). When the activation of specific Stat5 isoforms by the G-CSFR was examined, Stat5A and Stat5B are activated by the membrane proximal portion of the cytosolic domain associated with proliferative signaling (Chakraborty et al., 2000), whereas the Stat5p80 isoform is activated by Y704. Interestingly, only Stat5A and/or Stat5B formed heterodimers with Stat3; no Stat3:Stat5p80 heterodimers could be detected. This suggests a role for Stat5p80 in G-CSFR-mediated myeloid maturation. The recruitment and functional aspects of G-CSFR-mediated Stat1 activation remain to be explored. Members of the suppressor of cytokine signaling (SOCS) family negatively regulate signal transduction from a variety of cytokine and growth factor receptors through multiple reported mechanisms (Hilton, 1999). G-CSFR signaling has been shown to induce the up-regulation of SOCS-1, SOCS-2 and SOCS-3

mRNAs (Minamoto et al., 1997; Naka et al., 1997). The details of how these SOCS family members interact with G-CSFR signaling remain to be determined. The protein inhibitors of activated Stats (PIAS) family members PIAS-3 and PIAS-1 interact with phosphorylated Stat 3 and Stat 1, respectively (Hilton, 1999). The role of these two molecules with regards to G-CSFRmediated STAT activation has not been formally described but they may participate in buffering G-CSFR-mediated Stat 3 and Stat 1 homo- and heterodimer formation. The SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2 have been shown to participate in G-CSFR signal regulation. SHP-1 has been shown to inhibit G-CSFR- mediated myeloid differentiation (Ward et al., 2000), but not through a direct interaction with the G-CSFR itself. SHP-2 appears to interact with the G-CSFR directly through phosphorylated tyrosines Y704 and Y764 of the human G-CSFR and link to proliferation signaling through the Ras/MAP kinase system (Ward et al., 1998b, 1999b). Both the human and murine G-CSFR activate elements of the Ras/MAP (mitogen activated protein) kinase signal transduction system. Signal transduction through the p21 Ras pathway by the G-CSFR requires activation of Jak2 via the membrane proximal portion of the G-CSFR cytosolic domain (Barge et al., 1996). Connecting the activated G-CSFR to the Ras/MAP kinase pathway occurs via multiple reported mechanisms. One means to activate p21Ras by the G-CSFR is through recruitment of tyrosinephosphorylated Shc to phosphorylated Y764 of the human G-CSFR via an SH2 domain interaction (de Koning et al., 1996; Ward et al., 1998b). The Shc adapter molecule then interacts with SHP2, Grb2, an unidentified protein p145, or an unidentified protein p90. Also there has been demonstration of Grb2/p90 complexes recruited to phosphorylated Y764 (de Koning et al., 1996). SHP2/Grb2 complexes have also been demonstrated to form following G-CSFR dimerization, however, these complexes form independent of G-CSFR tyrosine phosphorylation (de Koning et al., 1996). What occurs subsequently to the recruitment and activation of these various adapter molecules remains to be completely delineated. There is evidence that Y764 of the human G-CSFR participates in the proliferative signaling response (de Koning et al., 1998)

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in connection with Shc recruitment and phosphorylation. Y763 of the murine G-CSFR has been linked to the activation of JNK, whereas Erk2 activation required only the membrane proximal 100 amino acids of the cytosolic domain (Rausch and Marshall, 1997). In this work Erk2 activation was associated with a proliferative response to G-CSF, whereas JNK activation was not required for a proliferative response. Later studies identify p38 activation as also being linked to Y763 of the murine G-CSFR in a manner similar to that for JNK (Rausch and Marshall, 1999). Low level p38 activation was observed when only the membrane proximal 100 amino acids of the murine G-CSFR were present. In terminally differentiated neutrophils, G-CSF-mediated activation of p38 has been associated with enhanced survival (Villunger et al., 2000), but the role of p38 activation in proliferating myeloid cells has not been established. Erk 5 is a member of the MAP kinase family and is activated by the human and murine G-CSFR in murine myeloid cell lines (Dong et al., 2001). In particular, the carboxy-terminal portion of the cytosolic domain is required for Erk5 activation with protein kinase C negatively regulating Erk5 activity (Dong et al., 2001). Its role in the cell lines studied appears to be that of promoting cell survival and proliferation. Phosphatidylinositol 3 kinase (PI3 kinase) is a family of proteins that produce phosphorylated inositol lipids that act as second messenger molecules regulating many cellular functions including proliferation and survival (Stein and Waterfield, 2000). The human G-CSFR activates PI3 kinase via an interaction with amino acids 682–715 of the cytosolic domain, presumably mediated by an interaction with phosphorylated Y704 and an SH2 domain of the p85 subunit of PI3 kinase (Hunter and Avalos, 1998; Stein and Waterfield, 2000). The result of G-CSF-mediated PI3 kinase activation is to suppress apoptosis and enhance G-CSF-mediated proliferation in a cell line model incapable of myeloid maturation via activation of the downstream target molecules Akt and Bad (Dong and Larner, 2000; Hunter and Avalos, 2000). SHIP is an inositol 5 phosphatase that degrades the inositol triphosphates generated by PI3 kinase and is recruited to the human G-CSFR via the adapter molecule Shc interacting with phosphorylated Y764 of the G-CSFR (Hunter and Avalos, 1998). In naturally occurring and mutant G-CSFR isoforms that lack Y764,

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SHIP activation is markedly attenuated or absent, resulting in prolonged activation of Akt and Bad, resulting in even greater proliferation in response to G-CSF stimulation (Hunter and Avalos, 1998, 2000; Dong and Larner, 2000). In addition to the JAK family of tyrosine kinases, other protein tyrosine kinases (PTKs) are recruited to and activated by ligand-induced dimerization of the G-CSFR. The Src-related PTK Lyn and the non-Srcrelated PTK Syk are activated by the human G-CSFR in human neutrophils (Corey et al., 1994; Kasper et al., 1999) and in the avian lymphoid cell line DT-40 (Grishin et al., 2000). Lyn appears to constitutively associate with the G-CSFR, while Syk is recruited to the receptor following ligand binding. The downstream effects of Lyn activation have been linked to proliferation through the PI3 kinase and MAP kinase pathways (Grishin et al., 2000), whereas the role of Syk in G-CSFR signaling has not been linked to a specific response. The non-Src-related PTK Tec is phosphorylated by ligand-activated G-CSFR and this has been associated with growth and differentiation signaling responses (Miyazato et al., 1996). The hematopoietic cell kinase Hck is also recruited to the murine G-CSFR in a ligand-dependent fashion (Ward et al., 1998a). Hck interacts directly with the G-CSFR through its SH2 domain, presumably with one or more of the phosphorylated tyrosine residues of the activated G-CSFR.

CURRENT AREAS OF ACTIVE RESEARCH G-CSF as a pro-inflammatory cytokine Following resuscitation from hemorrhagic shock, neutrophils act as the most crucial circulating cell inciting dysfunctional inflammatory events that begin as capillary leakage which progresses to small vessel plugging and eventual multiorgan failure (Barroso-Aranda et al., 1988; Anderson and Harken, 1990; Leff and Repine, 1990). Recent work in a rat model of hemorrhagic shock with ischemia followed by resuscitation demonstrates that G-CSF mRNA production is up-regulated in the liver, lungs and bowel with levels peaking by 4 h post-resuscitation (Hierholzer et al., 1997a, 1997b, 2001). Ischemic injury

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alone was not sufficient to produce G-CSF mRNA upregulation; resuscitation by circulatory system volume repletion and associated tissue reperfusion were requisite. In these same studies, the G-CSF mRNA levels correlated with the degree of tissue injury and neutrophilic granulocyte infiltration. G-CSF instillation into the lungs of rats reproduces the tissue edema and neutrophilic granulocyte infiltration seen in the lungs of rats subjected to hemorrhagic shock with reperfusion (Hierholzer et al., 1998). Furthermore, addition of G-CSF to resuscitation fluids increased lung neutrophil infiltration and damage (Meng et al., 2000). Current studies in G-CSF-deficient mice are confirming the crucial role of G-CSF in reperfusion injury following hemorrhagic shock. One aim of these studies is to develop agents that transiently block the effects of G-CSF on neutrophils then to test their efficacy in reducing dysfunctional inflammation following resuscitated hemorrhagic shock or other settings in which over-exuberant neutrophil activity is a contributing factor.

Class IV G-CSFR constitutive signaling in leukemogenesis Soon after the cloning of the class IV G-CSFR cDNA, it was demonstrated that it could only support the proliferative signaling functions associated with G-CSF stimulation (Dong et al., 1993; Fukunaga et al., 1993). A role for abnormal regulation of class IV G-CSFR expression as the mechanism that uncouples the proliferative and maturational signaling capacities of the G-CSFR in AML has been demonstrated in a model of G-CSF-mediated myeloid maturation (White et al., 1998, 2000). Recent work has demonstrated ligandindependent activation of selective elements of the MAP kinase pathway and PI3 kinase by the class IV G-CSFR isoform; no evidence of constitutive STAT protein activation was detected. Current research focuses on the mechanisms through which the class IV G-CSFR constitutively activates some, but not other signal transduction pathways linked to the G-CSFR and how this selective constitutive signaling contributes to leukemogenesis.

Stat3 isoform expression during G-CSF-mediated myelopoiesis Another active area of research is pursuing whether or not distinct Stat3 isoforms differentially regulate GCSF-mediated myeloid proliferation, anti-apoptosis and neutrophilic maturation. G-CSF is established as the primary growth factor stimulating expansion of myeloid progenitors and precursors and driving their maturation into circulating neutrophils. G-CSFRmeditated Stat3 activation has been identified as a crucial part of these processes (Chakraborty and Tweardy, 1998; de Koning et al., 2000; McLemore et al., 2001). As human CD34 cord blood mononuclear cells are driven to differentiate into neutrophils, the expression of Stat3 isoforms evolves from a Stat3apredominant to a Stat3b-predominant profile with the changeover occurring midway during the maturation pathway (White et al., 1998; Biethahn et al., 1999). Ongoing work examines how these two Stat3 isoforms differ in their transcriptional activation properties, the repertoire of genes they activate and the mechanisms involved in regulating their expression during myeloid maturation.

REFERENCES Adachi, S., Kubota, M., Lin, Y.W. et al. (1994). In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro. Eur. J. Haematol. 53, 129–134. Adkins, D.R. (1998). Anaphylactoid reaction in a normal donor given granulocyte colony-stimulating factor. J. Clin. Oncol. 16, 812–813. Akashi, M., Shaw, G., Hachiya, M. et al. (1994). Number and location of AUUUA motifs: role in regulating transiently expressed RNAs. Blood 83, 3182–3187. Anaguchi, H., Hiraoka, O., Yamasaki, K. et al. (1995). Ligand binding characteristics of the carboxyl-terminal domain of the cytokine receptor homologous region of the granulocyte colony-stimulating factor receptor. J. Biol. Chem. 270, 27845–27851. Anderson, B.O. and Harken, A.H. (1990). Multiple organ failure: inflammatory priming and activation sequences promote autologous tissue injury. J. Trauma 30, S44–S49. Aritomi, M., Kunishima, N., Okamoto, T. et al. (1999). Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme. Nature 401, 713–717. Armstrong, W.S. and Kazanjian, P. (2001). Use of cytokines in human immunodeficiency virus-infected patients: colony-stimulating factors, erythropoietin, and interleukin-2. Clin. Infect. Dis. 32, 766–773. Asano, M. and Nagata, S. (1992). Constitutive and inducible

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Subsection B. The Interferon Family Members

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23 Type I interferons [IFNa, b, d, j, x, s, IL-28A (IFNk2) IL-28B (IFNk3) and IL-29 (IFNk1)] Jeanne M. Soos and Brian E. Szente GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA

If we will only allow that, as we progress, we remain unsure, we will leave opportunities for alternatives. We will not become enthusiastic for the fact, the knowledge, the absolute truth of the day, but remain always uncertain . . . In order to make progress, one must leave the door to the unknown ajar. Richard P. Feynman (1918–1988)

INTRODUCTION

SUBTYPES OF TYPE I IFN

The activity for which the interferons, and in particular the type I interferons received their name, was identified by Isaacs and Lindenmann in 1957. In chick chorioallantoic membranes, they observed a marked inhibition of the replication of influenza virus, and thus began the field of interferon research (Isaacs and Lindenmann, 1957). Since that time, our understanding of the nature and biology of interferons has grown substantially to the point such that the cellular receptors for the interferons have been identified, and some of the intracellular signaling cascades of which they make use. Natural and recombinant interferons a and b are now approved as effective therapies for a number of conditions, including a variety of hematologic and non-hematologic malignancies, hepatitis and papilloma virus infections, and multiple sclerosis.

The first classification of the IFNs was defined based on the cellular sources of these proteins – namely leukocyte, fibroblast and immune interferons (Johnson et al., 1994). As the naming schemes were refined based on biology, the leukocyte and fibroblast IFNs were grouped together as type I IFNs and the immune IFN received the designation of type II IFN. The current IFN nomenclature is based primarily on the specific nucleotide sequences of individual IFN genes and groups of IFN genes. The leukocyte IFNs are now referred to as IFNa and IFNx (formerly IFNa-I and IFNa-II, respectively), while fibroblast IFN is now referred to as IFNb. The former immune IFN is now the potent immunomodulator known as IFNc. In humans, there are at least 18 non-allelic IFNa genes, five of which are pseudogenes and at least six IFN genes, five of which are pseudogenes (Table 23.1). The IFNa and x genes, together with a single IFNb gene, comprise an IFNa/b superfamily.

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

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TABLE 23.1 Biological properties of the type I interferons

Cellular source Number of genes Species restriction

Biologic effects Antiviral Antiproliferative Antitumor Major histocompatibility complex regulation Pregnancy signaling

Alpha a

Beta b

Tau s

Omega x

Leukocyte 15 human No, varying activity among species

Fibroblast 1 human Yes

Trophoblast 8 bovine No, varying activity among species

Leukocyte 1 human Yes

    

    

    

    

, possesses indicated effect; , lacks indicated effect.

The genes for the type I IFN genes are arranged in a genomic cluster on the short arm of human chromosome 9 (Diaz et al., 1994). Of particular interest is the fact that all of the type I IFN genes are intronless, indicating the ancient origins of this gene family. Despite their striking lack of introns, IFNs do have secretory signal peptide sequences that are cleaved prior to secretion, resulting in mature IFNs a, b and x ranging from 165 to 172 amino acids in length. Homology at the amino acid level between the IFNa and IFNx is higher than homology of either subtype with IFNb. The IFNa and IFNx are thought to have diverged more recently than IFNa and IFNb. All of the IFNa, x and b genes are believed to have arisen from a single ancestral gene (Weissman and Weber, 1986). In 2000, Oritani and colleagues identified a factor in the culture supernatants of a murine bone marrow stromal cell line (BMS2.4) which had an antiproliferative effect on B lymphocytes. Experiments led to the cloning of a gene for a soluble factor of 182 amino acids with a signal peptide of 21 amino acids which they named limitin. Limitin displays roughly 30% amino acid sequence identity with the other type I IFNs and likewise exerts it biological effects via interaction with the type I IFN-R complex (Oritani et al., 2000). Similar to IFNa and IFNb, limitin inhibits B lymphopoiesis both in vitro and in vivo. It furthermore suppresses the proliferation of T cells while enhancing the induction of CTL activity through augmentation of the perforingranzyme activity and induction of MHC Class I

expression also in a manner analogous to IFNa and IFNb (Takahashi et al., 2001). However, an important distinguishing feature of limitin is that it appears to not suppress the growth of myeloid and erythroid progenitors unlike the other type I IFNs (Oritani et al., 2000). Additionally, limitin has no apparent deleterious effects on the development of thymocytes either in vitro or in vivo. One additional interesting feature of the limitin gene is the presence of two initiation codons in the 5 portion of the cDNA, corresponding to two different reading frames which overlap by 34 bp. The second of these ATGs is the preferred start codon, and it is this start site that encodes the mature limitin protein. The presence of the second upstream ATG may imply a novel transcriptional regulation mechanism for this gene. Limitin appears to be produced constitutively by T cells and epithelial cells in the adult mouse (Oritani et al., 2003). As of early 2003, a human homologue of limitin remains to be identified. LaFleur and colleagues in 2001 reported the identification of a novel Type I IFN via analysis of a database of over 3 million human EST sequences. A single EST derived from a keratinocyte library was found to display homology to the type I IFNs. The gene which they identified represents a novel subclass of the Type I IFNs, and has been designated IFNj. This gene is located adjacent to the type I IFN gene cluster on the short arm of human chromosome 9. The IFNj gene codes for a protein of 207 amino acids including a 27 amino acid leader peptide, and is distinct from the other type I IFNs in that

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SUBTYPES OF TYPE I IFN

it contains a single intron (LaFleur et al., 2001). Expression analysis of a large number of cDNA libraries indicated and confirmed expression of IFNj predominantly in epidermal keratinocytes with lower (but detectable) expression in dendritic cells and monocytes. IFNj expression is induced in response to a number of the same stimuli that lead to expression of the classical type I IFNs including dsRNA, encephalomyocarditis virus infection, IFNb and IFNc. Similar to the other type I IFNs, IFNj signals via the type I IFN receptor and activates the ISRE signaling pathway. Genes induced in response to other type I IFNs are induced in response to IFNj as well, as evidenced by the increase in mRNA for MxA, PKR, 2-5oligoadenylate synthetase, IRF-1 and Stat1 in Daudi cells treated with 1 lg/ml IFNj for 6 hours (LaFleur et al., 2001). In late 2002 two groups independently reported the identification of a small family of three interferonlike cytokines through computational analyses of human genome sequence databases. The three family members designated alternately as IL-28A, IL-28B and IL-29 or IFN-k2, IFN-k3 and IFN-k1, respectively (Sheppard et al., 2003; Kotenko et al., 2003). IL-28A and IL-29 are paralogous sequences which share approximately 80% amino acid identity, while IL-28B, which is likely a recent duplication, bears 96% homology with IL-28A. This group of IFN-like sequences bears homology to both the type I IFNs (from 15–19% with IFNa) and the IL-10 family (11–13%), and may represent a critical evolutionary link between these two important immunomodulatory groups of proteins (Kotenko et al., 2003; Sheppard et al., 2003). Unlike the type I IFN cluster, the IL-28 and IL-29 genes are located on the long arm of human chromosome 19. They have a multiple exon structure which is more reminiscent of the IL-10 gene family than of the classical type I IFNs which have but a single exon. The 5 regulatory regions of these genes reportedly bear a number of sequence elements involved in the transcriptional regulation of the other type I IFN genes. This observation is borne out by the fact that poly(I)-poly(C) treatment or infection with viruses including encephalomyocarditis virus (EMCV), Sindbis virus, Dengue virus or vesicular stomatitis virus (VSV) leads to transcriptional activation of IL-28A, IL-28B and IL-29 variously in human peripheral blood mononuclear cells,

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monocyte-derived dendritic cells and a number of human cell lines (Kotenko et al., 2003; Sheppard et al., 2003). The antiviral activity which is the hallmark of the type I IFNs is also shared by these proteins as they were able to protect a number of cell types from viral challenge with either EMCV or VSV (Kotenko et al., 2003; Sheppard et al., 2003). The antiviral activity of IL-28A, IL-28B and IL-29 could not be inhibited by the addition of neutralizing antibodies directed against the type I IFN-R complex (Sheppard et al., 2003). Instead, a novel heterodimeric receptor complex was found to be essential for productive signaling in response to this family of IFN-like proteins. This novel receptor consists of a protein now designated as IL-28Ra (previously CRF2-12) and the second chain of the IL-10R, IL-10Rb (previously CRF2-4 or IL-10R2) (Kotenko et al., 2003; Sheppard et al., 2003) with both chains apparently required for full binding affinity. Binding of ligand to this receptor complex induces activation of Stat1, Stat2, Stat3 and Stat5 while further promoting ISGF3 complex formation. Among the IFN-inducible genes which require ISGF3 for their activation, IL-28A, IL-28B and IL-29 increased the message levels for 2-5oligoadenylate synthetase and the MxA protein as well as MHC Class I (Kotenko et al., 2003). Given the critical role that IFNs play at the intersection of innate and adaptive immunity, it will be of significant interest to determine the full therapeutic potential of this new family of IFN-like proteins. Two additional subtypes of the type I IFNs can be found: one in ruminant animals such as sheep and cattle and one in pigs. IFNs was first described as a factor produced by the developing trophoblast of sheep and cattle. During early pregnancy, IFNs functions to block the luteolytic effects of PGF2a, permitting the establishment of pregnancy by preserving the corpus luteum (Martal et al., 1979). IFNs was subsequently shown to possess antiviral activity and other biologic activities characteristic of the IFNs. The promoter region of the IFNs gene sequence contains unique elements not present in other type I IFN genes. IFNd is a porcine IFN that is secreted by the pig trophoblast during early stages of pregnancy (Lefévre and Boulay, 1993). The specific function of IFNd is unclear, although the timing of its release seems to mirror that of IFNc (Labonnardière et al., 1991; Lefévre and Boulay, 1993).

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TYPE I IFN STRUCTURE The three-dimensional crystal structures of IFNa, IFNb and IFNs have been solved (Senda et al., 1995; Radhakrishnan et al., 1996; Karpusas et al., 1997; Radhakrishnan et al., 1999). The type I IFNs are globular proteins that possess the four-helix bundle topology characteristic of the a-helical cytokine family. The presence of a fifth a helix tightly associated with the helical bundle identifies the structure of type I IFNs as distinct from other a-helical cytokines. Differing biologic activities among the IFNs may be related to altered affinities due to differences in structure. Comparisons between huIFNa2b, huIFNb, muIFNb and ovIFNs reveal a conserved structural core (Plate 23.1)(see Plate section). However, the crystal structures of IFNa and b revealed zinc-mediated dimers, while IFNs was a monomer. The largest structural variations occur at the N-terminus in the first and second a helices and the loop between these two helices. This structurally variable N-terminal region plays an important role in receptor binding and functional properties. These differences may account for the identification of different regions related to antiviral and antiproliferative activities in structure function studies of IFNs a2 and s (Pontzer et al., 1990, 1994).

INDUCERS OF TYPE I IFN In human tissues, low levels of IFNs can be detected even in the absence of a specific inducer (reviewed in Taniguchi and Takaoka, 2001). This in fact may serve as a natural surveillance system for the innate immune function (see below). A multitude of biological stimuli may trigger type I IFN production: (1) infection by microorganisms (including viruses, bacteria, mycoplasma and protozoa); (2) exposure to certain cytokines and growth factors (e.g. CSF-1, IL-1, IL-2 and TNFa); and (3) double-stranded nucleic acids such as the dsRNA produced during viral replication. IFN was discovered during examination of the phenomenon of viral interference. Viruses are in fact one of the primary natural inducers of type I IFNs. A number of other pathogenic microbes and their products can also elicit IFN production and release. Many bacteria, especially obligate intracellular organisms,

including Listeria monocytogenes and other parasites, induce type I IFNs during systemic infection (Remington and Merigan, 1969). Endotoxin, which is a lipopolysaccharide component of Gram-negative bacterial cell walls is a potent IFN inducer, as are the M proteins of group A streptococci (Weigent et al., 1986). Double-stranded RNA (dsRNA) is frequently used as a replicative intermediate by viruses. Both natural and synthetic double-stranded RNAs (dsRNAs) have been observed to function as efficient inducers of type I IFNs (Field et al., 1967; Tytell et al., 1967). Of the synthetic, double-stranded polynucleotides, the homopolymer pair poly-riboinosinic-ribocytidilic acid (polyI:C) is the most active and has been commonly used for IFN induction studies (for examples see Thacore and Youngner, 1973; Rothmann et al., 1985). Stimulation of murine bone marrow cells with either CSF-1 or IL-2 results in the production of murine IFNa/b (Moore et al., 1984; Reis et al., 1989). In human diploid fibroblasts, IL-1 and TNFa, can also induce the synthesis of human IFNb (Reis et al., 1989). Additionally, IFNc functions as an inducer of the release of both IFNa and IFNb from macrophages (Gessani et al., 1989; Cantell and Pirhonen, 1996). RANK ligand can also induce the expression of IFNb in bone marrow macrophages (Taniguchi and Takaoka, 2002). A surprising regulatory function of type I IFNs is the induction of expression of other type I IFN genes. A transcriptional activator, IRF-3, is expressed broadly and constitutively at high levels. Viral infection serves to activate IRF-3 via specific serine phosphorylation events (Yoneyama et al., 2002), leading to the synthesis and release of specific IFNs, predominantly IFN-a4 and IFN-b (Juang et al., 1998; Marie et al., 1998). In paracrine fashion, these newly released IFNs are free to act on neighboring cells. They do so by binding to the type I IFN receptor on neighboring cells, and in a Stat1-dependent manner, induce the expression of yet another transcriptional activator, IRF-7. When cells expressing IRF-7 are in turn infected with virus, IRF-7 is phosphorylated, and the cells respond by expressing other IFNa genes (i.e. subtypes other than IFN-a4) (Levy et al., 2002). Thus there exists a virusdependent amplification of the IFN response at the level of induction of different type I IFN subtypes.

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THE TYPE I IFN RECEPTOR In 1990, Uzé and colleagues cloned the major ligandbinding chain of the human type I IFN receptor, IFN-Ra (Uzé et al., 1990). The transfer of a cDNA clone for this protein into murine cells rendered them sensitive to human IFN-a8. IFN-Ra is a type I transmembrane protein of 557 amino acids in length, which has a predicted molecular mass of 63 kDa. The extracellular domain of IFN-Ra is 409 amino acids in length and contains two fibronectin type III repeats (Bazan, 1990). Recent data indicate that the ligand binding function of the IFN-Ra resides in the N-terminal of these two repeat domains (Chill et al., 2002). This structure is consistent with classification as a class II cytokine receptor (reviewed in Mogensen et al., 1999). The apparent electrophoretic mobility of IFN-Ra is approximately 110–130 kDa (Ling et al., 1995) indicating substantial glycosylation of this protein. The cytoplasmic domain of IFN-Ra contains only 100 amino acids with no intrinsic enzymatic activity. The second subunit of the type I IFN receptor, IFN-Rb was cloned in 1994, and was originally believed to have two forms: one soluble and one transmembrane (Novick et al., 1994). Coexpression of the transmembrane form with IFN-Ra, however, was unable to recapitulate type I IFN signaling. A third form of the IFN-Rb was cloned in 1995. It had an extended cytoplasmic domain, and was able to fully support signaling in response to type I IFN stimulation (Domanski et al., 1995; Lutfalla et al., 1995). There are in fact then three forms of the IFN-Rb subunit (one soluble version and two transmembrane versions, long and short), generated by alternative splicing of the same gene (Prejean and Colamonici, 2000). As alternatively spliced isoforms, the long and short forms of IFN-Rb are identical in their extracellular and transmembrane domains. The sequences are divergent in their cytoplasmic domains, although the first 16 amino acids here are also identical. The long form of the b subunit (bL) is composed of 515 amino acids and is the isoform competent for signaling. Its extracellular domain contains two sites for N-linked glycosylation. The cytoplasmic domain contains seven tyrosines, several of which are required for function (Nadeau et al., 1999), and six acidic domains (Domanski et al., 1995). The short form, IFN-RbS is a 331 amino acid protein with only two cytoplasmic

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tyrosines (Novick et al., 1994). The function of this isoform of IFN-Rb is unknown, although it is likely to act as a rheostat for modulating cellular responses to the type I IFNs. The soluble form of IFN-Rb is generated by the insertion of a stop codon following amino acid 236. This may function as a natural antagonist of the type I IFNs. IFN-Rb is a member of the same helical cytokine receptor family as IFN-Ra. These two genes are found in a genomic cluster on human chromosome 21, along with IL-10Rb and IFNGR2 (Lutfalla et al., 1995). There have been no reports as yet of mice deficient in the IFN-Rb gene. However, two strains of mice with a disrupted IFN-Ra gene have been reported (Müller et al., 1994; Hwang et al., 1995). These mice display a phenotype that is predominantly normal, with only minor perturbations in their immune responses when compared with wild-type mice. Upon challenge with sublethal doses of a virus such as vesicular stomatitis virus (VSV) however, they are unable to mount an efficient antiviral response and as a consequence, die rapidly.

IFN SIGNAL TRANSDUCTION One of the major pathways for the propagation of interferon signals involves the activation of the JAK/STAT signal transduction pathways (Plate 23.2) (see Plate section). The JAKs, or janus kinases, are a family of cytoplasmic non-receptor tyrosine PTKs with a structure unique among kinases (Ihle, 1995). Two members of the Jak kinase family, Tyk2 and Jak1, constitutively associate with the IFNRa and IFN-RbL subunits of the type I IFN receptor, respectively (Colamonici et al., 1994, 1995; Domanski et al., 1997). When type I interferons bind to their receptor, Tyk2 and Jak1 are activated by either trans- or autophosphorylation events. IFN-Ra and IFN-RbL are rapidly phosphorylated by Tyk2 and Jak1 on cytoplasmic tyrosines (reviewed in Platanias and Fish, 1999). The tyrosine phosphorylated receptors serve as docking sites for members of the Stat (signal transducer and activator of transcription) family of transcriptional co-activators, specifically Stat1a and Stat2 (reviewed in Prejean and Colamonici, 2000). Interestingly, although both IFNa and IFNb ultimately induce tyrosine phosphorylation of the receptor subunits, IFNb

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but not IFNa, induces the tight association of the IFN-Ra and IFN-RbL chains. The nature of the interaction of distinct type I IFN subtypes with the IFN receptor is thus qualitatively different, and may account for the variation in biological responses to IFN subtypes (Platanias et al., 1994, 1996; Abramovich et al, 1994; Croze et al., 1996). The activation of Jak kinases in response to ligation of the type I IFN receptor leads to the tyrosine phosphorylation of a number of Stat proteins, including Stat1, Stat2, Stat3 and Stat5 (reviewed in Platanias and Fish, 1999). Stat1 and Stat2 form a heterodimer, otherwise known as ISGF3a, that in turn associates with IRF-9 (p48 or ISGF3c) to form the mature ISGF3 transcriptional activator. The ISGF3 complex translocates to the nucleus and initiates gene transcription by binding to interferon stimulated response elements (ISREs), palindromic sequences found in the promoter regions of IFN-responsive genes (Darnell, 1997, 1998; Stark et al., 1998). Full transcriptional activation of ISREs by ISGF3 requires (1) serine phosphorylation of the Stat1a component by PKCd; and (2) PKCddependent activation of the p38 MAP kinase pathway. (Uddin et al., 2002) Activation of p38 MAP kinase is essential for IFNa-induced ISRE-dependent gene transcription, but not for the DNA binding activity of the ISGF3 complex (Uddin et al., 2000). Stat1:1 homodimers (AAF, IFNa-activated factor), Stat3:3 homodimers, Stat1:3 heterodimers (Darnell, 1997; Stark et al., 1998) and Stat5 homodimers (Meinke et al., 1996) also bind palindromic DNA elements similar to ISREs. These complexes also play an important, but less well characterized role in IFN-dependent gene transcription.

IFN-INDUCED GENES AND ACTIVITIES Protein kinase R (PKR) PKR is a cytoplasmic serine/threonine kinase which binds to and is activated by double-stranded RNA (dsRNA) (reviewed in Williams, 1999). The dsRNA binding activity resides in an N-terminal regulatory domain, while the C-terminus houses the kinase domain. Upon binding to dsRNA, which is a common replicative intermediate of many RNA viruses, PKR

dimerizes, autophosphorylates and becomes activated. PKR phosphorylates a number of cellular targets including the eukaryotic translation-initiation factor, eIF2a. Phosphorylation renders eIF-2a inactive, and leads to a generalized inhibition of protein synthesis. With the cellular protein synthesis machinery shut down, viral protein expression and viral replication are also negatively impacted. In addition to its effects on protein synthesis, PKR is also capable of mediating antiviral effects at other levels. PKR may exert some of its antiviral activity through the induction of apoptosis dependent in part on signaling via FADD (Takizawa et al., 1996; Der et al., 1997; Balachandran et al., 1998; Yeung et al., 1999; Gil and Esteban, 2000). PKR may also regulate IFN synthesis in some tissues and cell types. dsRNA activation of PKR can be a positive regulatory signal for the synthesis of IFNb via activation of the NFkB pathway (Kirchoff et al., 1995; Yang et al., 1995; Kumar et al., 1997; Chu et al., 1999; Gil et al., 2000).

2-5 Oligoadenylate synthetase system The 2–5 oligoadenylate synthetases are a group of IFN-inducible enzymes that catalyze the synthesis of oligomers (three to five units) of adenosine linked by 2 to 5 phosphodiester bonds, using ATP (and occasionally GTP) as the starting material (Kerr and Brown, 1978). The 2–5A oligoadenylate molecules bind with high affinity to ribonuclease L (RNaseL) and facilitates its dimerization and consequent activation. RNaseL, when activated, cleaves single-stranded RNAs, including mRNA, and contributes to the inhibition of protein synthesis (reviewed in Player and Torrence, 1998). RNase L was recently shown to cleave 28S ribosomal RNA in a site-specific manner, causing ribosomal inactivation and translational inhibition (Iordanov et al., 2000). Several of the different isozymes of 2–5 oligoadenylate synthetase directly impact both cellular proliferation and cellular survival (see below).

Mx proteins The Mx proteins are a highly conserved family of large GTPases which bear homology to dynamin, a large cytosolic protein with GTPase activity. Mx proteins are found in most, if not all, vertebrate species, including

THE CYTOKINES AND CHEMOKINES

IFN - INDUCED GENES AND ACTIVITIES

mammals, birds and fish (reviewed in Staehli et al., 1993; Arnheiter et al., 1995). These proteins act to inhibit virus replication through an as yet undefined mechanism. The murine Mx1 protein accumulates in the nuclei of IFN-treated cells and interacts with a number of nuclear factors, some of which are likewise IFN-inducible. Among the potential interacting partners for Mx1 are Sp100, Daxx and Bloom’s syndrome protein (BLM), all of which localize to nuclear subdomains termed promyelocytic leukemia protein nuclear bodies (PML NBs) (Engelhardt et al., 2001). Additionally, Mx1 may interact with SUMO1 (small ubiquitin-like modifier one) and SAE2 (subunit 2 of the SUMO-activating enzyme). Interestingly, it is the modification of proteins by conjugation to SUMO that allows for their localization to PML nuclear bodies (Regad and Chelbi-Alix, 2001). It is therefore possible that Mx proteins inhibit either the trafficking of viral particles or the activity of viral polymerases (Stranden et al., 1993). The human cytoplasmic protein MxA inhibits the replication of members of several RNA virus families, including the family Orthomyxoviridae, of which influenza is a member. In mice, mutant forms of Mx proteins lacking the ability to bind or hydrolyze GTP, fail to suppress virus replication (Staehli et al., 1988).

Other antiviral mechanisms Mice triply deficient for the three IFN-mediated antiviral pathways mentioned above (PKR, RNase L and Mx) have been generated (Zhou et al., 1999). These triple-knockout mice were more sensitive to viral infection than wild-type mice, as would be anticipated. However, these mice also mounted an appreciable antiviral response following IFN treatment, and did not display the dramatic susceptibility to infection observed in mice lacking either the IFNa/b receptors or Stat1. Thus, there are likely to exist other major antiviral pathways induced by IFN. One family of proteins which may be candidates for involvement in the effects described above is the family of adenosine deaminases. The three known members of the ADAR (for adenosine deaminases that act on RNA) family, ADAR1, ADAR2 and ADAR3, recognize dsRNA as a substrate and systematically deaminate adenosine residues within the RNA, converting them to inosine (Bass et al., 1989; Polson and Bass,

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1994; O’Connell et al., 1995; Patterson et al., 1995). This effectively unwinds the dsRNA due to the inability of inosine to pair with uracil. If and when these dsRNAs are translated, the inosines are ‘read’ as guanines. Since many viral RNAs utilize a dsRNA-based replicative intermediate this substitution functions effectively as a host-driven mutagenesis. There are in fact several reports of genomic substitutions consistent with ADAR activity (reviewed in Goodbourn et al., 2000). Furthermore, there is some evidence to suggest that an inosine-specific ribonuclease could act in concert with ADARs to specifically degrade modified viral RNAs (Scadden and Smith, 1997), although a candidate has yet to be cloned. The expression of at least one member of the adenosine deaminase family, ADAR1, is regulatable by IFNa (Patterson et al., 1995).

Antiproliferative functions of type I IFN Both PKR and RNaseL are integral mediators of the antiproliferative functions of IFNs. The amount of PKR in cultured cells appears to vary according to their state of growth and there is a direct correlation of this with the level of eIF2a phosphorylation (reviewed in Jaramillo et al., 1995). Even in the absence of viral dsRNA, PKR exhibits activity which appears to be related at least in part to physiologic stress. Other, non-dsRNA cellular inducers of PKR activation include PACT (Patel and Sen, 1998) and caspases-3, -7 and -8 (Saelens et al., 2001). Overexpression of PKR leads to growth suppression and apoptosis in a number of cell types (Koromilas et al., 1992; Chong et al., 1992; Dever et al., 1993), an effect which again is due to eIF2a phosphorylation. Activation of the 2–5 oligoadenylate synthetase/RNase L pathway also leads to growth inhibitory effects. Overexpression of a 40 kDa isoform of 2–5 oligoadenylate synthetase reduces growth rates of transfected cells (Chebath et al., 1987; Rysiecki et al., 1989; Coccia et al., 1990), and sustained expression of the P69 isoenzyme leads to dysregulation of cell cycling (Ghosh et al., 2000). The isoenzyme 9-2, which has homology to Bcl-2, is another member of the 2–5 OAS family. Overexpression of 9-2 has pro-apoptotic effects, and 9-2 has been demonstrated to bind to both Bcl-2 and Bcl-XL (Ghosh et al., 1999). Overexpression of RNase L, which is activated by 2–5 oligoadenylates, enhanced

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antiproliferative activity of IFN (Zhou et al., 1998), and conversely expression of a dominant negative mutant of RNaseL in murine SVT2 cells inhibited the antiproliferative effect of IFN on these cells (Hassel et al., 1993). IFNs can also exert a more direct negative regulatory effect on the cell cycle by specifically up-regulating the expression of a number of cyclin-dependent kinase inhibitors (CKIs). IFNa specifically up-regulate the levels of the CKI, p21Cip1/Waf1 (Chin et al., 1996, Subramaniam and Johnson, 1997; Subramaniam et al., 1998), which plays a crucial role in the progression from G1 into S phase by binding to and decreasing the activity of Cdk2 (reviewed in Harper et al., 1993; Gartel et al., 1996; Sangfelt et al., 1997). Type I IFNs also increase the expression of another CKI, p15Ink4b that can complex specifically with Cdk4 (Sangfelt et al., 1997). A third protein, p27Kip1, preferentially binds to cyclin E/Cdk2 complexes, dissociating the retinoblastoma-(Rb)-related pocket proteins p107 and p130 from these (Sangfelt et al., 1999). When p21, p15 and p27 levels are elevated, cyclindependent kinase activity is thus reduced and the phosphorylation of the retinoblastoma gene product (pRb) and the related pocket proteins (p107 and p130) are concomitantly suppressed (Sangfelt et al., 1999). Rb and the related pocket proteins, in their nonphosphorylated forms, interact strongly with the E2F family of transcription factors, thus inhibiting their activity (Iwase et al., 1997; Kirch et al., 1997; Furukawa et al., 1999). Phosphorylation of Rb (and p107 or p130), normally releases E2F transcription factors, and permits transition from G1 to S phase. Interestingly, p15 and a related protein p16Ink4a are frequently deleted in tumors, and may be a mechanism by which tumors can evade the growth suppressive effects of the type I IFNs (Sangfelt et al., 1999) Members of the p200 gene family, for example the p202 gene product, are potent repressors of the cell cycle (Kingsmore et al., 1989; Lembo et al., 1995; Gutterman and Choubey, 1999). p202 binds both hypophosphorylated Rb (Choubey and Lengyel, 1995), as well as E2F-1 and E2F-4, members of the E2F transcription factor family (Choubey et al., 1996; Choubey and Gutterman, 1997). Furthermore, p202 binds to complexes in which both E2F-4 and Rb (or the related proteins p107 and p130) are present (Choubey and Gutterman, 1997). p202 specifically

occupies the DNA-binding domain of E2F-4, thus abrogating its transcriptional activity. The murine p202a protein binds to and inhibits a number of transcription factors, including c-Fos, c-Jun, AP2, MyoD, myogenin, NF-kB p50 and NF-kB p65 (Min et al., 1996; Datta et al., 1998). p202a also interacts with c-Myc, preventing its heterodimerization with Max, and consequently inhibiting gene transcription (Wang et al., 2000). In addition to blocking the activity of transcription factors, p200 proteins may also exert a more direct effect on gene expression by directly binding to promoter elements. The human p202 homologue, IFI 16, contains a functional transcriptional repression domain (Johnstone et al., 1998).

Regulation of apoptosis The type I IFNs can have pro-apoptotic activities, although the distinct mechanisms for many of these remain to be elucidated. Many cells infected with viruses readily undergo IFN-dependent apoptosis (Tanaka et al., 1998), and antibodies to IFNs will prevent this in vitro. Some of the apoptotic events in virally infected cells are mediated through the actions of PKR and the 2–5 oligoadenylate synthetases (described above). In fact, PKR activation leads to the expression of both CD95/Fas and Bax, while cells deficient in the expression of FADD are resistant to PKRinduced apoptosis (Balachandran et al., 1998). IFN induces caspase-3 activation (Subramaniam et al., 1998), caspase-7 activation (Sanceau et al., 2000), and caspase-8 activation (Balachandran et al., 2000). IFNa also causes the release of cytochrome c from mitochondria, depolarization of mitochondrial membranes and cleavage of Bax in the B cell lymphoma line Daudi (Yanase et al., 2000). Finally, the type I IFNs may also promote apoptosis of DR4- and DR5expressing cells by increasing T cell expression of TRAIL (Kayagaki et al., 1999).

Immunomodulation The type I IFNs also exert a number of immunomodulatory effects. Several of these effects revolve around the modification of NK cell biology. IFNa-induced IL-15 drives the proliferation and maturation of NK cells (Ogasawara et al., 1998; Fawaz et al., 1999; Gosselin et al., 1999), while IFN can directly enhance

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the cytotoxic function of NK cells (reviewed in Reiter, 1993; Biron et al., 1999), in part by increasing perforin expression (Mori et al., 1998; Kaser et al., 1999). T cells are also influenced in a number of ways by the type I IFNs. The IFN-induced IL-15 stimulates the division of memory T cells (Tough et al., 1996; Zhang et al., 1998), while IFNa and b directly promote the survival of T cells activated in vivo (Marrack et al., 1999). As mentioned above, IFNa and b also increase TRAIL expression on CD4 and CD8 T cells alike (Kayagaki et al., 1999). IFNb directly inhibits T cell migration, primarily by preventing expression and/or release of matrix-degrading enzymes, such as gelatinase and MMP-9 (Leppert et al., 1996; Stuve et al., 1996). Additionally, type I IFNs down-regulates the expression of a number of other immunologically relevant proteins, including IFNc, IL-12 and IL-12R (Cousens et al., 1997), B7-1 and B7-2 (Arnason, 1996). IFNb may also induce monocytes to secrete IL-10 (Porrini et al., 1995).

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THERAPEUTIC APPLICATIONS OF TYPE I IFN IFNa have been FDA approved for multiple indications (Table 23.2). Currently, among viral diseases, condyloma accuminatum, chronic hepatitis B and chronic hepatitis C are treated by IFNa2 variants. In oncology, the IFNs are an important treatment for a number of solid tumors and hematological malignancies, including non-Hodgkin’s lymphoma, follicular lymphoma, melanoma, renal cell carcinoma, AIDS-related Kaposi’s sarcoma (KS), hairy cell leukemia and chronic myelogenous leukemia (CML).

Condyloma acuminatum Condyloma acuminatum is the most common disease for which the effectiveness of IFNa has been demonstrated. Non-antiviral therapies, such as surgery or cytodestruction, are effective in eliminating the lesions of Condyloma acuminatum, but recurrences are common, presumably because of viral latency. In a number of studies, IFNa was effective in eradicating all visible lesions in 60 to 70% of patients with Condyloma acuminatum who previously had recurrences

TABLE 23.2 Therapeutic indications for type I IFNs Disease

Interferon (trade name)

Condyloma acuminatum

IFN-a2a (Roferon A), IFN-a2b (Intron A), IFN-an3 (Alferon N), IFN-an1 (Wellferon)

Juvenile laryngeal papillomatosis

IFN-an1 (Wellferon)

Chronic hepatitis B

IFN-a2b (Intron A), IFN-an1 (Wellferon)

Chronic hepatitis C

IFN-a2a (Roferon A), IFN-a2b (Intron A), IFN-acon-1 (Infergen), Peg-IFN-a2b (PEG-Intron) IFN-an1 (Wellferon)

Chronic myelogenous leukemia

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Follicular lymphoma

IFN-a2b (Intron A)

Non-Hodgkin’s lymphoma

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Melanoma

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Multiple myeloma

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Kaposi’s sarcoma

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Hairy cell leukemia

IFN-a2a (Roferon A), IFN-a2b (Intron A), IFN-an1 (Wellferon)

Renal cell carcinoma

IFN-a2a (Roferon A), IFN-a2b (Intron A)

Multiple sclerosis

IFN-b1a (Avonex, Rebif), IFN-b1b (Betaseron) THE CYTOKINES AND CHEMOKINES

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after a variety of non-antiviral therapies (Tyring, 1988; Reichman et al., 1988, 1990). The amount of virus is thought to be sufficiently reduced so that the host’s immune system may prevent recurrences.

Chronic hepatitis B IFN treatment is effective for the treatment of chronic hepatitis B via multiple mechanisms including enhanced clearance of hepatocytes infected with hepatitis B and direct inhibition of viral replication. IFNa promotes conversion to low levels of viral replication. Low to non-detectable levels of hepatitis B e antigen and anti-e antigen antibodies are found in the sera in up to 15–20% of treated patients each year. In contrast, only about 5% of patients with chronic infection a year undergo spontaneous conversion from high level viral replication to low level replication in the absence of treatment. The approved IFNa treatment regimen for chronic hepatitis B is 5 million units per injection three times weekly for 4 to 6 months. While greater doses of IFNa demonstrated further improved conversion rates, dose-limiting toxicities associated with IFNa prevented escalation (Gow and Mutimer, 2001).

Hepatitis C As a single therapy, IFNa alone is rarely effective in curing hepatitis C infection. Identification of the hepatitis C variant responsible for infection is a good indicator of patient responsiveness to treatment (Hino et al., 1994). Responsiveness to IFN-a alone is less than 10% in patients infected with a hepatitis C resistant variant. The development of combination therapy using IFN-a and ribavirin, a nucleoside analog, has significantly improved responsiveness. The type of viral variant still greatly influences response rates (Poynard et al., 1998). Cure defined as sustained viral clearance, is observed in approximately 30–40% in patients treated with the combination of IFN and ribavirin. Recent studies suggest that pegylated IFNa alone and in combination with ribavirin may be a useful and even more effective therapy for hepatitis C (Reddy et al., 2001).

Chronic myelogenous leukemia IFNa demonstrated the antiproliferative activity of IFN on granulocyte precursors (Verma et al., 1979). One of the first studies of IFN-a in CML studied seven CML patients treated with IFNa (9–15  106 U i.m. qd) (Talpaz et al., 1983) in which five out of seven exhibited hematologic remission. Three years later a second study followed 17 early CML patients treated with rIFNa2a (5  106 U m2 i.m. qd) (Talpaz et al., 1986). In this study 14 patients responded to IFN therapy, with 13 complete hematologic remission (CHR) and one partial hematologic remission (PHR). Approximately 20–25% of patients showed long-term suppression of the Philadelphia chromosome. Multiple studies of CML patients that received IFNa2b therapy showed most patients achieving CHR (46 to 80% of patients) and cytogenetic responses were observed in smaller numbers (13 to 32% CR rates; 11 to 16% PR rates) (Alimena et al., 1988; Ozer et al., 1993; Kantarjian et al., 1996; Mahon et al., 1998). Patients with diagnosed CML for less than 1 year are more likely to respond to higher daily and sustained doses of IFNa2 therapy (Kantarjian et al., 1996). Thrombocytosis observed with myeloproliferative disorders, regardless of Philadelphia chromosome involvement can be regulated by IFNa2 therapy (Talpaz et al., 1989). The serious, clonal hypereosinophilic syndrome is also controllable (Butterfield and Gleich, 1994). Sustained IFN therapy modulates both leukocytosis and thrombocytosis in nearly all patients studied (Gilbert, 1998).

Follicular lymphoma Treatment of follicular lymphoma with IFNa began in the early 1980s (Ozer et al., 1998). Initial studies demonstrated a 50% response rate, however two following phase III trials of single alkylating agents, alone or in combination with IFNa, showed increased remission duration without overall survival improvement (Price et al., 1991; Peterson et al., 1997; Ozer et al., 1998). Subsequent trials examined the combination of IFN and cytoreductive chemotherapy (Smalley et al., 1992; Solal-Celigny et al., 1998). One of these studies demonstrated improved median progression-free survival and overall survival in the group of patients receiving IFN (Solal-Celigny et al., 1998).

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Non-Hodgkin’s lymphoma IFNa has also been developed as a maintenance therapy for patients with low-grade non-Hodgkin’s lymphoma (NHL). Patients who had undergone cytoreductive chemotherapy and demonstrated reduced tumor bulk were treated with IFNa (McLaughlin et al., 1993; Hiddemann et al., 1994; Hagenbeek et al., 1995; Unterhalt et al., 1995, 1996; Aviles et al., 1996; Ozer et al., 1998). Improvement in failure-free survival rates was observed in patients treated with the maintenance IFN regimen, but no significant increase in overall survival was seen. Another IFNa2b maintenance therapy study did show an increase in overall survival (Aviles et al., 1996). For B-cell lymphomas, IFNa2 is being developed in combination with other efficacious treatments for both low- and intermediate-grade non-Hodgkin’s lymphomas (Smalley et al., 1992; Solal-Celigny et al., 1993; Borden, 1994).

Melanoma Murine IFN was originally shown in 1980 to inhibit the B16 melanoma in vitro and the in vivo oncology tumor model (Bart et al., 1980). Early clinical studies using partially purified leukocyte IFN resulted in minimal improvement (Krown et al., 1984). The use of rIFNa2a at two different dosages 12  106 U m2 three times a week (tiw) (Creagan et al., 1984a), and 50  106 tiw i.m. (Creagan et al., 1984b), for a duration of 3 months showed response rates of 20%. IFN as a single agent in patients with metastatic melanoma demonstrated responses in approximately 15% and induced complete remission in 5% of patients (Kirkwood, 1995). Uninterrupted schedules of therapy indicated better outcomes compared with intermittent cyclic therapy (Kirkwood, 1995). IFNs are more effective against tumors of smaller mass than larger mass for metastases. IFNs are currently in use with IL-2 and chemotherapy for metastatic disease (Legha et al., 1998; Richards et al., 1999).

Kaposi’s sarcoma Treatment of HIV-associated Kaposi’s sarcoma with IFN has been in use since 1981 (Real et al., 1986). High doses on the order of 20  106 U d1 induced significant response rates, however multiple signs of toxicity

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was observed (Krown et al., 1983). In later studies (Lane et al., 1988), correlation of CD4 counts with improved chance of therapeutic responsiveness was demonstrated. Those patients possessing CD4 counts greater than 400/mm3 responded, while those with CD4 counts of less than 150 mm3 exhibited no response.

Hairy cell leukemia The first report of IFN response in patients with hairy cell leukemia was recorded in 1984 (Quesada et al., 1984). In early studies, IFNa and more specifically IFNa2a were efficacious in exerting partial and complete remisions. All blood hematologic indices either improved or normalized in patients who responded to IFN treatment (Quesada et al., 1986). A multicenter phase II study of 64 patients published in 1986 confirmed the drug’s efficacy in hairy cell leukemia (Golomb et al., 1986). IFNa2a and IFNa2b are approved for use in hairy cell leukemia, however upon completion of treatment, a greater percentage of patients exhibit disease relapse. While approved for use, other more effective therapies for hairy cell leukemia have taken the place of IFN.

Multiple sclerosis IFN-b is an FDA-approved treatment for relapsingremitting multiple sclerosis (MS). IFN-b1b (Betaseron) and IFN-b1a (Avonex) have both been shown to reduce the frequency and severity of clinical exacerbations in addition to reduction in disease activity (Euro, 1998; PRISMS, 1998). The mechanisms by which IFN-b exerts its efficacy likely include induction of IL-10 (Porrini et al., 1995; Rep et al., 1996, 1999), down-regulation of IFN-c and IFN-c-induced MHC class II expression (Ling et al., 1985; Satoh et al., 1995; Miller et al., 1996), inhibition of the costimulatory surface molecules B7–1/B7–2 (Arnason, 1996) and inhibition of T cell migration and proliferation of human T cells (Leppart et al., 1996; Stuve et al., 1996). Combined therapy of IFN-b with intravenous immunoglobulin has also been shown to be effective for treatment of Guillain-Barré syndrome (Schaller et al., 2001). IFN-s is also effective in preventing disease in an animal model for MS, known as experimental allergic

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encephalomyelitis (EAE) (Soos et al., 1995). IFN-s is as effective as IFN-b in EAE in the absence of the toxicities normally associated with IFN-b (Soos and Johnson, 1999). IFN-s is effective by both the oral and injectable routes of administration and can reverse ongoing paralysis in the chronic relapsing EAE model (Soos et al., 1997; Mujtaba et al., 1998). IFN-s has recently been shown to down-regulate MHC expression and exert antiviral activity against Theiler’s virus, a model for virus-induced demyelination (Tennakoon et al., 2001). IFN-s is currently in clinical trials for relapsing-remitting MS (Olek et al., 2001).

CONCLUSION In this review, we have presented a broad overview of some of the critical activities and therapeutic uses of the type I IFNs. This family of cytokines is the archetypical pleiotropes, and occupies a pivotal position at the crossroads of innate and adaptive immunity. The list of biological responses in which IFNs are involved continues to expand. Perhaps the most striking aspect of IFN biology then is that, despite the fact that this family of cytokines was discovered 45 years ago, their role in host defense has yet to be fully appreciated.

ACKNOWLEDGMENTS The authors would like to give their heartfelt thanks to Dr Howard M. Johnson of the University of Florida for his guidance, training and mentorship.

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24 Interferon-c Gregory H. Schreiber and Robert D. Schreiber Washington University School of Medicine, St Louis, MO, USA

There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes. The Doctor’s Dilemma, George Bernard Shaw

INTRODUCTION The interferons (IFNs) were originally described as an activity found in the supernatant of virally infected cells that directly ‘interfered’ with viral replication (Isaacs and Lindenmann, 1957). These proteins are now known to represent a family of cytokines and have been classified into two types based on structural and functional criteria, as well as the stimuli that elicit their expression. Type I IFNs are primarily induced in response to viral infection and have been divided into two groups: IFN-a, which is secreted largely by leukocytes, and IFN-b which is produced by fibroblasts. Type II IFN, now designated IFN-c, is synthesized primarily by T lymphocytes and natural killer (NK) cells following activation with immune and inflammatory stimuli rather than viral infection. During the last 15 years, the biology and biochemistry of IFN-c has been extensively studied. As a result, a great deal is now known about the events that lead to IFN-c production and the mechanisms by which The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

IFN-c manifests its biologic effects on cells. Whereas IFN-c promotes both innate and adaptive protective immune responses in the host against a variety of infectious agents and tumors, it also plays a central role in the development of immunopathologic conditions. IFN-c is therefore truly pleiotropic, not only in its effects at the individual cell level, but also in its effects on the intact host.

THE PROTEINS AND GENES OF THE INTERFERON-c SYSTEM Interferon-c genes and regulation The cDNAs for human and murine IFN-c were first cloned in 1982 by Gray and Goeddel, and bear no obvious sequence identity to cDNAs encoding type I IFNs (Gray et al., 1982; Gray and Goeddel, 1982, 1983). Whereas IFN-a is comprised of a family of more than Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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22 proteins, each encoded by a distinct gene, IFN-c is the product of a single gene. Using in situ hybridization techniques, the genes for human and murine IFN-c were localized to human chromosome 12 (12q24.1) and murine chromosome 10, respectively (Trent et al., 1982; Naylor et al., 1983, 1984). In both species, four exons and three introns comprise the 6 kb gene which encodes a 1.2 kb mRNA transcript. The cDNAs for human and murine IFN-c exhibit only 60% identity at the nucleotide level. The minimal promoter elements necessary to direct the regulated expression of IFN-c have not yet been clearly delineated, and are believed to vary depending upon both the cell lineage (Carter and Murphy, 1999) and the pathway that led to gene induction (Yang et al., 1999). When introduced into mice as a transgene, the expression of a fragment of human genomic DNA containing (a) 2.3 kb of 5 flanking sequence; (b) the IFN-c gene; and (c) 1 kb of 3 flanking sequence was found to parallel that of endogenous murine IFN-c (Young et al., 1989). This observation suggested that the elements necessary for tissue-specific and T cell mitogen-inducible in vivo expression of human IFN-c are present within this region. Two regulatory elements were identified within a 500-bp fragment 5 of the human IFN-c TATAA box that have PMA-inducible, enhancer-like activity when transfected into a murine T cell line (Ciccarone et al., 1990). These elements included a distal region capable of binding Fos, Jun and GATA-3 and a proximal NFIL-2-like element that can bind CREB/ATF-1, ATF-2, Oct-1 and AP-1 (Penix et al., 1993, 1996). Methylation of CpG dinucleotide sites within the 5 regulatory element or first intron of the IFN-c gene were found to correlate inversely with the ability of T cell populations to express IFN-c (Young et al., 1994; Melvin et al., 1995). When introduced individually into transgenic mice, each of the two 5 elements directed reporter gene expression in CD4 T cells, but only the distal element was active in CD8 T cells (Aune et al., 1997). Consensus binding sequence analysis (Campbell et al., 1996) and DNase footprint analysis (Sweetser et al., 1998) have also revealed several potential NFAT sites in the IFN-c promoter. Data supporting a physiologic role for two of these potential NFAT binding sites include the observation that IFN-c-reporter activity was decreased when dinucleotide mutations were made in these sequences

(Sweetser et al., 1998). In addition, multiple binding sites for NF-jB proteins (c-Rel, p50, p65) (Sica et al., 1992, 1997) have been identified in the IFN-c promoter, and IFN-c production was dramatically impaired in antigen-stimulated T cells from transgenic mice that expressed a dominant-inhibitor of NF-jB/Rel (Aronica et al., 1999). Furthermore, potential binding sites for Stat4 have been identified in both the 5 regulatory region and the first intron of the IFN-c gene (Xu et al., 1996, Barbulescu et al., 1998), and lymphocytes from Stat-4 deficient mice fail to upregulate IFN-c in response to IL-12 (Kaplan et al., 1996b). Recently, a novel transcription factor, T-box expressed in T cells (T-bet), was identified and reported to play a central role in T-helper cell subset differentiation and IFN-c secretion (Szabo et al., 2000). Analysis of the IFN-c gene revealed three potential binding sites for T-bet: two within the proximal promoter, and one in the third intron (Szabo et al., 2000). The functional significance of these sites however, has not yet been rigorously examined by mutagenesis. Finally, positive (Sweetser et al., 1998) and negative (Ye et al., 1996) regulatory roles have been proposed for the multifunctional DNA-binding protein Yin-Yang 1 (YY1) in the IFN-c promoter.

Interferon-c protein The IFN-c protein has been studied extensively. In humans, the 1.2 kb mRNA is translated into a 166 amino acid polypeptide (Gray et al., 1982; Rinderknecht et al., 1984). The amino terminal 23 residues constitute a typical hydrophobic signal sequence which when proteolytically removed, leaves a mature molecule of 143 amino acids, with a predicted molecular mass of 17 kDa (Figure 24.1A/B). During biosynthesis the peptides are variably glycosylated at positions 25 and 97, producing mature proteins of 17, 20, and 25 kDa (Kelker et al., 1984). In mice, the transcript is translated and processed into a mature 134 amino acid polypeptide that can also be glycosylated at two N-linked sites to give rise to mature polypeptides with molecular masses of 15.4, 20, or 25 kDa. While glycosylation is not required for IFN-c functional activity, it influences the circulatory half-life of the molecule (Kelker et al., 1983; Rutenfranz and Kirchner, 1988; Cantell et al., 1986). In both humans and mice, the fully glycosylated IFN-c

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569

A

B

A D F’

D’

E’ B

C

E

F

C’

A’

B’

NH2

NH2 COOH

COOH

FIGURE 24.1 Structure of IFN-c. (A) Amino acid sequence alignment of the mature forms of human (Swissprot P01579) and mouse IFN-c (pir IVMSG) using Allscript. The primary sequences are 36% conserved and identical residues are boxed. Secondary structure elements are shown above the sequences and every 10th residue is indicated. (B) Ribbon diagram of human IFN-c (in which the five carboxy-terminal residues are deleted) in the receptor unbound state showing the interdigitating helices of the two monomers. This structure was generated using Ribbons with data from PDB 1HIG. polypeptide is the predominant form. Mature IFN-c proteins in humans and mice share only 36% identity at the amino acid level. This primary amino acid sequence diversity is the basis for the strict species specificity that human and murine IFN-c display in binding to and inducing responses in human and murine cells, respectively. Under physiologic conditions, two IFN-c polypeptides self-associate to form a noncovalent homod-

imer. The IFN-c homodimer is a labile molecule that can be denatured by extremes of temperature (65 C) or pH (pH 4 or 9) (Chang et al., 1984). The dimeric nature of this cytokine was confirmed by the crystal structures of human, bovine and rabbit IFN-c (Figure 24.1B) (Ealick et al., 1991; Samudzi et al., 1991; Samudzi and Rubin, 1993). These structures indicate that IFN-c is primarily helical, with each monomer consisting of 6 a-helices (A–F) connected

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INTERFERON - c

by short linkers. The dimer is formed by the sharing of a-helices between the monomers. The two carboxy-terminal helices (E and F) from one chain associate with helices A, B, C and D from the paired chain. The interdigitating helices of the IFN-c homodimer result in extensive interactions between the two chains. The crystal structure of human IFN-c bound to the extracellular domain of its receptor has also been solved (Walter et al., 1995). When bound to its receptor, the IFN-c homodimer is a V-shaped structure that exhibits a two-fold axis of symmetry perpendicular to the plasma membrane between the two globular domains. This topology is also seen in the crystal structure of the related cytokine, IL-10 (Walter and Nagabhushan, 1995; Zdanov et al., 1995). Importantly, the two identical receptor binding sites of the IFN-c homodimer are required for the induction of a full IFN-c-dependent biologic response. Binding studies using IFN-c mutants and synthetic peptides have revealed critical roles for His 111, amino acids in the linker connecting a-helices A and B (the AB loop), and residues in the C terminus in mediating the binding between human IFN-c and its receptor (Lundell et al., 1991, 1994; Lunn et al., 1992; Griggs et al., 1992, Jarpe and Johnson, 1993). These results correlate well with the exposed residues on the human IFN-c homodimer that localize to the binding interface in the three-dimentional structure (Walter et al., 1995). Based on this crystal structure, 11 hydrogen bonds and salt bridges were identified in the interface. Additional residues in the interface that are likely to contribute to the interaction include Val 5, Ile 114 and Ala 118 (Walter et al., 1995).

Interferon-c biosynthesis Natural killer (NK) cells and defined subsets of T cells are the major cellular sources of IFN-c (Farrar and Schreiber, 1993; Boehm et al., 1997). These lymphocytes can be activated by both cell contact-dependent and -independent mechanisms. Recent data show that myeloid lineage antigen presenting cells (APCs) can also secrete IFN-c but the biologic function of this secretion has not yet been established (Frucht et al., 2001). NK cells are an important source of IFN-c especially during the innate phase of a developing immune

response. Unlike T and B lymphocytes, NK cells do not employ classical antigen receptors. Instead, they employ both activating and inhibitory receptors (such as NKG2D and Ly-49 or KIRs) to recognize their targets (Wang and Yokoyama, 1998; Smith et al., 2001). NK cell proliferation and effector functions are influenced by the delicate balance of signals transmitted through the immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosinebased inhibition motifs (ITIMs) that are present in the intracellular domains of the receptor subunits. Many inhibitory receptors bind to MHC class I and provide protection to cells that express normal levels of ‘self’ class I protein. Increased stimulation through activating receptors, or decreased signaling through inhibitory receptors due to the absence of MHC class I (‘missing self’), results in expression of NK cell effector functions, including the production of IFN-c. Cytokines provide a second mechanism by which NK cells can be activated to secrete IFN-c. This observation was initially made through the analysis of severe combined immunodeficiency (SCID) mice infected with the Gram-positive bacteria Listeria monocytogenes (Bancroft et al., 1991; Tripp et al., 1993). Upon interaction with bacterial products, tissue macrophages produce low levels of TNF-a and IL-12. These cytokines stimulate NK cells to secrete low amounts of IFN-c. NK cell-derived IFN-c then stimulates macrophages to increase the production of TNF-a and IL-12, which in turn leads to more IFN-c production by NK cells. This reciprocal stimulation forms a positive amplification loop that results in the rapid generation of substantial quantities of IFN-c and a large number of activated macrophages. The activated macrophages express potent nonspecific anti-microbial activity and function to contain the infection until an adaptive response is established. Furthermore, NK cell derived IFN-c helps to establish an appropriate cytokine milieu that favors the eventual generation of protective cell mediated immune responses. CD8 T cells and the TH1 subset of CD4 helper T cells are important cellular sources of IFN-c during the adaptive phase of immune responses. Two independent and pharmacologically distinct pathways for IFN-c production have been identified in studies using CD4 T cells: a T cell receptor (TCR) mediated antigen-dependent pathway which

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THE PROTEINS AND GENES OF THE INTERFERON - c SYSTEM

is cyclosporine-sensitive and a cytokine-induced, cyclosporine-insensitive pathway (Yang et al., 1999). Experimentally, IFN-c production can also be induced by stimuli that mimic activation through the TCR, including cross-linking antibodies, mitogens (such as concanavalin A or phytohemaglutinin), or pharmacologic agents (such as the combination of phorbol myristate acetate and calcium ionophore). In resting T cells, the IFN-c gene is not expressed and protein cannot be detected. However, after T cell activation, IFN-c message can be detected within 6 to 8 h, reaches maximum levels by 12 to 24 h, and then subsequently declines to baseline values (Farrar and Schreiber, 1993). IFN-c proteins are secreted immediately after synthesis and reach maximal extracellular levels 18 to 24 h after T cell stimulation. Previously differentiated TH1 and cytotoxic CD8 T lymphocytes 1 (Tc1 cells) also secrete robust quantities of IFN-c in response to stimulation with the combination of IL-12 and IL-18 (also called IFN-c-inducing factor, or IGIF) (Okamura et al., 1995a, 1995b) without the requirement for further stimulation through the TCR (Robinson et al., 1997; Yang et al., 1999; Carter and Murphy, 1999). IFN-c production in response to cytokine stimulation is prolonged in duration compared with that induced by TCR stimulation (Yang et al., 2001). IL-12 and IL-18 synergize to induce IFN-c production through mechanisms involving the transcription factors Stat4 and NF-jB. Activation of these transcription factors is not sufficient to induce IFN-c transcription in T cells; the synthesis of additional proteins including GADD45b is required (Yang et al., 1999, 2001). Synergistic induction of IFN-c production is also observed following stimulation through the TCR and the IL-12R (Zhang et al., 1999a). IL-23, an IL-12-like protein that shares the p40 subunit and signals through Stat4, can also induce IFN-c production, although to a lesser extent than does IL-12 (Frucht, 2002). It has long been accepted that the combination of IL-12 and IL-18 induces IFN-c production not only by CD4 and CD8 T cells, but also by cdT cells, NKT cells and NK cells (Farrar and Schreiber, 1993, Carnaud et al., 1999). More recent studies have reported that professional antigen presenting cells including B cells, macrophages, and both CD8 and CD8 dendritic cells can also secrete IFN-c in response to IL-12 and IL-18 (reviewed in Frucht et al.,

571

2001). As in T cells, IL-12-mediated signaling and the induction of IFN-c require the activation of Stat4, which is expressed in activated, but not resting myeloid cells (Frucht et al., 2000; Fukao et al., 2001). However, the physiologic function and relevance of IFN-c production by the latter cells remains undefined. IFN-c production is inhibited by IL-10 and TGF-b. IL-10 functions by preventing macrophage production of TNF-a and IL-12 by a poorly defined mechanism involving the transcription factor Stat3 (Riley et al., 1999; Takeda et al., 1999).

Interferon-c receptor IFN-c interacts with a distinct high affinity (Ka  1010–1011 M1) receptor comprised of two subunits that is expressed on the surface of nearly all cells (Figure 24.2) (Bach et al., 1997; Pestka et al., 1997). Ligand binding and therefore the ensuing biological responses, occurs in a strictly species-specific manner. This specificity requires that the ligand and the two polypeptide subunits of the receptor are all species-matched. The IFN-c receptor chains are both type I transmembrane proteins of the class 2 cytokine receptor family, which is characterized by the presence of tandem fibronectin type III (FBN-III) domains in the extracellular region. The larger subunit, IFNGR1 (IFN-c receptor-a chain, IFN-c-R1, or CDw119), is responsible for ligand binding, as well as ligand trafficking through the cell and is necessary but not sufficient for signal transduction. The smaller subunit, IFNGR2 (IFN-c receptor-b chain, IFN-c-R2, or accessory factor-1), is required for IFN-c signaling, but plays only a minor role in ligand binding. IFNGR1 is encoded by a 30 kb gene on human chromosome 6 and a 22 kb gene on murine chromosome 10 (Table 24.1) (Mariano et al., 1987; Pfizenmaier et al., 1988). Both genes are organized similarly and consist of seven exons. Exons 1–5 of the human gene encode the extracellular domain, exon 6 encodes the transmembrane domain and a portion of the membrane-proximal region of the intracellular domain, and exon 7 encodes the bulk of the intracellular domain. Transcription of the gene in each species produces a 2.3 kb mRNA. Following the proteolytic cleavage of the signal peptide (17 amino acids in humans and 26 amino acids in mice), the mature

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FIGURE 24.2 Polypeptide chain structure of the human IFN-c receptor. The IFN-c receptor consists of two species-matched polypeptides. IFNGR1 is required for ligand binding and signaling. IFNGR2 is required primarily for signaling and plays only a minor role in ligand binding. The intracellular domain of IFNGR1 contains three functionally important sequences: (1) a LPKS sequence required for IFNGR1 association with Jak1; (2) a LI sequence involved in effecting receptor-mediated ligand internalization/degradation; (3) a YDKPH sequence that, when phosphorylated, forms the docking site for latent Stat1. The intracellular domain of IFNGR2 contains a functionally important PPSIPLQIEEYL sequence required for Jak2 association.

human and murine proteins consist of 472 and 451 amino acids, respectively, and are arranged similarly in the cell membrane. Each is symmetrically oriented around a single 23 amino acid transmembrane domain. In both species, the extracellular domains consists of 228 amino acids including 10 cysteines and five N-linked glycosylation sites (Farrar and Schreiber, 1993). The core structure of the ligandbound IFNGR1 extracellular domain is a rod-like molecule that is folded into two domains, denoted D1 (membrane distal) and D2 (membrane proximal) (Walter et al., 1995). These two domains are separated

by an 11 amino acid linker and are oriented at an angle of 120 relative to one another. The intracellular domains of the human and murine IFNGR1 are 221 and 200 amino acids, respectively. Both are particularly rich in serine and threonine residues, and contain five (mouse) or six (human) tyrosine residues (Farrar and Schreiber, 1993). Notably, IFNGR1 lacks an intrinsic tyrosine kinase domain. Despite their structural similarities, the overall sequence identity between human and murine IFNGR1 is only 52% (50% identity between the extracellular domains and 55% identity between the intracellular domains). Due to cell specific differences in glycosylation, the molecular weights of mature human and murine IFNGR1 are approximately 90 kDa (range  80–95 kDa). Human and murine IFNGR2 subunits are also structurally similar at both the gene and protein levels. The human gene for IFNGR2 spans 33 kb on chromosome 21, while the mouse gene spans 17 kb on chromosome 16 (Rhee et al., 1996; Bach et al., 1997). In each species, the gene contains 7 exons. In humans, exon 1 encodes the signal sequence and exons 2–6 encode the extracellular domain. Exon 6 also encodes the transmembrane domain and part of the intracellular domain, while exon 7 encodes the remainder of the intracellular domain. Transcription of the gene produces messages of approximately 1.8 kb and 2 kb in human and murine cells, respectively (Bach et al., 1997). The human IFNGR2 protein contains a 21 amino acid signal sequence, a 226 amino acid extracellular domain, a single 24 amino acid transmembrane domain, and a relatively short intracellular domain of only 66 amino acids (Soh et al., 1994). Similarly, the murine IFNGR2 polypeptide consists of an 18 amino acid signal sequence, a 224 amino acid extracellular domain, a 24 amino acid transmembrane domain, and a 66 amino acid intracellular domain (Hemmi et al., 1994). Like IFNGR1, IFNGR2 also lacks intrinsic kinase activity. The overall sequence identity between human and murine IFNGR2 is 58%, although this value increases to 73% when just the cytoplasmic domains are compared. IFNGR2 has five (human) or six (murine) potential N-linked glycosylation sites, the modifications of which account for the observed molecular weights of approximately 62 kDa (range  60–67 kDa) for the mature proteins. The two IFN-c receptor subunits are, in general, coexpressed on the surfaces of all cells, except erythro-

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SIGNAL TRANSDUCTION THROUGH THE IFN c RECEPTOR

TABLE 24.1 Properties of the IFNGR1 and IFNGR2 subunits IFNGR1

IFNGR2

Property

Human

Murine

Human

Murine

Chromosomal localization Gene size (kb) Exons Primary sequence Signal peptide Mature form Intracellular conserved tyrosines Homology Domain structure Extracellular Transmembrane Intracellular Potential N-linked glycosylation sites Predicted Mr (kDa) Mr (kDa)

6 30 7

10 22 7

21 33 7

16 17 7

17 aa 472 aa

26 aa 451 aa

21 aa 316 aa

18 aa 314 aa

5

3

52%

58%

228 aa 23 aa 221 aa 5

228 aa 23 aa 200 aa 5

226 aa 24 aa 66 aa 5

224 aa 24 aa 66 aa 6

52.5 ~90

49.8 ~90

34.8 ~62

35.6 ~62

The cloning of human IFNGR1 is described by Aguet et al. (1988), murine IFNGR1 by Gray et al. (1989), Hemmi et al. (1989), Kumar et al. (1989), Munro and Maniatis (1989), human IFNGR2 by Soh et al. (1994) and murine IFNGR2 by Hemmi et al. (1994).

cytes. IFNGR1 is constitutively expressed at moderate levels on most cells (200–25 000 sites per cell). IFNGR2 is also constitutively expressed on cells but at very low levels (Bach et al., 1997).

SIGNAL TRANSDUCTION THROUGH THE IFNc RECEPTOR Janus protein tyrosine kinases and signal transducers and activators of transcription proteins Studies performed during the 1990s, that were directed toward elucidating the mechanism by which the IFNs transmit signals from the cell surface to the nucleus, revealedapreviouslyunrecognizedsignaltransduction pathway. Concordant results from a genetic approach to identify genes required in IFN response pathways and a separate biochemical and gene cloning approach to identify transcription factors that are activated by the IFNs, led to the identification of two novel groups of molecules(reviewedin Darnelletal.,1994)and(O’Shea et al., 2002). The first group, the Janus kinases or Jaks,

is a family of non-receptor protein tyrosine kinases. The second is a family of proteins that were found to serve dual roles as signal transducers and activators of transcription, and therefore are referred to by the acronym STATs. The observation that stimulation of the IFN-c receptor leads to the formation of a docking site for Stat1 bridged the understanding of events that occur outside the cell membrane to gene regulation events in the nucleus (Greenlund et al., 1994). Subsequent work performed in many laboratories has demonstrated that the Jak-Stat pathway of signal transduction is employed by many cytokine and growth factor receptors, and provides a rapid and direct signaling pathway from the cell membrane to the nucleus, without the requirement for second messenger molecules (Ihle, 1995; Leonard and O’Shea, 1998). The Janus kinase family consists of four proteins: Jak1, -2, -3 and Tyk2. Although there is no detailed structural information regarding the Jaks, these kinases share several unique characteristics. Seven Janus homology (JH) domains have been assigned based on sequence similarities within the family. The most carboxy-terminal domain (JH1), which contains all the consensus sequences associated with tyrosine kinases, has documented tyrosine kinase activity

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(Wilks et al., 1991). Adjacent to the catalytic domain is a pseudokinase (JH2) domain that lacks kinase activity. The precise function of this kinase-like domain is unclear, although it has been implicated in regulating the catalytic domain and in mediating interactions with substrate molecules (Luo et al., 1997; Fujitani et al., 1997; Chen et al., 2000; Saharinen et al., 2000; Yeh et al., 2000). The JH3 to JH7 domains in the amino terminus do not appear to represent independent functional domains. The tenuous similarity noted between the primary amino acid sequence of SH2 domains and a portion of the JH3 and JH4 domains (Harpur et al., 1992) has been extended by more recent analyses that combined sequence alignments with predicted structural alignments to model this region of the Jaks as an SH2 domain, capable (with the exception of Tyk2) of binding phosphotyrosine residues (Bork and Gibson, 1996; Kampa and Burnside, 2000; Al-Lazikani et al., 2001). Mutation in murine Jak2 of the invariant bB5 arginine of the SH2 domain, however, had no effect on the ability of IFN-c to activate Stat1 (Kohlhuber et al., 1997). Recent computational studies of the amino termini (JH4 to JH7) of the Jaks indicated that they are divergent members of the band four point one, ezrin, radixin, and moesin (FERM) homology domain family (Girault et al., 1999). This region of the Jaks plays a critical role in the specific and constitutive association of these kinases with cytokine receptor subunits (Frank et al., 1995; Chen et al.; Gauzzi et al., 1997; Richter et al., 1998; Cacalano et al., 1999) and has recently been shown to play a role in maintaining the integrity of the kinase domain (Zhu et al., 2001). In the case of the IFN-a/b receptor 1 subunit (IFNAR1), the erythropoietin receptor, and the oncostatin M receptor, this association is required for the Jaks to chaperone the receptor subunits to the cell surface, as well as to stabilize their expression once in the cell membrane (Gauzzi et al., 1997; Huang et al., 2001; Radtke et al., 2002). Following the interaction of cytokine receptors with their cognate ligands, the Jaks serve critical roles in phosphorylating the receptor and Stat molecules. The Stat family consists of seven distinct polypeptides: Stat1, Stat2, Stat3, Stat4, Stat5A, Stat5B and Stat6 (Schindler and Darnell, 1995; O’Shea, 1997). These proteins contain 750 to 850 residues that, beginning at the amino terminus, comprise the following domains: (1) an amino-terminal domain that is responsible for

Stat dimer–dimer interactions; (2) a coiled-coil domain that mediates additional protein interactions; (3) a DNA binding domain; (4) a linker domain that has a role in mediating transcription; (5) an SH2 domain; (6) a sequence containing a critical tyrosine that acts as an SH2 domain-ligand; and (7) a transcriptional activation domain that interacts with additional nuclear factors and, in some Stats, includes a site for serine phosphorylation (Horvath, 2000). The presence of the SH2 domain in the Stats makes them unique among transcription factors and is the key to their recruitment and activation. Sequence variations within these SH2 domains form the basis for the selective recruitment of distinct Stats to different tyrosine phosphorylated cytokine receptors, and allow for the subsequent specific pairing of two tyrosine-phosphorylated Stat proteins that form the functionally active transcription factor complex. The specificity of the interaction between cytokine receptors and Stat proteins is highlighted in studies of mice and cells that lack a particular Stat protein, such as Stat1 (Meraz et al., 1996; Durbin et al., 1996). Crystallographic analysis revealed that a phosphorylated Stat1 dimer forms a C-shaped clamp around DNA, with the SH2 domains forming the hinge (Chen et al., 1998). The central portion of the dimer forms an immunoglobulin fold and is responsible for DNA binding, although very few amino acids make direct contact with the DNA. Stat1 homodimers preferentially bind to a 9-nucleotide DNA motif termed gamma-interferon activated sites, or GAS elements, that contain the consensus sequence TTNCNNNAA (Darnell et al., 1994).

IFN-c receptor signaling via the Jak-Stat pathway Detailed structure/function analyses of the IFN-c receptor, along with analyses of the events that occur following the stimulation of this receptor, have facilitated the construction of a model of ligand-induced receptor activation that has become a paradigm for Jak-Stat signal transduction. In unstimulated cells, the IFN-c receptor subunits associate constitutively via their intracellular domains with inactive forms of Jak1 and Jak2 (Kotenko et al., 1995; Sakatsume et al., 1995; Bach et al., 1996; Kaplan et al., 1996a). Jak1 binding to IFNGR1 requires the four amino acid sequence, LPKS269, in the membrane proximal region of the 266

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IFNγ

IFNGR1 Jak1

Jak2

Jak2

Jak1

YY

Assembly of Active Receptor Complex

IFNGR2

IFNGR2

IFNγ IFNGR1

SHP-1, SHP-2

IFNγ

Active Jak1 + Jak2

Active Jak1 + Jak2

JAK Activation and Stat1 Docking Site Formation

SOCS

YY P P

IFNGR2 DownRegulation

IFNγ

Active Jak1 + Jak2

Stat1 Recruitment, Activation and Homodimer Formation

Active Jak1 + Jak2 P

Y

P

Active Stat1 P P Homodimer

Stat1

Y

Stat1

intracellular domain of IFNGR1, while Jak2 associates with the 12-amino-acid sequence, 263PPSIPLQIEEYL274, in the intracellular domain of IFNGR2 (numbers represent amino acid positions in the mature human proteins) (Figure 24.2). Signal transduction is initiated when a homodimeric IFN-c molecule binds to the IFN-c receptor and induces the activation of Jaks by mechanisms involving auto- and trans-phosphorylation (Figure 24.3). The activated kinases then phosphorylate a key tyrosine residue within the YDKPH444 sequence near the carboxy terminus of 440 the IFNGR1 chain, thereby forming the phosphorylated sequence on the receptor that is specifically recognized by the SH2 domain of Stat1 (Greenlund et al., 1994, 1995). Two Stat1 molecules then bind to the paired docking sites on the activated receptor complex and are themselves phosphorylated on a specific tyrosine residue (Y701) near their C terminus by the receptorassociated, activated tyrosine kinases (Schindler et al., 1992; Shuai et al., 1993). The two phosphorylated Stat1 proteins then dissociate from the IFNGR1 subunits and form a dimer through reciprocal phosphotyrosineSH2 domain interactions. During the activation process, Stat1 molecules are also phosphorylated on a specific serine residue (S727) through a mechanism that requires a kinase(s) distinct from the Janus tyrosine kinases. Activated Stat1 homodimers associate with the nuclear import receptor NPI-1 (Importin-a) through a novel conformational arginine/lysine-rich nuclear localization signal generated from residues contributed by each of the two Stat1 DNA binding domains of the dimer (Melen et al., 2001; Meyer et al., 2002). Together with Importin b p97, which associates via NPI-1, Stat1 translocates across the nuclear pore complex through an active process that requires the hydrolysis of GTP by Ran (Sekimoto et al., 1996; Meyer et al., 2002). Once inside the nucleus, activated Stat1 homodimers, also called gamma-activated factor (GAF), bind GAS elements and effect the transcription of IFN-cinduced genes (Darnell et al., 1994; Bach et al., 1997). The ability of Stat1 to bind DNA and alter gene expression is influenced by additional nuclear factors. Interaction of N-myc interactor (Nmi) with the Stat1 coiled-coil region for example, potentiates transcription following stimulation with IFN-c (Zhu et al., 1999) by recruiting additional coactivators such as the histone acetyl transferases and cAMP-response-element-

PIAS

NUCLEUS P

UbiquitinProteasome Degradation

S Active Stat1 P P Homodimer S P

Stat1 Serine Phosphorylation, Nuclear Translocation and Initiation of Gene Transcription

FIGURE 24.3 Defined IFNGR1 and IFNGR2 mutations in patients with Mendelian susceptibility to mycobaterial disease. Receptor chains are drawn to scale to show the relative size of the exons and position of each mutation. Mutations are grouped vertically by disorder. The signal sequence and transmembrane domains are shaded and stippled, respectively. Nucleotides are numbered beginning at ‘1’ of the mature protein. Functionally important sequences are indicated.

binding protein (CREB)-binding protein (CBP)/p300 (Zhu et al., 1999). CBP/p300 alone contacts Stat1 at both the N-terminus, as well as the C-terminus (Zhang et al., 1996). An interaction between Stat1 and the transcriptional activator specificity protein 1 (Sp1) has also been observed and was shown to lead to the synergistic activation of intracellular adhesion molecule-1 (ICAM-1) gene transcription (Look et al., 1995). Maximal transcriptional activity requires the phosphorylation of S727 within the C-terminal transcriptional activation domain of Stat1a (Wen et al., 1995). Serine phosphorylation at this position was found to increase the binding affinity of Stat1 for a number of nuclear proteins which may represent a complex of

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transcriptional coactivators for Stat1 (Zhang et al., 1998). Mini-chromosome maintenance 5 (MCM5), which is involved in the initiation of DNA replication, was among the proteins in this complex. Transient overexpression of MCM5 was found to enhance Stat1dependent reporter gene activity following IFN-c treatment (Zhang et al., 1998). Another study identified the breast and ovarian cancer susceptibility marker (BRCA1) as a transcriptional coactivator of serine phosphorylated Stat1 (Ouchi et al., 2000). The BRCA1 tumor suppressor was found to cooperate with Stat1 in selectively activating the cyclin dependent kinase inhibitor p21WAF, but not interferon regulatory factor-1 (IRF-1). The extent to which phosphorylation of S727 promotes transcription varies with the target promoter (Kovarik et al., 2001). The identity of the kinase that modifies Stat1 S727 in response to IFN-c remains unclear, and may vary among different cell types. This modification requires active Jak1 and Jak2 kinases (Zhu et al., 1997; Nguyen et al., 2001), and evidence suggests that tyrosine phosphorylation occurs more rapidly and positively influences serine phosphorylation (Kovarik et al., 1998). The sequence surrounding Stat1 S727 (PMSP) conforms to the mitogen-actived protein kinase (MAPK) consensus motif PXn(S/T)P (where S/T is serine or

threonine and n is 1 or 2). Independent studies have implicated a number of kinases, such as prolinerich tyrosine kinase 2 (pyk2) (Takaoka et al., 1999), p38 MAPK (Goh et al., 1999), double-stranded RNAactivated protein kinase (PKR) (Ramana et al., 2000), and PI3K/Akt (Nguyen et al., 2001) in effecting Stat1 serine phosphorylation. Whether some of these proteins represent components of a single pathway, and which kinase is directly responsible for phosphorylating Stat1 on serine 727, are currently areas of active investigation. Studies utilizing cells or mice with induced genetic deficiencies of different Jaks and Stats have demonstrated that most IFN-c-induced biologic responses require Jak1, Jak2, and Stat1 (Table 24.2) (Durbin et al., 1996; Meraz et al., 1996; Rodig et al., 1998; Parganas et al., 1998; Neubauer et al., 1998). These studies therefore establish the physiologic relevance of this IFN-c signaling model.

Regulation of IFN-c Jak-Stat signaling Role of phosphatases Several different mechanisms have been proposed to regulate IFN-c receptor signaling. Experiments

TABLE 24.2 Phenotypes of mice lacking components of the IFN-c signaling pathway Gene deleted

Knockout mouse phenotype

Reference (KO generation)

IFN-c

Viable and fertile, no gross abnormalities in young mice, increased susceptibility to selected viral and bacterial infection Viable and fertile, no gross abnormalities in young mice, increased susceptibility to selected viral and bacterial infection, as well as chemically and genetically induced tumor formation Viable and fertile, no gross abnormalities in young mice, increased susceptibility to selected viral and bacterial infection Viable but perinatal lethality due to neurologic deficits; SCID Embryonic lethality due to failure in erythropoiesis

Dalton et al., 1993

IFNGR1 IFNGR2 Jak1 Jak2 Stat1 SOCS-1 SOCS-3 PIAS1 PIASy

Viable and fertile, no gross abnormalities in young mice, increased susceptibility to selected viral and bacterial infection, as well as chemically induced tumor formation Viable but perinatal lethality due to severe IFN-c-dependent inflammatory disease, multiple hematopoietic abnormalities Embryonic lethality attributed to either marked erythrocytosis or placental defects, alteration of IFN-c signaling has not been reported Not reported Not reported THE CYTOKINES AND CHEMOKINES

Huang et al., 1993 Lu et al., 1998 Rodig et al., 1998 Parganas et al., 1998; Neubauer et al., 1998 Durbin et al., 1996 Meraz et al., 1996 Starr et al., 1998; Naka et al., 1998; Marine et al., 1999b Marine et al., 1999a; Roberts et al., 2001

SIGNAL TRANSDUCTION THROUGH THE IFN c RECEPTOR

demonstrating the transient phosphorylation of the IFN-c receptor, Jak, and Stat proteins inspired the straightforward hypothesis that IFN-c signaling is regulated rapidly and directly by specific protein tyrosine phosphatases that are constitutively expressed. Several groups have examined this idea, and some initial data have emerged. SHP-2, a ubiquitously expressed SH2 domain-containing protein tyrosine phosphatase, was reported to associate constitutively with IFNGR1 (You et al., 1999). IFN-c treatment of SHP-2/ fibroblasts led to enhanced tyrosine phosphorylation of both Jak1 and Stat1, resulting in decreased cell growth and enhanced cell death. Similarly, the related phosphatase, SHP-1, was found to negatively regulate Jak1 and Stat1 tyrosine phosphorylation in the IFNa/b signaling system (David et al., 1995). Surprisingly, in response to IFN-c, SHP-1 appears to promote Jak-Stat signaling (You and Zhao, 1997). Overexpression of the nonhematopoietic form of SHP-1 in human cervical carcinoma HeLa cells increased IFN-c-induced Stat1 activity by 20–30% as measured by DNA binding, while overexpression of an inactive form of SHP-1 reduced Stat1 activity by 30–50%. The observation that activated Stat1 homodimers in the nucleus recycle back into the cytoplasm as non-phosphorylated molecules implicates a nuclear protein tyrosine phosphatase(s) in the negative regulation of IFN-c signaling (Haspel et al., 1996; Haspel and Darnell, 1999). To date, however, the identity of the Stat1 phosphatase(s) has not been reported.

Role of SOCS family members Members of the suppressors of cytokine signaling or SOCS family of inhibitory proteins (also known as Stat-induced Stat inhibitors (SSI) or Jak-binding (JAB) proteins) provide a classical negative feedback mechanism to regulate cytokine signaling (Starr et al., 1997; Endo et al., 1997; Naka et al., 1997). Unlike the phosphatases described above, the SOCS proteins are usually undetectable in resting cells, but their transcription can be rapidly induced by Stats activated in response to a broad spectrum of cytokines (reviewed in Yasukawa et al., 2000 and Greenhalgh and Hilton, 2001). In turn, the SOCS proteins act to quench Jak-Stat signaling, often blocking the pathway that initially induced their production. Eight SOCS

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proteins have been identified: SOCS-1-7 and CIS. Structurally, these proteins all contain a central SH2 domain and a 40-amino-acid region at the C terminus called the SOCS box. The N-terminal region of the SOCS proteins is variable in length and sequence. SOCS-1 and SOCS-3 share an additional related sequence of 12 amino acids termed the kinase inhibitory region (KIR) located upstream of the SH2 domains (Nicholson et al., 1999; Yasukawa et al., 1999; Yasukawa et al., 2000). SOCS-1, SOCS-2 and SOCS-3 are well characterized. The mechanisms by which these proteins regulate cytokine signaling include inactivation of the Jaks and ubiquitination of signaling proteins, resulting in their degradation by the proteasome. CIS itself is ubiquitinated (Verdier et al., 1998) and thus may also function to target bound signaling proteins to the proteasome, although a role for CIS in directly competing with Stat5 for binding to the erythropoietin receptor has also been suggested (Matsumoto et al., 1997). Currently, little is known about the function or the mechanism of action of SOCS-4, SOCS-5, SOCS-6 or SOCS-7. Multiple SOCS proteins can be induced by IFN-c and two have been implicated in down-regulating signals from the IFN-c receptor in studies performed in vitro. While SOCS-1, SOCS-2 and SOCS-3 messages were all up-regulated in bone marrow cells following stimulation for 1 h with IFN-c (Starr et al., 1997), this pattern is not generalizable to all cell types. Rather, the subset of SOCS proteins induced by IFN-c varies with the tissue or cell line being examined. Once produced, these SOCS proteins can cross-regulate other cytokine signaling pathways. For example, the antagonistic effects of IFN-c on IL-4-mediated responses, including the inhibition of IL-4 mediated IgE production, may be in part due to IFN-c-induced expression of SOCS-1 (Venkataraman et al., 1999). To date, only SOCS-1 and SOCS-3 have been implicated in the negative regulation of IFN-c signal transduction (Song and Shuai, 1998; Stoiber et al., 1999; Fujimoto et al., 2000). A physiological role for SOCS-1 but not for SOCS-3 in negatively regulating IFN-c signaling is suggested by the phenotypes of mice rendered deficient for these proteins. SOCS-1/ mice appear normal at birth, but exhibit stunted growth and die between 2 and 3 weeks of age with a syndrome characterized by fatty degeneration and necrosis of the liver, macrophage infiltration of major organs, and multi-

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ple hematopoietic abnormalities including severe lymphopenia (Starr et al., 1998; Naka et al., 1998; Marine et al., 1999b; Metcalf et al., 1999). The phenotype in the liver is similar to that observed in neonatal mice in which the levels of circulating IFN-c were experimentally elevated either by the expression of an IFN-c transgene or by injection of the purified IFN-c protein (Gresser et al., 1981; Toyonaga et al., 1994). Indeed, slightly elevated levels of IFN-c were detected in serum from SOCS1/ mice (Marine et al., 1999b). The significance of this finding is highlighted by the further observations that the perinatal lethality and much of the abnormal pathology associated with SOCS-1 deficiency is averted by injecting these mice with neutralizing antibodies against IFN-c, or by crossing them with either IFN-c/ or Stat1/ mice (Alexander et al., 1999; Marine et al., 1999b; Naka et al., 2001). Deficiency in RAG2 also rescued the phenotype of SOCS-1/ mice, demonstrating that lymphocytes are required for the perinatal lethality, likely due to the role T lymphocytes play in secreting IFN-c. The slightly elevated level of IFN-c in the serum, however, is not sufficient on its own to account for the phenotype of SOCS-1/ mice because transgenic mice that produce comparable levels of IFN-c do not exhibit the same pathology (Young et al., 1997; Marine et al., 1999b). This difference can be explained by the hypersensitivity of SOCS-1-deficient mice and tissues to IFN-c. Uncontrolled IFN-c signaling in SOCS-1/ mice is evidenced by the constitutive activation of Stat1 and the increased expression of the IFN-cinducible genes IRF-1, iNOS and MHC class I in tissues from these mice. Functionally, SOCS1/ macrophages require 100-fold less IFN-c to clear infection with Leishmania major than do wild-type macrophages (Alexander et al., 1999). SOCS-1/ mice were found to exhibit an increased resistance to infection with Semliki forest virus (Alexander et al., 1999). A physiologic role for SOCS-3 in regulating IFN-cinduced signaling is not immediately apparent from the phenotype of SOCS-3-deficient mice. Two groups have found that deletion of SOCS-3 results in embryonic lethality at mid-gestation between 11–16 days (Marine et al., 1999a; Roberts et al., 2001). Further studies must be performed to explore the physiological role of SOCS-3 in regulating IFN-c signaling.

Role of Stat inhibitors In an effort to identify proteins that regulate Stat1, a partial cDNA clone encoding a polypeptide that interacts with Stat1 was identified in a yeast two hybrid screen (Liu et al., 1998a). Based on the ability of the full length protein to inhibit Stat1-mediated gene regulation, the newly identified molecule was termed protein inhibitor of activated stats, or PIAS1. By searching the EST database and screening a human library, four related clones were identified: PIAS3, PIASxa, PIASxb and PIASy (Chung et al., 1997; Liu et al., 1998a). The members of the PIAS family exhibit approximately 50% identity and contain highly conserved domains including a putative a-helical nuclear receptor LXXLL signature motif (a protein interaction module), a putative zinc-binding motif, a highly acidic region, and a serine/threonine rich domain (which is absent in PIASy) (Liu et al., 1998a; Shuai, 2000). PIAS1 and PIASy can bind activated Stat1 and have been implicated in regulating Stat1-mediated signaling responses induced by IFN-c. Immunofluoresence analysis demonstrated that both PIAS1 and PIASy are localized predominantly to the nucleus (Liu and Shuai, 2001; Liu et al., 2001). The binding of PIAS1 to Stat1 prevents the association of Stat1 with DNA (Liu et al., 1998b). Although the carboxy terminus of PIAS1 (amino acids 392–541) interacts directly with the amino terminus of Stat1 (amino acids 1–191), the amino-terminus of PIAS1 prevents interactions with monomeric, but not dimeric Stat1 (Liao et al., 2000). The specific recognition of the Stat1 dimer forms the molecular basis for the cytokine-dependent action of PIAS1. Further investigation into the nature of the Stat1/PIAS1 interaction revealed that methylation of arginine 31 (a conserved arginine among the Stats) in Stat1 prevents its association with PIAS1 (Mowen et al., 2001). This post-translational modification was shown to greatly increase the ability of Stat1 to bind DNA and regulate transcription in response to IFNa/b, raising the question as to whether stimulation with IFN-c also leads to the methylation of Stat1. Whereas PIAS1 interferes with the ability of Stat1 to bind DNA, PIASy functions as a transcriptional corepressor. Transcriptional repression is achieved without affecting the tyrosine or serine phosphorylation of Stat1, and requires the LXXLL protein interaction

THE CYTOKINES AND CHEMOKINES

INTERFERON - c REGULATED GENES

module of PIASy (Liu et al., 2001). When transfected into 293T cells, PIASy-AA, in which LXXLL is mutated to LXXAA, strongly enhanced Stat1-dependent gene activation (Liu et al., 2001). This dominant-negative effect of PIASy-AA may be due to its ability to compete with endogenous PIASy or other inhibitory molecules for binding to Stat1. Although PIAS1 and PIASy have overlapping roles, the observation that PIAS1 is enriched in the thymus where PIASy is barely detectable, while the reverse is true in the spleen, suggests that these inhibitors may be involved in the regulation of Stat1 in different tissues (Liu et al., 2001). Members of the PIAS family have been found to play a number of roles in addition to functioning directly as regulators of Stat-mediated gene transcription. Attempts to generate a stable cell line that overexpresses PIAS1 lead to the discovery that members of the PIAS family are proapoptotic (Liu and Shuai, 2001). The induction of apoptosis by PIAS1 was found to be independent of its ability to inhibit Stat1mediated gene activation, but requires the activation of JNK1. Recently, PIASy was shown to function as a small ubiquitin-related modifier (SUMO) E3 ligase, facilitating the addition of SUMO to lymphoid enhancer factor 1 (LEF1) (Sachdev et al., 2001). This post-translational modification served to regulate the subnuclear localization of LEF1. Future studies are necessary to address whether the PIAS proteins sumoylate Stats, and how the addition of SUMO might alter the function of Stats.

Role of ubiquitin–proteasome pathway Degradation of activated signaling molecules via the ubiquitin–proteasome pathway provides an additional mechanism by which cells down-regulate IFN-c-induced signaling. Pre-incubation of HeLa cells with the proteasome inhibitor MG132 was found to prevent the disappearance of phosphorylated Stat1 and prolong Stat1 DNA binding activity following IFN-c stimulation (Kim and Maniatis, 1996). Furthermore, multi-ubiquitinated, phosphorylated Stat1 was detected in vivo, implying that some time after activation, Stat1 is targeted for degradation by the proteasome. Results from a separate study support a role for proteolysis in deactivating the Jak-Stat pathway, but suggest that MG132 acts upstream of activated nuclear Stat1 by sustaining signal transduction and

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the de novo generation of Stat1 homodimers (Haspel et al., 1996). In this study, the authors concluded that internalization of the receptor and the subsequent proteolysis of the receptor and/or the ligand stops IFN-c signaling. Interestingly, a third group reported that the SOCS box of SOCS family proteins mediates interactions with elongins B and C, which may target SOCS-bound proteins (i.e. Jaks or cytokine receptor chains) to the proteasomal degradation pathway (Zhang et al., 1999b). In support of this finding, mice with a deletion of the SOCS box of the SOCS1 gene exhibited a prolonged onset of inflammatory disease and an increased responsiveness to IFN-c, consistent with a partial loss-of-function of SOCS-1 in negatively regulating IFN-c-induced signaling (Zhang et al., 2001).

Expression and regulation of IFN-c receptor subunits In some circumstances, cellular responses to IFN-c can also be regulated by the expression of IFN-c receptor subunits. When cultured under conditions that promote the development of IFN-c-producing CD4 TH1 cells (i.e. in the presence of antigen, APC, IL-12 and anti-IL-4) for as few as 5 days, naive murine splenic T cells lose their ability to respond to IFN-c (Pernis et al., 1995; Bach et al., 1996). This deficiency is the result of down-regulated expression of IFNGR2 mRNA and protein, a response that is likely to be controlled at the level of transcription. Two lines of evidence show that this process is not intrinsic to TH1 differentiation, but rather is a response to IFN-c itself: (1) IFNGR2 expression was retained on murine TH1 cells derived from IFNGR1-deficient mice; and (2) the addition of IFN-c to cultures of murine TH2 cells resulted in the down-regulation of IFNGR2 similar to that seen in TH1 cells (Bach et al., 1996). In long-term TH1 clones, IFNGR2 expression is permanently suppressed (Pernis et al., 1995). The molecular basis of the mechanism underlying this desensitization remains poorly understood. This process is not generalizable to T cells of other species or to other cell types. For example, human TH1 and TH2 clones cultured in the presence of IFN-c retain expression of IFNGR2 and respond to IFN-c (Novelli et al., 1997).

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Alternative IFN-c receptor signaling pathways Analysis of mice that lack an intact Stat1 gene has demonstrated that Stat1 plays an essential role in promoting many of the classical IFN-dependent responses (Meraz et al., 1996; Durbin et al., 1996). However, it has recently become clear that the JakStat pathway of signal transduction is not sufficient on its own to account for the full range of biological events that ensue following the binding of IFN-c to its receptor. Rather, the Jak-Stat1 model will likely serve as the scaffolding upon which other signaling pathways may be intertwined. For example, the growth-inhibitory effects of IFN-c may be mediated in part by Ras GTPase-activating protein 1 (Rap1) which is activated by IFN-c through a pathway that involves c-Cbl, CrkL, and the guanine exchange factor C3G (Alsayed et al., 2000). Additional signaling proteins activated by IFN-c include CrkII (Platanias et al., 1999), the Vav pro-oncogene (English et al., 1997), the src-family kinase, Fyn (Uddin et al., 1997), and the Stat family proteins Stat3 (Stephens et al., 1998) and Stat5 (Meinke et al., 1996). Finally, as discussed above, a number of serine/threonine kinases are activated in response to IFN-c, and it remains possible that one or more of these enzymes is involved in pathways distinct from the serine phosphorylation of Stat1. The strongest evidence in support of the functional relevance of alternative signaling pathways comes from recent findings that IFN-c can mediate select biological responses in the absence of Stat1. One study demonstrated that Stat1-deficient mice, while more sensitive to murine cytomegalovirus (MCMV) and Sindbis virus than wild-type mice, are more resistant to these viruses than mice lacking both the IFN-c and IFN-a/b receptors (Gil et al., 2001). This finding reveals an IFN-dependent, Stat1-independent signaling pathway that plays a physiologically relevant role in mediating protective host responses against at least two viral pathogens. In the absence of Stat1, IFN-c also takes on the characteristics of a growth factor in some cells. For example, IFN-c enhanced the proliferation of primary murine Stat1-deficient bone marrow cells by almost three-fold when co-cultured with the macrophage growth factor M-CSF (Gil et al., 2001). Under the same

conditions, IFN-c had a profound anti-proliferative effect on wild-type cells. Likewise, growth of a human transformed/immortalized Stat1-deficient fibrosarcoma cell line (U3A) was regulated by IFN-c (Ramana et al., 2000). The proliferative response to IFN-c was mediated in part by c-myc, which was induced in the absence of Stat1 in these fibroblasts.

INTERFERON-c REGULATED GENES Stat1-dependent The pleiotropic effects of IFN-c are mediated by complex patterns of cell type-specific gene regulation. The rapid signaling of the Jak-STAT pathway makes it an ideal system to coordinate the activation of immediate–early genes that provide the host with a rapid mechanism to respond to infectious agents. In fact, IFN-c rapidly induces, within 15 to 30 min, a large number of genes without the requirement for new protein synthesis (Kerr and Stark, 1991; Lewin et al., 1991). These genes are induced by the binding of Stat-1 homodimers to GAS elements within their promoters. Examples of IFN-c-induced, Stat-1dependent immediate–early genes include IRF-1, human guanylate binding protein (GBP-1) (Briken et al., 1995), and type I Fcc receptor (FccR-1), all of which encode proteins that participate in inflammatory and immune responses (Boehm et al., 1997). Additional genes whose transcription is rapidly altered (within 1 h) by IFN-c in a Stat1-dependent manner include the chemokines MIG and IP-10 which are induced, and GRO/KC which is repressed (Gil et al., 2001). Several IFN-c-regulated intermediate genes have also been identified. Transcription of these genes requires additional protein synthesis, such as that of the transcription factor IRF-1, and is seen within 6 to 8 h of stimulation. Examples of intermediate genes include those that encode MHC class I and class II proteins, which are critical components of adaptive immune responses. Studies using Stat1-, Jak1-, or Jak2-deficient mice have shown that these proteins play essential roles in the physiologic response to IFN-c (Meraz et al., 1996; Durbin et al., 1996; Rodig et al., 1998; Parganas et al.,

THE CYTOKINES AND CHEMOKINES

BIOLOGIC ACTIVITIES OF IFN - c

1998; Neubauer et al., 1998). In total, the expression of several hundred genes is enhanced or suppressed by IFN-c (Boehm et al., 1997; Der et al., 1998).

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BIOLOGIC ACTIVITIES OF IFN-c Antiviral activities

Stat1-independent Representational difference analysis (RDA) and microarray analysis were employed to identify IFN-c-regulated genes in bone marrow-derived macrophages (BMM) and fibroblasts derived from Stat1-deficient mice (Gil et al., 2001; Ramana et al., 2001). Whereas IFN-c stimulation led predominantly to the induction of gene expression in WT BMMs, it resulted primarily in the suppression of gene expression in Stat1-deficient BMMs (Gil et al., 2001). The total number of genes regulated by IFN-c, however, was comparable in WT and Stat1-deficient macrophages (216 in WT and 150 in Stat1-deficient BMMs). These genes can be classified into two types based upon the involvement of Stat1 in regulating their expression. Type I genes are regulated similarly in WT and Stat1-deficient BMMs, and are therefore truly Stat1-independent. Type I genes that are induced in BMMs following stimulation with IFN-c for 1 h include monocyte chemotactic protein 1 (MCP-1), provirus insertion in murine leukemia 1 (PIM-1) and SOCS-3. CXC-chemokine receptor 4 (CXCR-4) expression was suppressed irrespective of Stat1 expression. Type II genes are regulated by IFN-c only in Stat1-deficient cells. Type II genes that are induced in BMMs include IL-1b and arginase. The tumor suppressor Rb and A-X actin are suppressed by IFN-c only in Stat1-deficient BMMs. Further examination of the regulation of a handful of these genes revealed that the IFN-c-dependent, Stat1-independent pathway requires the IFN-c receptor (the Stat1 docking site on IFNGR1 is not required) and Jak1, but not PKR. Future studies are required to link specific signal-transduction molecules and transcription factors to Stat1-independent gene regulation.

The molecular basis of the antiviral effects of IFNs has been extensively studied since the mid-1980s. IFN-c promotes antiviral responses that are either intrinsic to the infected cell itself, or extrinsic in that they affect recognition and destruction of infected cells by components of either the innate or adaptive limbs of the immune response. Intrinsic mechanisms of resistance to viral infections are mediated by IFN-induced proteins within the infected cell itself. Type I and type II IFNs regulate a large and partially overlapping set of genes which obfuscate the assignment of specific antiviral functions to a particular protein. By studying IFNinduced genes individually however, a handful of proteins have been identified that are produced in response to IFN-c, and have direct antiviral effects on the cell in which they were synthesized. These proteins include inducible nitric oxide synthase (iNOS), members of the 2,5-oligoadenylate synthetase family (OASs), PKR, GBP-1 and adenosine deaminase (ADAR1) (Patterson et al., 1995; Anderson et al., 1999; Biron and Sen, 2001). Among these IFN-c-induced antiviral proteins, the OASs, PKR and iNOS are the best characterized. OASs are encoded by three sets of genes. Each gene is induced by IFN-c to varying degrees in different cell types (Samuel, 2001) and encodes one of three types of OAS proteins: small, medium or large (Rebouillat and Hovanessian, 1999). Within each class, alternative splicing yields multiple polypeptides with unique carboxy termini (Benech et al., 1985; Ghosh et al., 1991). These proteins all have the ability to polymerize adenosine triphosphate into short 2-5-linked oligoadenylates (2-5A), but require dsRNA as a cofactor, thus ensuring that these enzymes are only active under conditions of infection. Once produced, 2-5A activates ribonuclease-L (RNase L), a constitutively expressed endoribonuclease, by causing its dimerization. Activated RNase L in turn degrades single-stranded RNA, including mRNA and rRNA, and thus inhibits protein synthesis. Recently, the small OAS isozyme 9-2 was found to promote apoptosis in mammalian cells (Ghosh et al., 2001). This proapop-

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totic activity of 9-2 is independent of its enzymatic activity and requires the Bcl-2 homology 3 (BH3) domain that is present in the carboxy-terminus of 9-2 but is not found in other OAS isozymes. PKR (known in earlier literature as double-stranded RNA-dependent kinase, P1 kinase, p68 kinase, or eukaryotic protein synthesis initiation factor-2 kinase) is a serine/threonine kinase with antiviral activity that is induced primarily by type I IFNs, but can also be induced by IFN-c (Thomis et al., 1992; Boehm et al., 1997). Similar to the OASs, PKR is normally latent, but is activated through autophosphorylation during viral infections primarily by binding to double-stranded RNA structures. PKR has a number of substrates including the a subunit of protein synthesis initiation factor 2 (eIF2a) (Samuel, 2001). The phosphorylation of eIF-2a at S51 inhibits translation by impairing the eIF-2B-catalyzed guanine nucleotide exchange reaction. PKR can also inhibit viral replication by promoting apoptosis of the host cell either directly through a pathway that involves the activation of the death substrate poly (ADP-ribose) polymerase (PARP) and can be blocked by Bcl-2 (Lee et al., 1997), or indirectly by inducing the expression of pro-apoptotic genes includingthedeath receptor, Fas (Balachandran et al., 1998). Extrinsic antiviral mechanisms induced by IFN-c are largely those that direct the development of the innate or specific limbs of the host immune response to recognize and destroy the infected cell. IFN-c promotes antigen processing and presentation and plays a key role in the induction of cellular and humoral immune antiviral responses. These actions will be discussed in more detail in later sections.

Antiproliferative activities IFN-c exerts an antiproliferative effect on a wide variety of cells, most often by inducing G1 arrest (Harvat and Jetten, 1996; Kominsky et al., 1998; Ruemmele et al., 1998; Supriya et al., 1998). IFN-cdependent inhibition of cellular proliferation has been observed in human fibrosarcomas and murine fibroblasts, but not in mutagenized human cells that lack Stat1 or in murine cells derived from Statdeficient mice (Chin et al., 1996; Bromberg et al., 1996; Gil et al., 2001). Enforced expression of wildtype Stat1, but not Stat1 S727A, restored the anti-

proliferative activity of IFN-c in these cells. This result suggests that Stat1 activation promotes growth arrest by regulating the expression of proteins that directly control cell cycle progression. Consistent with this idea, IFN-c induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21WAF1/CIP1 and p27Kip1. These inhibitors were found to associate with the cyclin-dependent kinases (CDK) CDK2 and CDK4 and interfere with their activation by CDK-activating kinase (CAK), thereby preventing the hyperphosphorylation of the retinoblastoma protein (Rb) and entry into the S phase of the cell cycle (Harvat and Jetten, 1996; Mandal et al., 1998).

Macrophage activation and innate immunity As the predominant physiologic macrophage activating factor (MAF), IFN-c plays a crucial role in promoting nonspecific host-defense mechanisms against a number of pathogens (Schreiber et al., 1983; Nathan et al., 1983; Buchmeier and Schreiber, 1985). In vitro and in vivo studies have demonstrated that macrophages activated by IFN-c have the capacity to nonspecifically kill a variety of intracellular and extracellular parasites, as well as neoplastic cells (Schreiber et al., 1985; Bancroft et al., 1989b; Liew et al., 1990a). Studies using several different in vivo murine infection models have demonstrated the physiologic significance of IFN-c’s role in macrophage activation and host defense against microbial pathogens. Mice pretreated with neutralizing antibodies to IFN-c lose the ability to resist a sublethal challenge with a variety of microbial pathogens such as Listeria monocytogenes (Buchmeier and Schreiber, 1985; Bancroft et al., 1989a), Toxoplasma gondii (Suzuki et al., 1988), or Leishmania major (Nacy et al., 1985). Furthermore, mice with targeted disruptions in the genes encoding IFN-c, IFNGR1, IFNGR2, or Stat1 die when challenged with sublethal doses of microbial pathogens such as Mycobacterium bovis, Listeria monocytogenes and Leishmania major (Dalton et al., 1993; Huang et al., 1993; Meraz et al., 1996; Lu et al., 1998). IFN-c-activated macrophages produce a variety of toxic substances, such as reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). These powerful oxidants bestow macrophages

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with cytostatic or cytotoxic activity against bacteria, viruses, fungi, protozoa, helminths and tumor cells (MacMicking et al., 1997). Professional phagocytes including monocytes, macrophages and neutrophils kill ingested pathogens in part by the production of reactive oxygen species derived from superoxide ions produced by an activated membrane-bound NADPH-dependent oxidase (reviewed in Babior, 1999). This multisubunit enzyme remains inactive in unstimulated cells due to the physical separation of its core phagocyte oxidase components, with p40PHOX, p47PHOX and p67PHOX residing as a complex in the cytosol and flavocytochrome b558, a heterodimer of p22PHOX and gp91PHOX localized in the membrane. Following exposure to the appropriate stimuli, p47PHOX becomes heavily serine phosphorylated and the entire cytosolic complex migrates to the outer membrane where it associates with cytochrome b558. Together with the guanine nucleotide-binding proteins Rac2 and Rap1A, this complex forms the active oxidase that catalyzes the reaction of oxygen with NADPH to form superoxide (O2). In the process of phagocytosis, part of the outer membrane becomes the phagocytic vesicle with the active oxidase oriented such that it injects toxic products into the vesicle lumen. These products include a mixture of corrosive oxidants including oxidized halogens, free radicals and singlet oxygen that are derived from superoxide. In addition to playing a role in activating phagocytic cells, IFN-c enhances the transcription of gp91PHOX, p67PHOX, and superoxide dismutase (SOD) which catalyzes the formation of hydrogen peroxide from superoxide anions (Boehm et al., 1997). The importance of NADPH oxidase is apparent from the recurrence of bacterial and fungal infections in patients with chronic granulomatous disease (CGD), a disorder that results from a deficiency in one of the core NADPH components: CGD due to a mutation in p40PHOX has not been reported (Segal et al., 1978; Royer-Pokora et al., 1986; Dinauer et al., 1987; Segal, 1987; Babior, 1999). Nitric oxide (NO) is produced in activated macrophages largely by the inducible form of NO synthase (iNOS or NOS2) (MacMicking et al., 1997). The gene encoding iNOS is transcriptionally activated following stimulation by IFN-c in combination with a second signal, such as TNF-a, IL-1, or LPS, that activates the transcription factor NF-jB. The iNOS

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enzyme catalyzes two sequential reactions with NADPH in which one molecule of L-arginine is oxidized to yield one molecule each of L-citrulline and NO. Interestingly, NO has been shown to prevent the formation of the active NADPH-dependent oxidase (Fujii et al., 1997). NO itself, however, can kill targets by one of two mechanisms. It can form an iron–nitrosyl complex with Fe-S groups of aconitase and complex I or complex II, causing the inactivation of the mitochondrial electron transport chain. Alternatively, NO can react with superoxide to form peroxynitrite, which decays rapidly once protonated to form the highly toxic compound hydroxyl radical. Evidence that NO is responsible for macrophage killing of certain intracellular parasites comes from a number of studies with Leishmania. Mice pretreated with the Larginine analogue N-monomethyl-l-arginine (NMMA), an iNOS inhibitor, were unable to resolve footpad infection with Leishmania parasites (Liew et al., 1990b). Furthermore, mice deficient in iNOS are highly susceptible to infection with microbial pathogens (Nathan, 1995; MacMicking et al., 1997; Shiloh et al., 1999).

Antigen presentation One of the major immunoregulatory roles of IFN-c is its ability to promote adaptive immune responses by influencing both the number of MHC molecules on the cell surface, as well as the repertoire of peptides presented by the MHC. In response to stimulation with either IFN-c or IFN-a/b, expression of the IRF-1 transcription factor is enhanced by a Stat1-dependent process and IRF-1, in turn, enhances expression of MHC class I proteins on a variety of cell types (Chang et al., 1992; Reis et al., 1992; Boehm et al., 1997). This event promotes the development of CD8 T-cell responses. In addition to directly inducing the MHC class I a-chain and b2-microglobulin, IFN-c promotes antigen presentation by regulating the expression of many intracellular proteins that are required for the generation of peptides that bind to MHC class I proteins (Boehm et al., 1997; Pamer and Cresswell, 1998). IFN-c plays a key role in modifying the activity of the proteasome, a multisubunit enzyme complex that generates the peptides displayed by MHC class I proteins. It does so in part by modulating the expression of the enzymatic proteasome subunits. IFN-c increases the expression of the genes encoding the

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inducible subunits LMP2, LMP7 and MECL1 while decreasing expression of the genes encoding the constitutive subunits x, y and z, thereby altering the composition and specificity of the proteasome (Gaczynska et al., 1994; York and Rock, 1996; Pamer and Cresswell, 1998). IFN-c also enhances the production of nonenzymatic proteasome components, such as the a and b subunits of PA28 (i.e. the 11S regulator of the proteasome), which function to regulate proteasome activity (Groettrup et al., 1996). Purified 20S and 26S proteasomes from IFN-c-treated cells show an increased capacity to cleave peptides after hydophobic and basic residues. Thus IFN-c also alters the proteasome in a way that changes the type of antigenic peptides produced (Gaczynska et al., 1994). IFN-c further enhances MHC class I antigen presentation by promoting the loading of peptides onto MHC class I molecules. IFN-c increases the expression of the peptide transporters associated with antigen processing -1 and -2 (TAP-1 and TAP-2), which transfer peptides that have been generated in the cytoplasm by the proteasome into the endoplasmic reticulum where they can bind to nascently produced MHC class I proteins (Pamer and Cresswell, 1998). Moreover, IFN-c increases the expression of heat shock proteins, such as gp96, which may play a role in transferring peptides within the cell from the TAPs to MHC class I molecules and between cells from nonprofessional antigenpresenting cells to a subset of macrophages (Suto and Srivastava, 1995). Taken together, IFN-c plays an important role in enhancing cellular immunogenicity by increasing the quantity and the repertoire of peptides displayed on MHC class I proteins. IFN-c is unique among the interferons in its ability to induce the expression of MHC class II proteins on certain cells (such as mononuclear phagocytes, endothelial cells and epithelial cells), and thereby regulate CD4 T-cell responses (Mach et al., 1996; Boehm et al., 1997). This is not the case with B lymphocytes however, in which IFN-c not only fails to enhance MHC class II expression, but also blocks IL-4-mediated MHC class II induction (Mond et al., 1986) probably by the induction of SOCS-1 that then acts to inhibit IL-4 receptor signaling (Venkataraman et al., 1999). The class II transactivator, CIITA, is an IFN-c-regulated gene product that functions as a master regulator of MHC class II expression and controls the transcrip-

tion of not only class II a- and b-chains, but also the invariant chain (Ii) and the DMA- and DMB-chains (Cresswell, 1994; Wolf and Ploegh, 1995). CIITA functions as a coactivator by bridging DNA bound proteins, such as the RFX complex to the basal transcription machinery (Reith et al., 1999). CIITA itself is regulated by at least four independent promoters including promoters I and III that direct constitutive expression in dendritic cells and B cells, respectively (MuhlethalerMottet et al., 1997). IFN-c-induced activated Stat1 homodimers and IRF-1, together with the ubiquitously expressed upstream stimulatory factor-1 (USF-1) protein, enhance CIITA transcription by binding to elements in promoter IV (Muhlethaler-Mottet et al., 1997, 1998; Piskurich et al., 1999). Patients with one form (type II) of the bare lymphocyte syndrome lack MHC class II expression on their cells due to the absence of CIITA, and exhibit a significant reduction in CD4 T cells (Harton and Ting, 2000). Type I IFN, TNF, bacterial endotoxin and immune complexes can either inhibit or enhance IFN-c’s ability to induce MHC class II proteins. For example, pre-exposure of mononuclear phagocytes to type I IFNs or LPS induces a state of unresponsiveness to IFN-c (Pace et al., 1983; Esparza et al., 1983). In contrast, treatment of cells with IFN-a, IFN-b, or LPS either together with, or following IFN-c treatment, leads to enhanced class II expression. Therefore, the composition of a cell’s microenvironment influences MHC class II expression in response to IFN-c (Farrar and Schreiber, 1993).

T helper cell development A critical process in developing immune responses in mice and humans is the differentiation of naive T helper (TH0) cells into one of two functional subsets (Mosmann et al., 1986; Parronchi et al., 1991; Romagnani, 1991). TH1 cells promote cell-mediated immunity, a required response for clearing microbial and viral infections and mediating tumor rejection. In contrast, TH2 cells aid in the development of a humoral immune response to parasites, such as helminths and promote allergic reactions through their effects on B cells that lead to the production of IgE. The cytokine profiles secreted by these cells are the basis for their different functional roles, and are used to define the two subsets. TH1 cells characteristically

THE CYTOKINES AND CHEMOKINES

BIOLOGIC ACTIVITIES OF IFN - c

produce IFN-c and lymphotoxin, while TH2 cells are defined by their capacity to secrete IL-4, IL-5, IL-9, IL-10 and IL-13. Since IFN-c is the hallmark TH1 cytokine, this discussion will focus on TH1 cells. The cytokine milieu present during antigen stimulation of TH0 cells greatly influences their differentiation into mature TH1 or TH2 effector cells. In vitro and in vivo studies have demonstrated that macrophage and dendritic cell-derived IL-12 is pivotal in driving T cells to the TH1 pole (Hsieh et al., 1993b; Gately et al., 1998). Mice deficient in either the gene for the p40 subunit of IL-12 or the IL-12 signaling protein, Stat4, are unable to efficiently generate TH1 cells and display reduced delayed-type hypersensitivity responses (Magram et al., 1996; Kaplan et al., 1996b; Thierfelder et al., 1996). In humans but not in mice, type I IFNs also activate Stat4 and can substitute for IL-12 in promoting TH1 development (Cho et al., 1996; Rogge et al., 1998). In human cells, the carboxy terminus of activated Stat2 allows Stat4 to be recruited to the IFN-a/b receptor (Farrar et al., 2000b). However, the insertion of a minisatellite into this region of murine Stat2 prevents type I IFN-mediated activation of Stat4 and the induction of interferon-mediated TH1 development in these animals (Farrar et al., 2000a). Although there is synergy between IL-12 and IL-18 in inducing IFN-c secretion from TH1 cells, IL-18 does not play a role in driving TH1 differentiation (Robinson et al., 1997). Furthermore, while IL-23 signals through Stat4, it specifically acts on memory T cells and therefore is not likely to influence TH1 development (Oppmann et al., 2000). IFN-c plays an important role in TH1 development. In vitro, antibody neutralization of IFN-c greatly reduces the development of TH1 cells and augments the development of TH2 cells (Hsieh et al., 1993a). Importantly, however, administration of exogenous IFN-c either in vitro or in vivo does not drive a TH1 response (Hsieh et al., 1993a). Therefore, IFN-c is necessary but not sufficient for TH1 development. The role of IFN-c in TH development has been shown to be due to its effects at the level of both the APC and the CD4 T cell. The effects of IFN-c on macrophages were elucidated in studies that used transgenic mice lacking IFN-c sensitivity specifically in the macrophage compartment. IFN-c-insensitive macrophages were unable to support efficient TH1 development due to a severely reduced capacity to

585

produce IL-12 (Dighe et al., 1995). IFN-c has also been shown to have direct effects on developing TH cells themselves. IFN-c maintains expression of the b2 subunit of the IL-12 receptor on T cells thereby preserving sensitivity of these cells to IL-12 and promoting their development into a TH1 phenotype (Szabo et al., 1997). At the same time IFN-c blocks development of the TH2 phenotype through two mechanisms. First, it inhibits the synthesis of IL-4 from undifferentiated, antigen-stimulated T cells, thereby inhibiting production of the key cytokine required for TH2 cell development (Szabo et al., 1995). Second, it prevents TH2 expansion by directly inhibiting the proliferation of TH2 cells (Gajewski and Fitch, 1990). The antiproliferative effects of IFN-c are not observed on TH1 cells because these cells do not express the IFNGR2 subunit (Pernis et al., 1995; Bach et al., 1995). Thus, IFN-c simultaneously promotes cell-mediated immunity through facilitating TH1 cell formation and inhibits development of humoral immunity through blockade of TH2 cell expansion. The transcription factor T-bet is a powerful regulator of TH1 development and IFN-c production. Remarkably, retroviral gene transduction of T-bet into polarized TH2 cells redirected these lymphocytes to produce IFN-c in response to stimulation with PMA and ionomycin (Szabo et al., 2000). IFN-c, in turn, synergizes with TCR engagement leading to the production of T-bet (Lighvani et al., 2001). Therefore, IFN-c and T-bet form an autocrine loop whereby IFN-c regulates the transcription factor, T-bet, that promotes the production of IFN-c. In a separate study, T-bet was shown to enhance the expression of IL-12Rb2, thus allowing cells to respond to IL-12 and activate Stat4, which promotes the outgrowth of TH1 cells (Mullen et al., 2001). CD4 T cells from T-bet-deficient mice failed to differentiate into the TH1 lineage and defaulted to the TH2 subset when cultured under conditions that normally drive TH1 development (Szabo et al., 2002). Furthermore, T-bet-deficient mice on the normally resistant C57BL/6 background were unable to resolve infection with Leishmania major and produced very little IFN-c (Szabo et al., 2002).

Humoral immunity IFN-c plays a complex role in regulating humoral immunity, exerting its effects either indirectly by reg-

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INTERFERON - c

ulating the development of specific T-helper cell subsets (as described above) or directly at the level of the B cell. In the latter case, IFN-c is predominantly responsible for regulating three specialized B-cell functions: (1) B-cell development and proliferation; (2) immunoglobulin (Ig) secretion; and (3) Ig-heavy chain switching. IFN-c has been shown to negatively regulate B-cell differentiation by inhibiting IL-4-dependent induction of MHC class II protein expression (as described above) and proliferation of B cells stimulated with anti-Ig and IL-4. In contrast, IFN-c enhances proliferative responses in human B cells activated with antiIg. IFN-c can also enhance or inhibit Ig secretion by either murine or human B cells. In this process, however, IFN-c’s effects depend on the differentiation state of the B cell, the timing of IFN-c stimulation, and the nature of the activating stimulus. The best-characterized B-cell-directed effect of IFN-c is its ability to influence Ig heavy chain switching. Ig class switching is significant because the different Ig isotypes promote distinct effector functions in the host. Immunoglobulin E (IgE) is the only isotype that can bind high affinity Fc-e receptors on mast cells and basophils and thereby promote immediate-type hypersensitivity and allergic reactions. Murine IgG2a fixes complement and can bind (in monomeric form) to FccR-I on murine macrophages, a high affinity Fc receptor upregulated during IFN-c-induced macrophage activation. Activated murine macrophages can efficiently use antibodies of the IgG2a isotype to mediate antibodydependent cellular cytotoxicity. IgG3 is an isotype that can self-aggregate, a process that may enhance its opsonic activity. Along with IgG2a, IgG3 can also bind to the NK-cell-IgG receptor CD16/CD32 and effect NK-mediated antibody-dependent cellular cytotoxicity. By favoring the production of IgG2a and IgG3 and inhibiting the production of IgE isotypes, IFN-c can facilitate the interaction between the humoral and cellular effector limbs of the immune response and enhance host defense against certain bacteria and viruses. In vitro, IFN-c is able to direct immunoglobulin class switching from IgM to the IgG2a subtype in LPSstimulated murine B cells and to IgG2a and IgG3 in murine B cells that have been stimulated by activated T cells (Snapper et al., 1988, 1992).

Moreover, IFN-c blocks IL-4-induced class switching in murine B cells to IgG1 or IgE (Snapper and Paul, 1987). The validity of these observations has been stringently tested in experiments in which Ig subclass production was monitored in mice that were injected with immunoglobulin-D (IgD)-specific antiserum to achieve polyclonal activation of B cells. Mice treated in this manner produced large quantities of IgG1 and IgE. When IFN-c was administered to the mice before anti-IgD treatment, however, they produced high levels of IgG2a, and decreased levels of IgG1. Thus IFN-c is clearly an important regulator of Ig class switching in vivo (Snapper, 1996).

Tumor immunity IFN-c plays an important role in the immunemediated rejection of established tumors (reviewed in Ikeda et al., 2002). A clear demonstration for the role of IFN-c in tumor rejection came from studies using the Meth A tumor cell line (Dighe et al., 1994). Meth A is a methylcholanthrene (MCA)-induced BALB/c fibrosarcoma that grows aggressively when transplanted subcutaneously into naive syngeneic mice and eventually kills the host (Old, 1985, 1990). Treatment of Meth A-bearing mice with sublethal doses of bacterial endotoxin (LPS), however, induces the rejection of progressively growing transplanted tumors. IFN-c’s role in LPS-mediated tumor rejection was revealed by the observation that neutralizing mAb specific for murine IFN-c blocked LPS-induced tumor regression (Dighe et al., 1994). Similar to LPS, IL-12 possesses potent anti-tumor activities that include slowing tumor growth, reducing the rate of metastasis, and in some cases, effecting complete tumor regression. All of the aforementioned effects of IL-12 are ablated with neutralizing antibodies to IFN-c (Brunda, 1994; Nastala et al., 1994). Therefore, IFN-c is a critical component of the mechanisms by which both LPS and IL-12 mediate the rejection of established tumors. IFN-c also participates in the elimination of primary tumors that are either chemically induced or that arise spontaneously. IFN-c-insensitive mice or mice lacking the IFN-c gene are more sensitive than genetically matched, wild-type mice to chemical carcinogens such as methylcholanthrene, and form tumors more rapidly and in greater frequency

THE CYTOKINES AND CHEMOKINES

IN VIVO DYSFUNCTIONING OF IFN - c SIGNALING

compared with their wild-type counterparts (Kaplan et al., 1998; Shankaran et al., 2001; Street et al., 2001). Importantly, the enhanced susceptibility to MCAinduced tumor formation was similar in mice deficient for IFNGR1 (IFN-c signaling only) or Stat1 (IFN-c, as well as IFN-a/b signaling) (Shankaran et al., 2001). This result suggests that IFN-c plays the major role in providing Stat1-mediated tumor protection. The increased MCA-induced tumor formation observed in IFN-c-insensitive mice was comparable to that observed in mice that lack lymphocytes due to the targeted disruption in recombination-activating gene-2 (RAG2) (Shankaran et al., 2001). Furthermore, no significant increase in carcinogen-induced tumor development was observed in mice deficient for both Rag2 and Stat1 (RkSk) compared with mice deficient in Stat1 alone, indicating an overlap between the antitumor mechanisms mediated by lymphocytes and IFN-c signaling. Enhanced spontaneous tumor development in IFN-c-insensitive mice was shown in two different model systems. First, mice deficient in both the p53 tumor suppressor gene and IFN-c signaling developed primary tumors more rapidly than did mice lacking p53 alone (Kaplan et al., 1998). More importantly, the two groups of mice displayed differences in the types of tumors that formed. While all of the IFNc-sensitive p53/  129Sv/Ev mice developed lymphoid tumors, 35% of the p53/  IFN-cR/ mice and 38% of the p53/  Stat1/ mice formed nonlymphoid tumors (such as teratomas, hemangiomas and chondrosarcomas) without concomitant lymphoid tumors. The second model of spontaneous tumor formation involved the examination of aged, unmanipulated, wild-type, RAG2/ and RkSk mice (Shankaran et al., 2001). This study found that RkSk mice developed more spontaneous cancers (which were largely intestinal, lung and breast adenocarcinomas) than did the RAG2/ mice. Taken together, these results revealed that IFN-c and lymphocytes play important and interdependent roles in protecting the host against tumor development. Although these results established the importance of IFN-c in regulating tumor growth and rejection, they did not identify the cellular target of IFN-c’s antitumor actions. IFN-c-sensitive Meth A tumor cells transfected with a dominant-negative form of IFNGR1 were not rejected when the tumor bearing

587

mice were treated with LPS (Dighe et al., 1994). Furthermore, the IFN-c-insensitive tumors neither primed naive mice for induction of Meth A immunity, nor were they rejected in mice with pre-established immunity to the wild-type tumor cell. Additional transplantation studies took the opposite experimental approach in which cells derived from MCA-treated IFNGR1/ mice were reconstituted with wild-type IFNGR1 (Kaplan et al., 1998). This reconstitution converted the highly tumorigenic cells into immunogenic cells that were rejected when transplanted into syngeneic wild-type mice, but not in immunodeficient RAG1/ mice. Together, these results demonstrate that the tumor cell itself is a key target of IFN-c’s actions and that IFN-c functions at least in part to enhance the immunogenicity of the tumor cell. The effects of IFN-c on the tumor are most likely quite pleiotropic. IFN-c is known to have potent antiproliferative effects on some tumors and can have apoptotic effects on others (Bromberg et al., 1996; Kumar et al., 1997). IFN-c can induce the production of angiostatic chemokines such as IP-10, MIG and/or I-TAC by tumors and the surrounding tissue resulting in blockade of new blood vessel development within the tumor leading to ‘death by starvation’ of portions of the tumor (Luster and Leder, 1993; Coughlin et al., 1998; Qin and Blankenstein, 2000). Finally, IFN-c has been found to enhance the immunogenicity of tumor cells by up-regulating the MHC class I antigen processing and presentation pathway in murine sarcomas (Shankaran et al., 2001). Importantly, a similar effect can be induced in IFN-c-insensitive tumors by enforced expression of specific MHC class I pathway components such as TAP-1 (Shankaran et al., 2001). This latter result demonstrates that IFN-c’s effects on tumor immunogenicity are sufficient to cause immunologic tumor rejection.

IN VIVO DYSFUNCTION OF IFN-c SIGNALING Mice with disrupted genes of interferon-c signaling proteins The physiologic consequences of the global in vivo inactivation of IFN-c-mediated responses in mice were first identified using neutralizing monoclonal

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antibodies specific for IFN-c (Buchmeier and Schreiber, 1985). Subsequently, the physiologic role of IFN-c has been more fully elucidated using mice with targeted disruptions in the genes encoding the IFN-c cytokine itself (Dalton et al., 1993), IFNGR1 (Huang et al., 1993), IFNGR2 (Lu et al., 1998), or Stat1 (Meraz et al., 1996; Durbin et al., 1996). As a group, these mice display a greatly impaired ability to resist infection by a variety of microbial pathogens including Listeria monocytogenes, Leishmania major and several mycobacterial species including Mycobacterium bovis and Mycobacterium avium. Importantly, mice lacking either IFNGR1 or IFNGR2 are able to mount a curative response to many viruses, while mice lacking the IFN-a/b receptor or Stat1, and cells from mice lacking Jak1, are not. These results demonstrate that under physiologic conditions, the majority of the antiviral effects attributed to the IFN system are mediated by type I interferons (Muller et al., 1994). The physiologic role of IFN-c cannot be experimentally addressed in mice with an engineered global deficiency in either Jak1 or Jak2 due to the early lethality of these animals. Jak1-deficient mice fail to suckle, and die within hours of birth due to a suspected neurological defect (Rodig et al., 1998). Jak2-deficient mice die at embryonic day 12.5 due to a failure of erythropoiesis (Parganas et al., 1998; Neubauer et al., 1998). Assays performed using cells generated from Jak1- or Jak-2 deficient mice, however, demonstrated that these kinases are obligatorily required for all the classical IFN-c-induced responses that were examined.

MSMD, while salmonellosis affects less than half of these cases (Casanova, 2001). Recent genetic analysis has identified five genes that are mutated in individuals with this syndrome. Various mutations in these genes define nine disorders (Casanova and Abel, 2002). Impaired response to IFN-c accounts for seven disorders that result from mutations in IFNGR1 (four disorders), IFNGR2 (two disorders) and Stat1 (one disorder). The other two disorders are caused by impaired IFN-c production due to mutations in IL-12 p40 or IL-12-receptor-b chain. In cases where the disorder is caused by an impaired response to IFN-c (but not impaired IFN-c production), the type of mutation (loss-of-function or hypomorphic) determines the cellular phenotype (complete or partial signaling defect), the histological phenotype (immature or mature granulomas), and the clinical outcome (Lamhamedi et al., 1998). The most severe MSMD disorders result from complete IFNGR1 or IFNGR2 functional deficiency (Figure 24.4 and Table 24.3). Genetic analysis of these patients’ families revealed that these disorders are inherited in an autosomal recessive manner; both homozygous and compound heterozygous patients have been identified. Of the four IFNGR1 disorders, complete deficiency in IFNGR1 accounts for two disorders that are distinguished from one another based on the molecular defect responsible for their inability to function. One disorder is caused by mutations that

IFNGR1 LPKSLI

Identification of human patients with defects in IFN-c signaling or production

1416

I II

III

45 8

Mendelian susceptibility to mycobacterial disease (MSMD) (OMIM 209950) is an extremely rare human disease that encompasses multiple disorders, all of which are mechanistically related by impaired IFN-cmediated immunity. Surprisingly, unlike patients with a classical primary immunodeficiency or mice that lack IFN-c sensitivity, individuals with MSMD are not susceptible to a broad range of bacterial, fungal, protozoal and viral infections. Instead, Bacillus Calmette–Guérin (BCG) vaccines and environmental non-tuberculous mycobacteria (NTM) are the leading causes of severe disease in individuals with

YDKPH

-51 1

IV

V

10

1 23 6 7

VI

VII

14 11 12

13

9 15 16 17 18 19

IFNGR2 PPSIPLQIEEYL -63

1

I II

316

III 20

IV

V

VI VII 22

21

FIGURE 24.4 Model of IFN-c signaling. The details of this model are described in the text.

THE CYTOKINES AND CHEMOKINES

TABLE 24.3 Defined IFN-c receptor mutations in patients with MSMD Immaturea

Maturea

Description

Zygosity

Reference

1 2 3 6

22delC 107ins4 131delC 2001G→A

30delC 56ins4 80delC 1491G→A

HM CHa HM CHa

Holland, 1998 Altare, 1998 Jouanguy, 1996 Altare, 1998

7

2012A→G

1502A→G

HM

Holland, 1998

11 12

S116X 3731G→T

S99X 3221G→T

HM CHb

Newport, 1996 Roesler, 1999

13

561del4

510del4

FS (1 nt deletion) FS (4nt insertion) FS (1 nt deletion) Disruption of splicedonor site (in intron 2) 1 nt 3 of exon 2 (nt 149) Disruption of the spliceacceptor site (in intron 2) 2 nt 5 of exon 3 (nt 150) Nonsense mutation Disruption of splicedonor site (in intron 3) 1 nt 3 of exon 3 (nt 322) FS (4 nt deletion)

CHb HM

Roesler, 1999 Rosenzweig, 2002

V61Q V63Q C77Y 295del12 301del3

V44Q V46Q C60Y 244del12 652del3

Missense Missense Missense In-frame deletion of 12 nt In-frame deletion of 3 nt

CHc HM HM HM CHc

Jouanguy, 2000 Allende, 2001 Jouanguy, 2000 Jouanguy, 2000 Jouanguy, 2000

I87T

I70T

Missense

HM

Jouanguy, 1997

811del4 817insA 818delT 818del4

760del4 766insA 767delT 767del4

HT HT HT HT

Sasaki, 2002 Dorman, 1999 Jouanguy, 1999 Jouanguy, 1999 Arend, 2001 Sasaki, 2002

E278X

E261X

FS (4 nt deletion) FS (1 nt deletion) FS (1 nt deletion) FS (4 nt deletions representing 2 to 5 indistinguishable mutations) Nonsense

HT

Villella, 2001

278delAG 791delG

215delAG 728delG

FS (2nt deletion) FS (1nt deletion)

HM HM

Dorman, 1998 Dorman, 2000

R114C

R93C

Missense

HM

Doffinger, 2000

Location Complete IFNGR1 deficiency Preclude surface expression

Preclude ligand binding 4 5 8 10 14 Partial recessive IFNGR1 deficiency May reduce ligand binding 9 Partial dominant IFNGR1 deficiency Mutant receptor chains unable to transduce signals 15 16 17 18

19 Complete IFNGR2 deficiency Preclude surface expression 20 22 Partial recessive IFNGR2 deficiency May reduce bindng to IFNGR1 21

FS, frame shift; nt, nucleotide; HM, homozygous; HT, heterozygous; CHa, CHb, and CHc represent pairs of compound heterozygous mutations. a The numbering system differs between laboratories which number either from the beginning of the signal sequence of the immature protein or the amino terminus of the mature protein.

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give rise to a premature stop codon upstream of the coding sequence for the transmembrane domain resulting in the production of truncated receptor proteins that are not retained on the cell surface. Genetic analysis of the nine kindreds (17 patients) with this disorder identified nonsense and splice mutations, as well as nucleotide insertions or deletions that result in a frame shift mutation and ultimately preclude cell surface expression of IFNGR1 (Doffinger et al., 2000a). The second complete IFNGR1 functional deficiency is caused by mutations in the coding sequence of the extracellular domain of the protein that prevent the receptor from binding its ligand, IFN-c. Five mutations, including missense mutations and small inframe deletions, have been identified in five children from four unrelated families (Jouanguy et al., 2000; Allende et al., 2001). In one case, the child of nonconsanguinous parents was found to be compound heterozygous for a missense mutation that results in the substitution of glutamine for valine at position 61 (V61Q) and a small in-frame deletion which removes glutamic acid at position 218 (652del3) (according to convention, mutations identified in the human IFN-c receptor chains are numbered beginning with the first methionine) (Jouanguy et al., 2000). Null recessive IFNGR2 mutations have been identified in two unrelated children (Dorman and Holland, 1998, 2000). These mutations are frameshift deletions that both result in the absence of IFNGR2 on the cell surface, and therefore define one disorder (Casanova and Abel, 2002). Functional assays using cells from children with complete IFNGR1 or IFNGR2 deficiency show a total absence of response to IFN-c (Doffinger et al., 2000a). Inoculation of these children with live BCG vaccine invariably results in disseminated BCG infection and is generally fatal, although some patients have been successfully treated by aggressive and prolonged antibiotic therapy. Survivors and affected individuals who were not BCG-vaccinated are selectively susceptible to the early-onset of severe mycobacterial infection from environmental exposure. Clinical infection is seen with even the least virulent species of mycobacteria, such as Mycobacterium smegmatis and Mycobacterium peregrinum, which otherwise have never been reported to cause disease in humans (Casanova and Abel, 2002). Lepromatous-like granulomas in infected tissues are characteristic of these individuals who are unable to

generate effective cell-mediated immunity. Unless a bone marrow transplant is undertaken, individuals with IFN-c receptor deficiency die during childhood; even then, this treatment is associated with considerable morbidity and mortality (Jouanguy et al., 2000; Casanova, 2001). Less severe mycobacterial disease occurs in patients with mutations in IFN-c signaling components that lead to the reduction rather than ablation of receptor signaling. The pathologic causes of these deficiencies define four MSMD disorders: (1) partial recessive IFNGR1 deficiency; (2) partial recessive IFNGR2 deficiency; (3) partial dominant IFNGR1 deficiency; and (4) partial dominant Stat1 deficiency (Figure 24.4 and Table 24.3). A recessive mutation in IFNGR1 was identified in a Portuguese family in which one child developed disseminated BCG infection and a sibling who had not been vaccinated with BCG developed clinical tuberculosis (Jouanguy et al., 1997). Both patients were homozygous for a point mutation in exon three of the IFNGR1 gene that produced an isoleucine-tothreonine amino acid substitution (I87T) in the extraceullar domain. Stat1 activation by the mutant receptors was found to require 100- to 1000-fold higher concentrations of IFN-c than that required by normal receptors. Similarly, a 20-year-old patient with a history of BCG and Mycobacterium abscessus was found to have a homozygous missense mutation in the extracellular domain of IFNGR2 (R114C) (Doffinger et al., 2000b). Although a mechanism was not determined, a causal relationship between this mutation and a weak cellular response to IFN-c was demonstrated by molecular complementation. The generation and characterization of IFN-cinsensitive cells and transgenic mice that overexpress a truncated form of IFNGR1 (Dighe et al., 1993, 1994), pointed to a molecular basis for a third partial IFN-c signaling disorder identified in humans. As a group, patients with this disorder were found to inherit partial IFN-c insensitivity in an autosomal dominant manner. The initial cohort of such individuals examined consisted of 19 patients from 12 unrelated kindreds. Genetic analysis revealed that these patients were heterozygous for one of multiple overlapping IFNGR1 frameshift mutations that result in a IFNGR1 receptor subunit that is truncated in the intracellular domain (Jouanguy et al., 1999). Eleven of these muta-

THE CYTOKINES AND CHEMOKINES

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REFERENCES

tions are indistinguishable and are designated 818del4, while one mutant, 818delT, is distinct. Subsequently, mutations at position 818 have been identified in five additional families (Arend et al., 2001; Sasaki et al., 2002). Interestingly, the fact that at least two (17 mutations to date) independent mutations of the same type were found at position 818 of IFNGR1 makes this region the first small deletion hotspot to be identified in the human genome (Jouanguy et al., 1999). Other autosomal dominant mutations that result in truncated IFNGR1 chains include the insertion of a single nucleotide at position 817 (817insA) (Dorman et al., 1999) and a small deletion of four nucleotides at position 811 (811del4) (Sasaki et al., 2002). Both mutations result in frameshifts and premature stop codons. In addition, a nonsense mutation at nucleotide position 832 leads to a premature stop codon at position 278 (E278X) (Villella et al., 2001). These truncated IFNGR1 subunits are expressed on the cell surface, incorporated into the IFN-c receptor complex, and bind IFN-c with a normal affinity, but function as dominant-negatives due to their lack of recruitment sites for Jak1 and Stat1. Accumulation of these truncated receptors on the cell surface, due to the absence of the 269LI270 (numbering based on the mature protein) intracellular recycling motif, further enhances the dominant-negative effect of these mutants. The cell surface expression of the wild-type IFNGR1 permits a limited response to IFN-c, and therefore this signaling defect is partial and the clinical phenotype of individuals with this disorder is milder than that of children with complete IFNGR1 deficiency. Recently, a partial dominant mutation in Stat1 was identified in two patients with a history of mycobacterial disease (Dupuis et al., 2001). Although unrelated, both individuals were heterozygous for a T→C mutation at nucleotide position 2116 of the Stat1 coding sequence, resulting in a serine to leucine substitution at position 706 (S706L). This mutation was found to impede Stat1 phosphorylation on tyrosine 701 resulting in a loss-of-function that is dominant for Stat1 homodimers, but recessive for IFN-a-induced ISGF3 (Stat1/Stat2/p48) (Dupuis et al., 2001). Two MSMD disorders result from reduced IL-12 production or responsiveness that in turn leads to defects in IFN-c production. Functional IL-12 p70 is a heterodimer composed of p35 and p40 subunits that

binds to high affinity receptors containing IL-12Rb1 and IL-12Rb2, which are expressed on the surfaces of T and NK cells. Deficiency in the p40 subunit of IL-12 defines one disorder which, interestingly, is the first identified human disease that results from a cytokine defect (Altare et al., 1998b). Two homozygous mutations have been identified in the IL-12B gene which encodes the p40 subunit of the heterodimer (Altare et al., 1998b; Picard et al., 2002). One mutation is a large loss-of-function deletion that was identified in two kindreds and the other is a loss-offunction frameshift insertion that was identified in patients from four families. The second MSMD disorder was identified in patients from a total of nine families, and is caused by recessive mutations in the gene encoding IL-12Rb1 that produce truncated receptors that lack a transmembrane domain and are not retained on the cell surface (Altare et al., 1998a, 2001; de Jong et al., 1998; Aksu et al., 2001; Sakai et al., 2001). Compared with lymphocytes from normal individuals, cells from patients with either IL-12-related disorder secrete less IFN-c. This defect is responsible for the high incidence of mycobacterial disease seen in these patients, although IL-12-independent, IFN-c-mediated immunity probably accounts for the milder clinical and histological phenotype that is generally observed. Unlike with IFN-c-receptor deficiencies, the clinical outcome of patients with IL-12 deficiencies vary from case to case such that there is no apparent correlation between the IL-12B and IL-12Rb1 genotypes and the clinical phenotype (Casanova and Abel, 2002).

CONCLUSIONS During the past two decades, our understanding of the biochemistry, cell biology and physiology of IFN-c has grown exponentially. IFN-c is currently one of the best understood members of the cytokine family. Yet, a definitive use of IFN-c as a therapeutic remains elusive. Clearly, the issue of targeting this cytokine to the right cells at the right time is a major obstacle that still needs to be addressed. In addition, the capacity to promote specific types of protective IFN-c-induced biologic responses while inhibiting unwanted immunopathologic effects is still beyond

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our reach. However, as the field moves further into the fine details of IFN-c’s functions and mechanisms of action, it is likely that new insights will be gained that will form a new foundation for the therapeutic use of this pleiotropic cytokine.

ACKNOWLEDGMENTS We thank Dr Chris Nelson for creating Figure 24.1. We also thank Gavin Dunn and Dr S. Ruby Chan for their thoughtful comments and critical reading of the manuscript. Work from Robert Schreiber’s laboratory quoted in this review was supported by grants from the National Institutes of Health (CA43039 and CA76464), the Cancer Research Institute, and the Ludwig Institute for Cancer Research.

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regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein. J. Biol. Chem. 273, 28185–28190. Villella, A., Picard, C., Jouanguy, E. et al. (2001). Recurrent Mycobacterium avium osteomyelitis associated with a novel dominant interferon gamma receptor mutation. Pediatrics 107, E47. Walter, M.R. and Nagabhushan, T.L. (1995). Crystal structure of interleukin 10 reveals an interferon gamma-like fold. Biochemistry 34, 12118–12125. Walter, M.R., Windsor, W.T., Nagabhushan, T.L. et al. (1995). Crystal structure of a complex between interferongamma and its soluble high-affinity receptor. Nature 376, 230–235. Wang, L.L. and Yokoyama, W.M. (1998). Regulation of mouse NK cells by structurally divergent inhibitory receptors. Curr. Top. Microbiol. Immunol. 230, 3–13. Wen, Z., Zhong, Z. and Darnell, J.E. Jr. (1995). Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82, 241–250. Wilks, A.F., Harpur, A.G., Kurban, R.R. et al. (1991). Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol. Cell. Biol. 11, 2057–2065. Wolf, P.R. and Ploegh, H.L. (1995). How MHC class II molecules acquire peptide cargo: biosynthesis and trafficking through the endocytic pathway. Annu. Rev. Cell. Dev. Biol. 11, 267–306. Xu, X., Sun, Y.L. and Hoey, T. (1996). Cooperative DNA binding and sequence-selective recognition conferred by the STAT amino-terminal domain. Science 273, 794–797. Yang, J., Murphy, T.L., Ouyang, W. and Murphy, K.M. (1999). Induction of interferon-gamma production in Th1 CD4 T cells: evidence for two distinct pathways for promoter activation. Eur. J. Immunol. 29, 548–555. Yang, J., Zhu, H., Murphy, T.L. et al. (2001). IL-18-stimulated GADD45 beta required in cytokine-induced, but not TCRinduced, IFN-gamma production. Nat. Immunol. 2, 157–164. Yasukawa, H., Misawa, H., Sakamoto, H. et al. (1999). The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J. 18, 1309–1320. Yasukawa, H., Sasaki, A. and Yoshimura, A. (2000). Negative regulation of cytokine signaling pathways. Annu. Rev. Immunol. 18, 143–164. Ye, J., Cippitelli, M., Dorman, L. et al. (1996). The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element. Mol. Cell. Biol. 16, 4744–4753. Yeh, T.C., Dondi, E., Uze, G. and Pellegrini, S. (2000). A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-alpha signaling. Proc. Natl Acad. Sci. USA 97, 8991–8996. York, I.A. and Rock, K.L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annu. Rev. Immunol. 14, 369–396. You, M. and Zhao, Z. (1997). Positive effects of SH2 domaincontaining tyrosine phosphatase SHP-1 on epidermal growth factor- and interferon-gamma-stimulated activation of STAT transcription factors in HeLa cells. J. Biol. Chem. 272, 23376–23381.

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You, M., Yu, D.H. and Feng, G.S. (1999). Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. Mol. Cell. Biol. 19, 2416–2424. Young, H.A., Komschlies, K.L., Ciccarone, V. et al. (1989). Expression of human IFN-gamma genomic DNA in transgenic mice. J. Immunol, 143, 2389–2394. Young, H.A., Ghosh, P., Ye, J. et al. (1994). Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-gamma gene. J. Immunol. 153, 3603–3610. Young, H.A., Klinman, D.M., Reynolds, D.A. et al. (1997). Bone marrow and thymus expression of interferongamma results in severe B-cell lineage reduction, T-cell lineage alterations, and hematopoietic progenitor deficiencies. Blood 89, 583–595. Zdanov, A., Schalk-Hihi, C., Gustchina, A. et al. (1995). Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon gamma. Structure 3, 591–601. Zhang, F., Nakamura, T. and Aune, T.M. (1999a). TCR and IL-12 receptor signals cooperate to activate an individual response element in the IFN-gamma promoter in effector Th cells. J. Immunol. 163, 728–735. Zhang, J.G., Farley, A., Nicholson, S.E. et al. (1999b). The conserved SOCS box motif in suppressors of cytokine signal-

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25 Interleukin-10 YaoZhong Ding, Shuang Fu, Dmitriy Zamarin and Jonathan Bromberg Mount Sinai School of Medicine, New York, NY, USA

A firm Union will be . . . a barrier against domestic faction and insurrection Alexander Hamilton, Federalist essay No. 9; 1787

INTRODUCTION Interleukin-10 (IL-10) is a key regulator of immune responses. Because of its ability to turn off cytokine production by T cells, IL-10 was originally described as cytokine synthesis inhibitory factor (CSIF) (Fiorentino et al., 1989). Subsequent in vitro studies showed that IL-10 can directly inhibit TH1 and TH2 IL-2 production and IL-5 production at the level of the T cell (de Waal Malefyt et al., 1993; Schandene et al., 1994). T cell stimulation in vitro in the presence of IL-10 can lead both to long-term anergy and the production of a negative regulatory T cell subset (Groux et al., 1996, 1997). Further investigations demonstrated that the immunosuppressive effects of IL-10 are more often at the level of the antigen presenting cell (APC) and not directly at the level of the T cell (Fiorentino et al., 1991). Thus, IL-10 inhibits monocyte and macrophage synthesis of IL-a1, IL-1b, IL-6, IL-8, IL-12, TNFa, GM-CSF and reactive oxygen and nitrogen intermediates; dendritic cell stimulation of The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

IFNc production; APC B7 expression; and antigen presentation to TH1, but not TH2 cells, while inducing IL-1 receptor antagonist production in neutrophils (Moore et al., 1993) (Table 25.1). IL-10 also suppresses epidermal Langerhans cell APC functions (Chang et al., 1994; Beissert et al., 1995), chemokine expression by monocytes (Berkman et al., 1995), and the bactericidal response of macrophages to IFNc (Murray et al., 1997). IL-10-treated dendritic cells induce peptide antigen and alloantigen specific tolerance (Steinbrink et al., 1997). Additional studies demonstrated that IL-10 inhibits the immune function of other cell types, too. Thus, IL-10 inhibits NK cell production of IFNc, ICAM-1 expression on activated vascular endothelial cells, and T dependent responses of B cells (Pecanha et al., 1993; Tripp et al., 1993; Eissner et al., 1996). In sum, the predominant effect of IL-10 has been shown to suppress multiple immune responses through individual actions on T cells, B cells, APCs, and other cell types, and to skew the immune response from TH1 to TH2. TH1

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TABLE 25.1 IL-10 signaling in different cell types Cell type

Stats

Interaction with other pathways

Regulation

Effect on the cell

Monocyte

Stat1, Stat3

Inhibits IKK activity and NFkB DNA binding; Activates PI3K and p70 S6K that may be involved in proliferation Inhibits p38 MAPK to modulate ARE dependent TNF mRNA translation

Inhibited by IL-1, TNFa, LPS signaling, SOCS1, SOCS3

Inhibits proliferation Inhibits production of inflammatory cytokines Inhibits IFN-induced gene expression by inhibiting Stat1 phosphorylation Inhibits p19INK4D expression Inhibits CD40-mediated activation of ERK1/2 Inhibits induction of IL-1R Inhibits COX2 expression Up-regulates expression of CD95 and CD95L, thus promoting apoptosis, Up-regulates FccRI Up-regulates SOCS-3

Dendritic cell

Stat1, Stat3

Inhibits activation of SAPK/JNK, p38MAPK, and ERK2

Antagonized by TNFa and CD40 ligand.

B cell

Stat1, Stat3, Stat5

Not described

Not described

Represses TNF-induced changes Modulates CCR expression, Induces differentiation and proliferation Induces expression of CD32/16, CD2, LECAM-1, heat-stable antigen, and c-fos Induces Bcl-2 expression and prevents apoptosis

T cell

Stat1, Stat3

Inhibits CD28 tyrosine phosphorylation and PI3K recruitment

Not described

Enhances TGF?R expression on activated T cells Prevents release of Th1 cytokines, blocks proliferation Enhances Bcl-2 expression and prevents apoptosis

Neutrophil

Stat1, Stat3

Not described

SOCS proteins

Inhibits mobilization of specific granules to membrane Suppresses production of superoxide

NK cell

Stat1, Stat3

Not described

Not described

Enhances cytotoxicity and proliferation

Mast cell

Stat1, Stat3

Not described

Not described

Inhibits kit expression Reduces TNF induction

Melanoma cell

Not described

Not described

Induces proliferation Down-regulates MHC class I and II complex expression

Oligo dendrocyte

Not described

Not described

Inhibits iNOS expression Promotes survival

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GENE AND PROTEIN STRUCTURE

In various disease states or models, IL-10 has been shown to effectively treat the cytokine syndrome and toxicity caused by anti-CD3 mAb or endotoxin by inhibiting the production of proinflammatory cytokines. Autoimmune models of rheumatoid arthritis, thyroiditis and collagen-induced arthritis and a model of herpetic stromal keratitis all suggest negative regulatory roles for IL-10 in limiting inflammation and immunopathology (Katsikis et al., 1994; Mignon-Godefroy et al., 1995; Kasama et al., 1995; Daheshia et al., 1997). Administration of IL-10 can prevent the development of autoimmune diabetes and prolong syngeneic islet survival in autoimmune diabetic recipients (Rabinovitch et al., 1995; Zheng et al., 1997). Similar adoptive transfer results have also been obtained with IL-10-transduced T cell clones in experimental autoimmune encephalomyelitis and Leishmania infection models (Hagenbaugh et al., 1997; Mathisen et al., 1997). IL-10-deficient knockout mice have highly polarized TH1 responses and develop a severe colitis related to chronic stimulation by enteric antigens (Rennick et al., 1997). A number of inconsistent and unexpected findings also suggested that IL-10 has actions which are more complex than originally proposed. IL-10 can act as a proliferative co-factor for immature and mature thymocytes stimulated by IL-2 plus IL-4 (MacNeil et al., 1990). IL-10 can inhibit T-dependent B cell responses, but not T cell-independent responses (Pecanha et al., 1993). In fact, IL-10 can act as a B cell growth factor (Go et al., 1990) and support the autocrine growth of B cell lymphomas (Beatty et al., 1997). IL-10 stimulates the development of systemic autoimmune disease in NZB/W F1 mice, which is mediated primarily by B cells, while anti-IL-10 mAb delays the onset of autoimmunity (Ishida et al., 1994). IL-10 can induce the expression of E-selectin on vascular endothelium (Vora et al., 1996), which would be expected to promote and sustain inflammatory responses. Likewise, the TH2 polarization induced by IL-10 enhances the development of granulomata and chronic inflammation (Wynn et al., 1997). IL-10 administration failed to prolong islet allograft survival and accelerated islet destruction and increased granzyme B gene expression, suggesting a role for IL-10 in CTL induction (Zheng et al., 1995). Mice with a mIL-10 transgene regulated by an insulin promoter had a pronounced leukocytic infiltrate of CD4 and CD8 T cells, B cells

and macrophages, along with activation of the vascular endothelium. Transgenic IL-10 expression in these mice did not prevent or delay autoimmune or alloimmune disease (Wogensen et al., 1993, 1994; Lee et al., 1994). These studies all suggest proinflammatory functions for IL-10 under some circumstances. In sum, IL-10 is generally considered an immunosuppressive cytokine, but IL-10 may have immunostimulatory or immunosuppressive effects depending on the assay, cell types involved, or other concomitant immune events.

GENE AND PROTEIN STRUCTURE IL-10 is produced by activated T cells, B cells, monocytes/macrophages, mast cells and keratinocytes (Moore et al., 1993). While human IL-10 (hIL-10) is encoded by a single 3.5-kb exon, the mouse IL-10 (mIL-10) gene contains five exons that span 5.1 kb of the genomic DNA, and both genes are located on chromosome 1. Detailed analysis of the mIL-10 gene revealed that there are possible transcriptional control sequences present in noncoding regions of the mIL-10 genomic sequence. For example, a TATA box (30 bp from the 5-end), NF-jB-like recognition sites, an IL-6 responsive element, and IFN inducible sites are all present (Kim et al., 1992). Less is known about hIL-10 gene structure and its regulation, although there are associations between promoter polymorphisms and disease states (Shin et al., 2000). The hIL-10 and mIL-10 cDNA sequences have 80% homology and encode peptides of 178 amino acids, including hydrophobic leader sequences. The mature N-terminus of hIL-10 is Ser19 while the N-terminus of mIL-10 is Gln22, and the proteins have 73% amino acid identity. Both proteins have been shown to exist in solution as noncovalent homodimers. hIL-10 is an 18 kDa nonglycosylated polypeptide; while mIL-10 is heterogeneously N-glycosylated at a site near its N-terminus, resulting in a mixture of 17, 19, and 21 kDa species. The glycosylation of mIL-10 is not required for its biological activity because a mutant lacking the N-linked site is active, and recombinant mIL-10 expressed in Escherichia coli retains all known biological activities (Moore et al., 1993; Ding et al., 2000).

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The crystal structure reveals that human IL-10 is a tight noncovalent homodimer and each monomer contains two intra-chain disulfide bonds, between residues 12 and 108, and between residues 62 and 114 (hIL-10 numbering). Each monomer consists of six a-helices termed A (residues 18–41, hIL-10 numbering), B (residues 48–58), C (residues 60–82), D (residues 87–108), E (residues 118–131) and F (residues 133–159), which tightly associate with the other monomer (A, B, C, D, E, F) to form two interpenetrating domains. Each of the domains are composed of six a-helices (A, B, C, D, E, F), four originating from one monomer (A, B, C, D) and two from the other (E, F). The overall topology of the helices bears close resemblance to IFNc. The two most notable differences between IL-10 and IFNc dimers are the size and orientation of the domains. In hIL-10, the helix bundles are perpendicular to one another, while in IFN-c the domains are oriented at an angle of about 60 . This topological similarity between IL-10 and IFNc may be important in interpreting the relationship that exists between their biological functions. Studies on the crystal structure of IL-10 bound to a soluble IL-10R1 complex reveal that there are two primary sites for IL-10 and IL-10 receptor interaction. The IL-10/IL-10R1 site I interface has a total of 27 residues from IL-10 that contact IL-10R1, and residues are donated from two discontinuous peptide segments that correspond to helix A, the AB loop, and helix F. The IL-10/IL-10R1 site II interface consists of interactions from the N-terminus and DE loop of IL-10 (Zdanov et al., 1995, 1996; Walter and Nagabhushan, 1995; Josephson et al., 2001).

VIRAL IL-10 There are several viruses, including Epstein–Barr virus (EBV), equine herpes virus type 2, orf virus (OV), and human cytomegalovirus that encode homologues of IL-10 which have functions similar to cellular IL-10 (Moore et al., 1990; Rode et al., 1993; Fleming et al., 1997; Kotenko et al., 2000). It was postulated that these viruses might take advantage of these IL-10-like products to suppress the production of early immunoregulatory cytokines, leading to ineffective immune responses. BCRF1, the EBV homologue of IL-10, is termed viral

IL-10 (vIL-10). Viral IL-10 exhibits high DNA and amino acid sequence homology to both mIL-10 and hIL-10. Viral IL-10 is expressed in the late phase of the lytic cycle of EBV and has 85% amino acid identity to hIL-10 and has 71% homology with hIL-10 at the DNA sequence level (Moore et al., 1990, 1993). vIL-10 was shown to have identical immunosuppressive properties to cIL-10, inhibiting IFNc production (Hsu et al., 1990), MHC class II expression (de Waal Malefyt et al., 1991), T cell proliferation (Del Prete et al., 1993), and B cell IgE production (Punnonen et al., 1993), further reinforcing the notion that IL-10 is an immunosuppressive cytokine. vIL-10, however, lacks several of the immunostimulatory activities of cellular IL-10 on certain cells. vIL-10 neither effectively enhanced class II MHC expression on mouse B cells, nor costimulated mouse thymocyte or mast cell proliferation (Go et al., 1990; MacNeil et al., 1990). In vivo, it has been shown that cIL-10 can be immunosuppressive or immunostimulatory, while vIL-10 is mostly immunosuppressive. Thus, experiments in which immunogenic and allogeneic mouse tumor cells were infected with recombinant retroviruses expressing mIL-10 or vIL-10 cDNAs, showed that mIL-10 accelerated and vIL-10 impeded tumor rejection (Suzuki et al., 1995). Experiments also showed that rejection of mouse heart allografts was substantially inhibited when the grafts were induced to express vIL-10, but not mIL-10 (Qin et al., 1996). These observations suggest that vIL-10 has conserved only a subset of activities of cIL-10, perhaps as a pathogenetic mechanism. These data also suggest the existence of at least two functional domains, only one of which has been conserved by EBV. The most significant difference in the amino acid sequence of viral and cellular IL-10 is found at the N terminus. The crystal structure of vIL-10 shows its structure is similar to that of human IL-10, forming an intercalated dimer of two 17 kDa polypeptides. Viral IL-10 exhibits novel conformations of the N-terminal coil and of the loop between helices A and B compared with hIL-10. It was suggested these differences might account for the different activities between vIL-10 and cellular IL-10 (Zdanov et al., 1997). Viral IL-10 binds to the IL-10R at least 1000-fold less avidly than cIL-10 (Liu et al., 1997), but like cIL-10 requires the presence of both IL-10R1 and IL-10R2 to induce

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IL - 10 EXPRESSION

signaling and immune responses (Ding et al., 2001). Mutagenesis of cIL-10 and vIL-10 reveals that the single amino acid isoleucine at position 87 of cIL-10 is required for its immunostimulatory function. Substitution of isoleucine in cIL-10 with alanine, which corresponds to the vIL-10 residue, abrogates immunostimulatory activity for thymocytes, mast cells and alloantigenic responses, while preserving immunosuppressive activity for inhibition of IFNc production and prolongation of cardiac allograft survival. Conversely, substitution of alanine with isoleucine in vIL-10 converts it to a cIL-10 like molecule with immunostimulatory activity. The mechanism of this switch is mostly due to changes in receptor binding affinity (Ding et al., 2000). Human cytomegalovirus encodes a viral IL-10 homologue designated cmvIL-10, and has only 27% identical at protein level with hIL-10. It is able to bind to the human IL-10 receptor and compete with human IL-10 for binding sites, and requires both subunits of the IL-10 receptor complex to induce signaling and biological function (Kotenko et al., 2000). A new cellular homologue of interleukin-10, AK155, was identified by transformation of T lymphocytes with Herpesvirus saimiri. AK155 protein shows 24.7% amino acid identity and 47% amino acid similarity to hIL-10, and has been mapped to the human chromosome 12q15. The biological function of this homologue is not known (Knappe et al., 2000). There is also a new molecule that is structurally related to IL-10 and named IL-22, which shares the same IL-10R2 with IL-10. Little is known about the biological activities of IL-22, and it has been described to up-regulate acute phase reactant production by hepatocytes (Dumoutier et al., 2000, 2001; Kotenko, 2001). There are some other forms of IL-10 that have been identified. It has been reported that a carboxylterminal peptide of human IL-10 (amino acids 152–160) has the ability to mimic some of the immunosuppressive activities of IL-10, while a NH2-terminal hIL-10 peptide (amino acids 1–9) did not reveal cytokine synthesis inhibitory properties, but induced mast cell proliferation (Gesser et al., 1997). A mononeric form of human IL-10 has also been engineered, by inserting six amino acids (GGGSGG) into the D-E loop, connecting the swapped secondary structural elements between Asn116 and Lys117. The mononeric form (IL-10M1) is stable under native condi-

607

tions, and structurally similar to one domain of IL-10. IL-10M1 binds to sIL-10R1 with a 1:1 stoichiometry and is biologically active (Josephson et al., 2000).

IL-10 EXPRESSION IL-10 was originally described as CSIF, produced by mouse TH2 cells in response to ConA or antigen stimulation, and inhibited IFN-c production by TH1 cells (Fiorentino et al., 1989). Further experiments demonstrate that IL-10 is expressed in a variety of cells, including T cells, B cells, macrophages/monocytes, NK cells, keratinocytes, eosinophils, mesangial cells, epithelial cells, and tumor cells. Activated TH0, TH1, and TH2-like CD4 T cells, as well as CD8 T cells, all express IL-10; CD4CD45RA cells produce low levels of IL-10, whereas CD4CD45RA ‘memory’ cells produce higher levels of IL-10 (Yssel et al., 1992; Del Prete et al., 1993; Sad et al., 1995). IL-10 has been implicated in the induction of regulatory or suppressor TH cell subsets and it also appears to contribute to their effector function (Groux et al., 1997). Normal B cells stimulated with LPS and B lymphomas constitutively express IL-10 (Benjamin et al., 1992; O’Garra et al., 1990). Monocytes activated by LPS produce high levels of IL-10 in a dose-dependent fashion (de Waal Malefyt et al., 1991). NK cells stimulated with IL-2 produce IL-10, and IL-12 can synergize with IL-2 in inducing IL-10 mRNA expression and protein synthesis (Mehrotra et al., 1998). Keratinocytes are also capable of secreting IL-10, and hapten can enhance IL-10 production (Enk and Katz., 1992). Normal peripheral blood eosinophils synthesize IL-4, IL-8 and IL-10 (Nakajima et al., 1996). Kidney mesangial cells express IL-10 mRNA and secret IL-10 protein (Fouqueray et al., 1995). Normal bronchial epithelial cells constitutively produce IL-10 (Bonfield et al., 1995). Human carcinoma cell lines are capable of secreting IL-10, which can be stimulated by IFN-c and IL-4 (Gastl et al., 1993; Kim et al., 1995). IL-10 production is induced by many pathogens which can directly activate monocytes/macrophages (bacterial wall components, parasites, fungi, HIV) (Sieling et al., 1993; Borghi et al., 1995; Vecchiarelli et al., 1996; Brigino et al., 1997), B cells (EB virus) (Burdin et al., 1993), or T cells (human T cell leukemia virus type 1) (Mori et al., 1996). In the course of severe

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infections and stress conditions, cytokines, hormones and arachidonic acid derivatives are released and upregulate IL-10 synthesis in monocytes, macrophages and T cells (Le Moine et al., 1994, 1996; Marchant et al., 1994a; Derkx et al., 1995). Elevated IL-10 expression is associated with some human diseases, such as septicemia (Marchant et al., 1994b), bacterial meningitis (Frei et al., 1993), malaria (Peyron et al., 1994), rheumatoid arthritis (Katsikis et al., 1994), lepromatous leprosy (Yamamura et al., 1991), visceral leishmaniasis (Ghalib et al., 1993), and lymphatic filariasis (King et al., 1993). IL-10 protein is also detected in many tumors, such as Burkitt’s lymphoma (Benjamin et al., 1992), AIDS lymphoma (Emilie et al., 1993), non-Hodgkin’s lymphoma (Blay et al., 1993), multiple myeloma (Merville et al., 1992), melanoma (Becker et al., 1994), ovarian cancer and other intra-abdominal cancers (Gotlieb et al., 1992). High levels of IL-10 are also produced by CD4 hostreactive T cell clones isolated from SCID patients transplanted with allogenic fetal hematopoietic stem cells. Both donor T cells and host accessory cells contribute to the production of IL-10, which may inhibit the in vivo activity of host reactive T cells (Bacchetta et al., 1994).

The IL-10 promoter contains several elements participating in the regulation of IL-10 gene expression, such as a cAMP-response element (CRE), NF-kB binding sites, a glucocorticoid response element (GRE), and an AUUUA motive (Moore et al., 1990; Kim et al., 1992; Eskdale et al., 1997). Hormones, PGE2, and other cAMP elevating factors increase cytoplasmic cAMP level, activate protein kinase A (PKA), and lead to phosphorylation of cAMP-response element binding protein (CREB); CREB binds to the CRE in the IL-10 promoter, increasing IL-10 expression. LPS, TNF-a, reactive oxygen species, UV light, EBV and HTLV-1 are NF-kB activators; NF-kB binds to the NF-kB binding site in the IL-10 promoter, and induces IL-10 synthesis. Glucocorticoids bind to the GRE in the IL-10 promoter and induce IL-10 expression. Adenosine-uridine (AU) instability elements are present in the 3-untranslated regions of numerous mRNAs to target rapid degradation of mRNA. Six AUUUA motives located in the IL-10 mRNA 3-untranslated regions destabilize IL-10 mRNA and inhibit IL-10 production (Brown et al., 1996).

IL-10 REGULATION

Because of its potent immunosuppressive and antiinflammatory properties and because of its widespread expression in multiple cell types, IL-10 likely plays an important role in many human disease states, including inflammation, autoimmunity, angiogenesis and transplant rejection.

IL-10 production is regulated by other cytokines. IL-4, IL-13, and IFN-c inhibit IL-10 production in monocytes activated by LPS (Chomarat et al., 1993; de Waal Malefyt et al., 1993). IL-10 itself also strongly inhibits IL-10 mRNA synthesis, suggesting IL-10 has autoregulatory activities (de Waal Malefyt et al., 1991, 1993). In contrast, IL-1, IL-2, IL-3, IL-6, IL-7, IL-12, and IL-15 induce IL-10 production in monocytes, T cells, NK cells, B cells and mast cells (Tilg et al., 1995; Daftarian et al., 1996; Jeannin et al., 1996; Marietta et al., 1996; Cohen et al., 1997; Dorholz et al., 1997; Foey et al., 1998). TNF-a induces IL-10 production in monocytes and mouse liver (Wanidworanun and Strober, 1993; Barsig et al., 1995). IFN-a induces IL-10 production in human T cells and monocytes (Aman et al., 1996; Rep et al., 1996). TGF-b induces IL-10 production in macrophages, mesangial cells, and liver cell lines (Fouqueray et al., 1995; Maeda et al., 1995; Ishizaka et al., 1996).

IL-10 AND HUMAN DISEASE STATES

IL-10 AND INFLAMMATION The ability of IL-10 to inhibit the production of proinflammatory cytokines (such as TNF-a and IL-1) (de Waal Malefyt et al., 1991), as well as to induce the production of anti-inflammatory agents (such as IL-1 receptor antagonist, IL-1RA) (Cassatella et al., 1994; Jenkins et al., 1994), suggests that IL-10 may inhibit inflammation mediated by TH1 cells in vivo. IL-10 has protective effects in experimental endotoxemia, and rescues mice from LPS-induced toxic shock, which is correlated with reduced levels of serum TNF-a (Gerard et al., 1993; Marchant et al.,

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IL - 10 AND EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS ( EAE )

1994b). IL-10 inhibits the production of TNF-a and MIP-2, regulates hemodynamic parameters, leukocyte–endothelial cell interactions, and microvascular permeability and reduces mortality in experimental endotoxemia (Standiford et al., 1995; Hickey et al., 1998). Mice treated with anti-IL-10 from birth or IL-10-deficient mice are more susceptible to endotoxin-induced shock than normal mice (Ishida et al., 1993; Berg et al., 1995). Human volunteers receiving IL-10 after endotoxin challenge suffer fewer systemic symptoms and less cytokine production.

IL-10 AND INFLAMMATORY BOWEL DISEASE (IBD) IBD is a group of chronic intestinal inflammatory disorders, generally divided into ulcerative colitis (UC) and Crohn’s disease (CD). The etiology of IBD is not clear, disregulation of the intestinal immune system may lead to the pathogenesis of IBD (Fiocchi, 1998). IL-10-deficient and IL-10R2-deficient mice develop chronic intestinal inflammatory disorders, and are successfully treated by administration of IL-10 (Kuhn et al., 1993), indicating the role of IL-10 in the development of IBD, and the effect of IL-10 in the treatment of IBD. Transfer of CD4CD45RBhigh T cells (which produce IFN-c and TNF-a) into SCID mice causes IBD (Powrie et al., 1993), and administration of CD4 CD45RBlow T cells (which produce IL-4 and IL-10) or mIL-10 prevent the development of IBD (Powrie et al., 1994). CD4CD45RBhigh T cells isolated from IL-10 transgenic mice do not induce IBD, but inhibit IBD induced by wild-type CD4CD45RBhigh T cells (Hagenbaugh et al., 1997). Furthermore, intraperitoneal injection of adenovirus producing IL-10 successfully prevents experimental colitis in rats (Barbara et al., 2000), and oral treatment with mIL-10 reduces colitis in mice and prevents colitis in IL-10-deficient mice (Steidler et al., 2000). A double-blind, randomized, multicenter trial in humans has been conducted to evaluate the safety, tolerance and pharmacokinetics of IL-10 in the treatment of Crohn’s disease. The results indicate that intravenous bolus injection of IL-10 over 7 days is safe, well tolerated, and results in clinical improvement (van Deventer et al., 1997). Another trial shows

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that subcutaneous administration of rhuIL-10 for 28 days to mild to moderately active Crohn’s disease patients is safe, well tolerated, and clinically efficacious; 23.5% patients receiving 5 lg/kg rhuIL-10 achieve clinical remission and endoscopic improvement at the end of treatment (Fedorak et al., 2000).

IL-10 AND AUTOIMMUNITY IL-10 is an immunosuppressive molecule, protects against autoimmunity, and exerts diverse effects on autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), and insulindependent diabetes mellitus (IDDM).

IL-10 AND EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) EAE is a myelin protein-specific, CD4 TH1 cellmediated, inflammatory disease of the central nervous system, and is an animal model for multiple sclerosis (MS) (Owens and Sriram, 1995). IL-10 as a TH2 immunoregulatory cytokine down-regulates TH1 responses and macrophage functions. However, the effect of IL-10 in the treatment of EAE is controversial, and various reports have yielded conflicting results. Initial studies show that IL-10 mRNA in the spinal cord dramatically rises with the recovery of EAE, and intracerebral synthesis of IL-10 is also increased during the recovery phase of EAE (Kennedy et al., 1992). IL-10-deficient mice develop more severe EAE and do not achieve spontaneous recovery, suggesting an important role of IL-10 in EAE (Samoilova et al., 1998). IL-10 prevents the induction of EAE in Lewis rats and SJL mice upon systemic administration during the initial phase of the disease (Rott et al., 1994; Nagelkerken et al., 1997). IL-10 decreases the relapse of EAE when given together with TNF-a, and neutralization of IL-10 increases the incidence and the severity of the relapse (Crisi et al., 1995). Adenovirusmediated IL-10 gene transfer inhibits development and prevents subsequent relapse of EAE (Cua et al., 2001). Murine IL-10 transgenic mice are protected from EAE induced by PLP immunization (Bettelli et al., 1998). Human IL-10 transgenic mice are highly

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resistant to EAE, mediated by suppression of autoreactive T cell function (Cua et al., 1999). In contrast, intravenous injection of IL-10 exacerbates the disease in an adoptive transfer model of EAE (Cannella et al., 1996). Intracranial injection of IL-10 or IL-10 plasmid does not inhibit EAE (Croxford et al., 1998). Adoptive transfer of a myelin basic protein (MBP) specific T cell hybridoma transduced with IL-10 also does not suppress EAE (Shaw et al., 1997). Hence, the role of IL-10 in EAE may be related to the route and timing of cytokine administration.

IL-10 AND INSULIN-DEPENDENT DIABETES MELLITUS (IDDM) IDDM is a chronic autoimmune disease in humans, resulting from the destruction of insulin-secreting pancreatic b cells by an autoimmune response directed against certain b-cell constituents (autoantigens) (Bach, 1994). The non-obese diabetic mice (NOD) spontaneously develop autoimmune diabetes at 15 to 20 weeks of age, and have been used as animal models for IDDM (Kikutani et al., 1992). Many experiments show that IL-10 plays paradoxical roles in the immunopathogenesis of experimental IDDM. Systemic administration of IL-10 to adult NOD mice delays the onset of diabetes, indicating the potential therapeutic effect of IL-10 in autoimmune diabetes. Daily subcutaneous treatments of IL-10 to 9- and 10-week-old NOD mice delay the onset and decrease the incidence of diabetes (Pennline et al., 1994). Combined therapy with IL-4 and IL-10 suppresses TH1 cytokine production in the islet grafts and inhibits IDDM recurrence in syngeneic islettransplanted NOD mice (Rabinovitch et al., 1995). IL-10/Fc protein, a long-lived noncytolytic fusion protein, completely prevents the occurrence of diabetes in 5- to 25-week-old NOD mice, and the mice remain disease-free for a long time after cessation of IL-10/Fc therapy (Zheng et al., 1997). Intramuscular plasmid injection provides long-term systemic delivery of cytokines. When an IL-10 expression plasmid is injected into the skeletal muscles of NOD mice, the incidence of diabetes is significantly reduced (Nitta et al., 1998). Intravenous injection of IL-10 plasmid

also prevents autoimmune insulitis in NOD mice (Koh et al., 2000). In contrast, transgenic expression of IL-10 on b cells accelerates the development of diabetes in NOD mice, suggesting a proinflammatory effect of IL-10 in the pathogenesis of autoimmune diabetes (Wogensen et al., 1994). In IL-10 transgenic NOD mice, the incidence of diabetes in the first and second generations is greatly increased, the onset of diabetes is much earlier than in the non-transgenic NOD mice, and histological analysis shows severe insulitis and prominent ductal proliferation (Moritani et al., 1994). IL-10 transgenic NOD mice backcrossed with NOD.B6 Idd3 Idd10 mice, which have diabetes-resistance alleles at Idd3 and Idd10 on chromosome 3 and have a very low frequency diabetes, results in IL-10 transgenic backcross mice which develop diabetes (Lee et al., 1997). As for EAE, the role of IL-10 in the pathogenesis of autoimmune diabetes is not clear at this time. Its effects may depend on timing, location and dose of cytokine, along with contributions from other regulatory events.

IL-10 AND ANGIOGENESIS IL-10 inhibits the production of a variety of cytokines, and is a potent inhibitor for angiogenesis in various cancers. IL-10 down-regulates the production of macrophage-derived angiogenic factors, such as VEGF, IL-1b, TNF-a, IL-6 and matrix metalloproteinase-9 (MMP-9), inhibits angiogenesis, and hence inhibits tumor growth and metastasis (Huang et al., 1996). IL-10 stimulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production, inhibits MMP-2 and MMP-9 production, inhibits angiogenesis in human prostate cancer cells in vitro, and significantly increases mouse survival after implantation with IL-10- and TIMP-1-expressing tumors in vivo (Stearns et al., 1999a, 1999b). Transferring the IL-10 gene into Burkitt’s lymphoma cells significantly reduces tumor formation in SCID mice, with IL-10 inhibiting VEGF-induced neovascularization and proliferation of microvascular endothelial cells (Cervenak et al., 2000). Local expression of vIL-10 in thrombosed veins decreases thrombosis-associated inflammation and inhibits the expression of cell adhesion molecules, such as P- and E-selectin (Henke et al., 2000).

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IL - 10 RECEPTOR EXPRESSION AND REGULATION IN DISEASE STATES

The role of IL-10 in angiogenesis was also studied in a hindlimb ischemia model in IL-10/ and IL-10/ mice. In IL-10/ mice, angiogenesis in the ischemic hindlimb is greatly increased, and VEGF expression is also increased. After transferring IL-10 cDNA into IL-10/ mice, angiogenesis and VEGF expression are all significantly inhibited, suggesting the effect of IL-10 on angiogenesis via down-regulation of VEGF expression (Silvestre et al., 2000). Further experiments show MMP-2 and MMP-9 are increased in the ischemic hindlimb in IL-10/ mice, and the MMP inhibitor BB-94 abolishes the increase in vessel density and blood perfusion index, suggesting a role for MMP in angiogenesis (Silvestre et al., 2001).

IL-10 AND TRANSPLANT REJECTION Many experiments have demonstrated that IL-10 promotes TH2 cells, but inhibits TH1 cells, inhibits antigen presenting cell functions, and inhibits the production of IL-2, IFN-c and other cytokines by T cells (Del Prete et al., 1993). Further studies show that IL-10 inhibits the production of IL-2, IL-2 receptor and IFN-c in human mixed lymphocyte culture (Danzer et al., 1994), inhibits allogeneic T cell response to human epidermal Langerhans cells (Peguet-Navarro et al., 1994), and protects target cells from allospecific cytotoxic T cells (Matsuda et al., 1994). These results suggest that IL-10 may play a protective role in transplant rejection, although as for autoimmune disease the role and effect of IL-10 are diverse and at times seemingly contradictory. IL-10 prolongs allograft survival in non-vascularized and vascularized transplantation in mice and rats (Qin et al., 1995, 1997; Debruyne et al., 1998; David et al., 2000; Mulligan et al., 2000). Adenovirusmediated IL-10 gene transfer reduces allograft rejection in sheep corneal transplants (Klebe et al., 2001). Cationic lipid-mediated hIL-10 gene transfer prolongs survival of allogenic hepatocytes in rats (Fabrega et al., 1996). Intracoronary infusion of an adenoviral vector expressing IL-10 efficiently prolongs graft survival in rabbits (Brauner et al., 1997). Injection of rhIL-10 down-regulates cytokine-induced neutrophil chemoattractant and prolongs liver allograft survival in rat orthotopic liver transplantation (Zou

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et al., 1998). Intraportal injection of an adenoviral vector expressing IL-10 significantly prolongs survival in orthotopic liver transplantation (Shinozaki et al., 1999). IL-10/Fc fusion protein inhibits macrophagemediated immune responses and prolongs pancreatic islet xenograft survival (Feng et al., 1999). In contrast, infusion of IL-10 in murine recipients of allogeneic bone marrow grafts accelerates graft rejection and increases GVHD-induced mortality (Blazar et al., 1995). mIL-10 inhibits the proliferation of donor spleen cells in MLR assay, but cannot reduce morbidity and mortality from GVHD (Emmanouilides et al., 1996). Postoperative intraperitoneal administration of mIL-10 simulates B cell and T cell alloimmune responses, and exacerbates allograft rejection in heterotopic vascularized heart transplant in mice (Qian et al., 1996). Subconjunctival or intraperitoneal administration of mIL-10 does not prolong corneal allograft survival and even accelerates rejection (Torres et al., 1999). Viral IL-10, encoded by Epstein–Barr virus BCRF I open reading frame, has the immunosuppressive activity of cellular IL-10, but lacks the immunostimulatory activity, and is a more potent immunosuppressant (Hsu et al., 1990; Moore et al., 1990). Plasmid-mediated or adenovirus-mediated vIL-10 gene transfer prolongs allograft survival in nonvascularized cardiac transplantation (Qin et al., 1997, 1998). Retrovirus-mediated vIL-10 gene transfer also prolongs allograft survival, whereas murine IL-10 gene transfer does not prolong graft survival (Qin et al., 1996). Similar results were also observed in tumor rejection: vIL-10 inhibits the process of immune rejection in tumors and vIL-10 transduced tumors still grow, whereas mIL-10 causes tumor rejection and inhibits tumor growth (Suzuki et al., 1995). Hence, vIL-10 may act as a more effective therapeutic agent in transplant rejection.

IL-10 RECEPTOR EXPRESSION AND REGULATION IN DISEASE STATES The IL-10 receptor belongs to the class II cytokine receptor family, other members of which include the receptors for both type I and II interferons, tissue

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factor and the orphan receptors CRF2-8 and CRF2-9. Several recently discovered members of the cytokine receptor class II family have been shown to be structurally related to the IL-10 receptor. These include the receptors for IL-20 and IL-22 (Blumberg et al., 2001; Dumoutier et al., 2001; Kotenko et al., 2001a). However, while signaling through these receptors seems to activate the same signal transduction components, they lead to different cellular responses, suggesting the presence of additional pathways and regulatory levels. RNA blot analysis of human tissues revealed that IL-10R is expressed at high levels in spleen, thymus and PBMC, and at much lower levels in nonhematopoietic tissues (Liu et al., 1994). As a result, IL-10 receptor expression has mainly been characterized in cells of hematopoietic lineage, such as dendritic cells, B cells, T cells, NK cells, neutrophils, monocytes and macrophages. While known for its anti-inflammatory effects, IL-10 in addition has been shown to induce a variety of responses in different cell types. In dendritic cells, it blocks cellular maturation and inhibits the expression of surface molecules involved in activation of TH1 cells. In macrophages it inhibits the synthesis of TNFa, IL-1, IL-6, IL-8, IL-12 and GM-CSF induced by IFNc or LPS and downregulates the expression of MHC class II molecules and production of reactive oxygen and nitrogen intermediates (Donnelly et al., 1999). In NK cells, signaling from IL-10 receptor augments IL-2-induced proliferation and increases cytotoxic activity of the cells (Carson et al., 1995). In B cell chronic lymphocytic leukemia cells, IL-10 has been shown to inhibit proliferation and induce differentiation (Jurlander et al., 1997). IL-10 receptors are also expressed intracellularly, in specific granules of human neutrophils. Stimulation of neutrophils with proinflammatory mediators, such as TNFa and GM-CSF, results in mobilization of the granules to the plasma membrane, causing higher levels of receptor expression in the membrane (Elbim et al., 2001). This suggests an important role for IL-10 in the regulation of inflammatory responses in neutrophils. Other cells, while not expressing the receptor, possess the signal transduction components necessary for receptor signaling. Murine pro-B-cells (Ba/F3) stably transfected with IL-10R1 chain can proliferate and differentiate in response to IL-10 stimulation (Ho et al., 1995).

IL-10 receptor expression is involved in mediation of inflammatory response by other cells as well. Functional IL-10 receptors were found to be expressed by oligodendrocytes in the brain (Molina-Holgado et al., 2001). Treatment of oligodendrocytes with IFNc or LPS reduces cellular proliferation and survival due to induction of nitric oxide synthase-mediated inflammation. IL-10 suppresses iNOS expression and reduces inflammation, improving cellular survival and proliferation. This suggests a possible protective role for IL-10 in demyelinating diseases. IL-10 receptor expression was also shown to be present in human epidermal cells (Michel et al., 1997). In acute exanthematic psoriatic epidermis, a disorder characterized by epidermal hyperproliferation and inflammation, the expression of IL-10 receptor is dramatically downregulated, suggesting a possible role for IL-10 in regulation of inflammatory responses in epidermis. In addition to the cells constitutively expressing the IL-10 receptor, certain cells express the receptor in response to inflammatory stimuli. Treatment of murine fibroblasts with LPS induced IL-10 receptor mRNA expression and appearance of the receptor on the membrane surface within 24 h (Weber-Nordt et al., 1994). In dendritic cells, IL-10 receptor expression is inhibited upon stimulation with TNFa or soluble CD40L, but up-regulated when stimulated with IL-10. In psoriatic epidermal cells, IL-10R expression is down-regulated by the proinflammatory cytokine IL-8 (Michel et al., 1997). While in many cells IL-10 activates a signaling pathway that initiates anti-inflammatory response, it serves as a growth factor for other cells, as seen in certain IL-10R-expressing tumors, such as melanoma cells. IL-10 in these cells does not only induce proliferation, but also down-regulates the expression of both classes of MHC complexes, enhancing resistance of the tumor cells to cell-mediated immune response (Yue et al., 1997). These facts must be taken into consideration when planning anti-inflammatory therapy with IL-10.

IL-10 RECEPTOR STRUCTURE The IL-10 receptor is a heterodimer composed of subunits known as IL-10 receptor 1 (IL-10R1) and IL-10

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receptor 2 (IL-10R2), which was formerly known as orphan receptor CRF2-4. The cDNA for mouse IL-10R1 was first isolated from murine macrophage (J774) and mast cell (MC/9) lines (Ho et al., 1993). RNA blot analysis in these cells revealed mIL-10R mRNA to be approximately 3.6 kb long. The human IL-10R1 gene was mapped to chromosome 11q23.3 (Taniyama et al., 1995). Human IL-10R1 messenger RNA is around 3.6 kb long and encodes a polypeptide of approximately 110 kDa (Liu et al., 1994). The predicted amino acid sequences of murine and human IL-10R1 are 60% identical and 73% similar. While the human IL-10 receptor is speciesspecific, murine IL-10 receptor binds both human and murine IL-10 with comparable affinity. The Kd of binding is in the 50–200 pM range (Tan et al., 1993). Sequence analysis and predicted structure revealed that IL-10 receptor is related to interferon receptors (Ho et al., 1993). The polypeptide was shown to be 578 amino acids long, with a putative signal peptide sequence of 21 amino acids, a 215-amino acid extracellular domain, a transmembrane segment of 25 amino acids, and a cytoplasmic domain of 317 amino acids. While the calculated molecular mass is approximately 61 kDa, the observed size is around 110 kDa, suggesting glycosylation on at least one extracellular site of the six identified (Liu et al., 1994). Structurally, the extracellular portion of mIL-10R consists of two homologous segments of approximately 110 amino acids (Ho et al., 1993). A soluble form of the extracellular domain of hIL-10R1 (sIL-10R1) has been cloned and expressed (Tan et al., 1995). The soluble receptor was shown to be capable of binding hIL-10 and inhibiting its biological activity. From the analysis of this binding, the stoichiometry of the ligand-IL-10R1 interaction was determined. Crystallographic analysis of the sIL-10–R1/IL-10 interaction revealed a complex consisting of two IL-10 dimers and four soluble IL-10R1s (Josephson et al., 2001). The stoichiometry of the vIL-10:hIL-10R complex has been determined to be the same. While the domains of sIL-10R1 are structurally similar to IFNcR1, the interdomain angle in sIL-10R1 is around 90°, which is more similar to cytokine class I receptors, rather than 120° observed for cytokine class II receptors. The topology of each domain is closely related to FBN-III modules that form a sandwich from two antiparallel beta sheets.

RECEPTOR SIGNALING Functionally important regions of the intracellular domain of the murine IL-10R were identified using deletion and substitution mutagenesis approaches. Two tyrosine residues (Y427 and Y477) have been found to be important in recruitment and activation of Stat3, but not Stat1 or Stat5 (Weber-Nordt et al., 1996). Synthetic peptides encompassing either one of the two tyrosine residues co-precipitate with Stat3. Tyrosines 427 and 477 in the murine receptor correspond to the tyrosines 446 and 496 in their human counterpart. The sequences surrounding each of the conserved tyrosines have also been found to be highly conserved between murine and human receptors. For the murine IL-10 receptor, the sequence around tyrosine 427 it is FQGYQKQTR and around tyrosine 477 it is AAGYLKQES. For the human receptor, the sequence around tyrosine 446 is FQGYLRQTR and around tyrosine 496 it is AKGYLKQD. Three distinct regions, responsible either for inhibition or induction of proliferation, or for antiinflammatory pathway, have been identified in the cytoplasmic domain of the IL-10R1 (Ho et al., 1995; Riley et al., 1999). The first region, containing the two important tyrosines, is required for proliferation, which is mediated by recruitment and activation of Stats. Substitution of individual tyrosine residues with phenylalanine had no effect on proliferation or antiinflammatory pathway, suggesting that the tyrosines are functionally redundant (Weber-Nordt et al., 1996; Riley et al., 1999). However, substitution of both tyrosines ablated both proliferative and antiinflammatory responses, suggesting that Stat recruitment is important for activation of both pathways. The second cytoplasmic domain region of functional importance is the C-terminal sequence of approximately 30 amino acids, deletion of which had no effect on proliferation of B cells, but ablated the antiinflammatory pathway in LPS-stimulated macrophages (Ho et al., 1995; Riley et al., 1999). This sequence was shown to contain at least one serine residue that is functionally important. Substitution of C-terminal serine residues with alanines led to the same effect as deletion of the C-terminal sequence. Finally, there is a membrane-proximal region, which is most likely involved in suppression of signaling, since removal of this region resulted in

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much higher sensitivity to IL-10 stimulation (Ho et al., 1995). While the first chain of the IL-10R is capable of binding the ligand on its own and possesses Statrecruitment sites, it requires a second chain for initiating a signaling pathway. The second chain of the receptor was originally identified as an orphan cytokine receptor CRF2-4. The gene was mapped to chromosome 16 in mice and chromosome 21 in humans (Gibbs and Pennica, 1997). The human gene spans more than 30 kb and encodes a 325 amino acid protein, which is 69% identical with its 349 amino acid long murine counterpart (Lutfalla et al., 1993; Gibbs and Pennica, 1997). It is a transmembrane protein, with a 19 amino acid leader, extracellular portion of 201 amino acids, 29 amino acid transmembrane domain, and 76 amino acid intracellular portion. Its relatively close location to the IFNaR gene (less than 35 kb away) and its homology in structure to the interferon receptors suggested that the receptor belongs to the interferon-class II cytokine receptor family and possibly plays a role in interferon signaling. It was later shown that CRF2-4 receptor is an integral component of the IL-10 receptor complex. CHO cells transfected with IL-10R1 chain, while being capable of binding IL-10, were unable to initiate the IL-10 signal transduction pathway (Kotenko et al., 1997). Co-expression of the CRF2-4 with IL-10R1 renders CHO cells sensitive to IL-10. Immunoprecipitation experiments have shown that CRF2-4 associates with IL-10R1 in the cells, further confirming its role. In order to characterize the role of CRF2-4 in signaling, the transmembrane and intracellular domains of CRF2-4 and IL-10R1 were substituted by transmembrane and intracellular domains of IFNc receptor II and I, respectively. Expression of this chimeric receptor complex in COS cells rendered the cells capable of initiating an IFNc response (measured by induction of an MHC class I reporter gene) in response to IL-10 stimulation. Unlike IL-10R1, CRF2-4 is constitutively expressed in most cells and tissues and has not been found to be regulated by any stimuli. Interestingly, it was shown that CRF2-4 also functions as a second chain of the receptor for IL-22, an IL-10 homologue (Kotenko et al., 2001b). The importance of CRF2-4 receptor was further demonstrated in CRF2-4 knockout mice (Spencer

et al., 1998). These mice are developmentally normal, however they do not respond to IL-10 and develop chronic colitis and splenomegaly, similar to the IL-10 knockout models. The mice showed normal responses to interferon, suggesting that CRF2-4 is not obligatory for interferon signaling.

JAK-STAT ACTIVATION AND TRANSCRIPTIONAL REGULATION Stimulation of T cells and monocytes with IL-10 leads to assembly of the IL-10 receptor complex consisting of the ligand dimer, two IL-10R1 chains, and two IL-10R2 chains, which results in induced proximity between their respective associated Janus kinase family members (Josephson et al., 2001). IL-10R1 has been shown to associate with Jak1, while IL-10R2 is associated with Tyk2 (Kotenko et al., 1996). Receptor assembly leads to tyrosine phosphorylation of members of Janus kinase family Jak1 and Tyk2, but not Jak2 or Jak3 (Finbloom and Winestock, 1995). Pathway specificity, however, is primarily dictated by tyrosinebased motifs in receptor components, while Jaks have been shown to be redundant in function (Stahl et al., 1995). Upon assembly of the receptor complex, Janus kinases trans-phosphorylate each other and the intracellular domain of the IL-10R1, leading to recruitment of Stats, predominantly Stat3 (Finbloom and Winestock, 1995). Electrophoretic mobility shift assays revealed that in all cell populations expressing IL-10 receptor, IL-10 stimulation results in activation of Stat1 and Stat3, while in pro-B-cells expressing IL-10 receptor, Stat5 is also activated (Weber-Nordt et al., 1996). Neutrophils, monocytes, dendritic cells, mast cells and NK cells have been shown to primarily activate Stat3 and Stat 1 upon IL-10 stimulation (Carson et al., 1995; Weber-Nordt et al., 1996; Mirmonseff et al., 1999; Corinti et al., 2001; Crepaldi et al., 2001). Phosphorylation of Stat3 on Tyr-705, Stat1 on Tyr-701, and Stat5 on Tyr-694 have been found to be prerequisites for Stat dimer formation, nuclear translocation, binding to cognate DNA sequences, and regulation of target gene expression (Donnelly et al., 1999). Gel-shift experiments revealed formation of distinct homo- and heterodimeric transcriptionally active Stat complexes which assemble on specific Stat consensus

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elements of a selected gene promoter (Wehinger et al., 1996). Three complexes containing Stats 1, 3 and 5 bound to the GRR sequence of the FccRI promoter. A single gel-shift complex containing Stat5 homodimers was detected with a probe from the b-casein gene promoter. Finally, three distinct DNA-binding complexes containing Stat1 and Stat3 homo- and heterodimers were detected with a probe from the c-fos promoter. Despite the fact that several cytokines, such as IL-10 and IFNc, activate identical Stat proteins, they promote distinct cellular responses (Meraz et al., 1996). Knockout of the Stat1 gene in mice abolishes cellular response to interferons, rendering mice highly susceptible to microbial and viral infection. The response to IL-10, however, is not affected. In contrast, treatment of Stat3-deficient macrophages with IL-10 failed to inhibit LPS-induced TNFa production (Riley et al., 1999). EMSAs performed on IL-10-treated macrophages from Stat1 knockout mice revealed formation of only Stat3 homodimers. This suggests that in wildtype cells the IL-10 response is most likely primarily mediated by Stat3 homodimers, while the significance of Stat1-Stat3 heterodimers and Stat1 homodimers is not completely understood (Meraz et al., 1996). It is possible that these complexes play a functional role in cells other than macrophages, where other signaling pathways may play prominent roles. Stat3 activation by itself, however, is not sufficient for the IL-10-dependent inhibition of LPS-induced TNFa production, since IL-6, which also induces Stat3, does not inhibit TNFa production. While both IL-10 and IL-6 stimulate B cell proliferation, only IL-10 is able to induce strong anti-inflammatory response in macrophages, suggesting that there is a difference in receptor signaling between the two cytokines and that additional components and pathways might be involved (Riley et al., 1999).

INTERACTION WITH OTHER SIGNALING PATHWAYS There is evidence to suggest that IL-10 signal transduction pathway through Jak-Stat interacts with other pathways as well. The inflammatory mediators IL-1, TNFa and LPS have been shown to block the activation of Stat DNA binding and tyrosine phosphoryla-

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tion by IL-6 and IL-10 (Ahmed and Ivashkiv, 2000). The inhibition is rapid and is not affected by actinomycin D treatment, suggesting that it is independent of new gene induction. Inhibition of IL-6 signaling was found to be mediated by the p38 family of stressactivated protein kinases (SAPK), induced by TNFa in dendritic cells (Sato et al., 1999; Ahmed and Ivashkiv, 2000). Experiments involving truncated IL-6 receptors suggested that the target of inhibition for IL-6 signaling by SAPKs is contained within the membraneproximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor, which contains the p38MAPK consensus phosphorylation site. It is possible that inhibition of IL-10 signaling could be carried out in a similar way, since the membrane-proximal region of the cytoplasmic domain of IL-10 receptor was also previously identified as an inhibitory region, deletion of which increased sensitivity to IL-10 (Ho et al., 1995). In addition to JNK/SAPK, TNFa induces tyrosine phosphorylation of ERK2 and p38MAPK (Sato et al., 1999). Dual stimulation with TNFa and IL-10, however, abolished TNFa-induced changes and resulted in decreased phosphorylation of all MAPKs. IL-10 and TNFa in a dose-dependent fashion may thus modulate the properties of dendritic cells. The inflammatory pathway in monocytes is also induced via CD40 on the monocyte surface. Binding of CD40 to CD40 ligand on activated T cells results in activation of signaling in monocytes through extracellular signal-regulated kinases 1 and 2 (ERK 1/2) MAPKs, with subsequent production of pro-inflammatory cytokines. Pre-treatment of monocytes with IL-10 inhibits CD40-mediated activation of ERK 1/2 kinase activity by decreasing levels of phosphorylated ERK 1/2 without affecting levels of ERK expression (Suttles et al., 1999). There is evidence that the IL-10 pathway interacts with the PI3K pathway as well. In primary monocytes and the D36 cell line, IL-10 stimulates PI3K and activates p70 S6 kinase (Crawley et al., 1996; Williams et al., 2000). LY294002 and wortmannin (PI3K inhibitors) and rapamycin (S6 kinase inhibitor) inhibit proliferation, but not anti-inflammatory signaling and TNFa production, suggesting that PI3K and S6 kinase may be involved in a proliferation pathway. The mechanisms of PI3K and S6 kinase activation through IL-10 signaling, however, are still not clear.

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The interaction of the IL-10 pathway with other pathways not only takes place at the level of the receptor, but also at the level of transcriptional activation. Signaling by many proinflammatory cytokines and costimulatory proteins was demonstrated to be regulated by the transcription factor NFjB (Schottelius et al., 1999). NFjB is a heterodimer composed of p65 and p50 subunits, which resides in the cytoplasm as an inactive complex bound to the inhibitor protein IjB. In response to proinflammatory extracellular stimuli like TNFa and IL-1, IjB is phosphorylated by IjB kinase (IKK), which targets IjB for degradation and releases NFjB. NFjB is then able to migrate to the nucleus and activate transcription. IL-10 has been shown to inhibit TNFa-induced NFjB activity by blocking NFjB activity at two levels: through the suppression of IKK activity and through the inhibition of NFjB DNA binding activity (Clarke et al., 1998; Schottelius et al., 1999). The inhibition of DNA binding is not due to impairment in NFjB nuclear translocation, since even when the levels of NFjB are high in the nucleus, there is inhibition of NFjB DNA binding activity by IL-10. The inhibition is possibly through phosphorylation of p65/p50 subunits or interaction with other inhibitory nuclear proteins. IL-10 has been shown to suppress inflammatory signaling at the level of translation (Kontoyiannis et al., 2001). Stimulation of murine macrophages with IL-10 showed that IL-10 inhibited TNFa production by reducing translation of its mRNA. Treatment with actinomycin D revealed that IL-10 does not alter mRNA stability. Further experiments showed that IL-10 signaling results in inhibition of the p38/MAPK kinase pathway. The p38/MAPK cascade activates a number of downstream kinases, including the serine/threonine kinase MAPK-activated protein kinase 2 (MK2). MK2-deficient macrophages show reduced levels of TNFa production without changes in mRNA levels following LPS stimulus. MK2 has been shown to target the 3 AU rich elements (ARE) of TNF mRNA to activate its translation. Mice that lack ARE sequences in the TNF mRNA show reduced levels of TNFa protein production, indicating that ARE sequences must play a role in regulation of TNFa mRNA translation (Kontoyiannis et al., 2001). This is thus another example pointing out the possible connection between the IL-10 signaling pathway and the MAPK pathway.

SOCS REGULATION IL-10 signaling through Jak-Stat pathway has been shown to lead to induction of SOCS proteins involved in negative regulation of signaling. SOCS (suppressor of cytokine signaling) is a family of proteins containing an SH2 domain and SOCS box that mediate suppression of cytokine signal transduction pathways (Chen et al., 2000). One of the mechanisms of inhibition is through binding to the Jak kinases through the SH2 domain and inhibiting their kinase activity, as seen for SOCS-1. In addition, SOCS-1 protein has been shown to suppress signaling downstream of receptor tyrosine kinases. Levels of SOCS proteins appear to be controlled transcriptionally and posttranscriptionally. IL-10 is an efficient stimulus for SOCS3/CIS3 induction in neutrophils and monocytes (Cassatella et al., 1999). Incubation of monocytes with IL-10 resulted in inhibition of gene expression for several IFN-induced genes, such as IP-10, ISG54 and ICAM-1. This was correlated with suppression of IFNa-induced assembly of Stat factors to specific promoter motifs (ISRE and GAS elements) on IFNa- and c-inducible genes and inhibition of tyrosine phosphorylation of Stat1. Because vanadate, a protein tyrosine phosphatase (PTP) inhibitor, had no effect on inhibition of Stat1 phosphorylation, the effect induced by IL-10 is probably not through SHP-1 or other PTP. Induction of SOCS proteins may also be involved in inhibition of interferon signaling. IL-10 has been found to suppress IFNa-activated Stat1 in the liver, possibly through induction of SOCS2, SOCS3 and CIS expression (Ito et al., 1999, Shen et al., 2000). There is evidence to suggest that IL-10 signaling is autoregulated through induction of SOCS-1 protein inhibiting Jak activity at the IL-10 receptor. This is supported by the fact that promoter elements of the SOCS-1 gene contain Stat3-binding sites (Chen et al., 2000). Other possible levels of regulation are through activation of protein tyrosine phosphatases such as SHP-1. However, neither activation of SHP-1, nor its association with IL-10 receptor has been reported. The IL-10 pathway may also be regulated by other pathways. For example, in addition to Stat3 tyrosine phosphorylation, phosphorylation of Stat3 at serine 727 plays a role in regulation of transcription (Lim and Cao, 2001). Active MEK kinase1 (MEKK1) is a

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AREAS OF ACTIVE RESEARCH AND SUMMARY

MAPKKK that activates c-Jun NH2-terminal kinase signaling pathway and is induced by growth factors, cytokines and environmental stress. MEKK1 was found to carry out phosphorylation of Stat3 at ser727 in vitro, while in vivo, MEKK1 activated Src and Jak kinases, which phosphorylated Stat3 at tyr705. MEKK1 thus may be involved in modulation of IL-10 signaling by growth factors and environmental stress.

DOWNSTREAM GENES Signaling through the IL-10 receptor activates a number of genes that mediate both anti-inflammatory and proliferative effects induced by IL-10. Activated/ memory T cells become refractory to TGFb-mediated inhibition of proliferation through the downregulation of the TGFbRII. TGFbR expression and inhibitory function can be restored on activated/ memory T cells by addition of IL-10 (Cottrez and Groux, 2001). IL-10 has been shown to downregulate the expression of MHC class I and II complexes and ICAM-1, thus inhibiting HLA-dependent antigen presentation and T cell activation. In monocytes, IL-10 has also been shown to inhibit proliferation directly by up-regulating expression of p19ink4D and p21cip (O’Farrell et al., 2000). The p19ink4D promoter was determined to have two Stat3 binding sites. Blocking Stat3 signaling with dominant negative Stat3 or mutant IL-10 receptor prevents induction of p19ink4D, however it has no effect on p21cip expression, suggesting that p21cip is regulated through other signaling mechanisms. The antiproliferative effect of IL-10 on macrophages is also exerted through upregulation of the CD95 receptor and ligand on the monocyte cell surface, leading to apoptosis, and through down-regulation of c-kit protooncogene expression, a protein tyrosine kinase that plays a role in hematopoiesis (Mirmonsef et al., 1999; Schmidt et al., 2000). In T cells, IL-10 has been shown to induce anergy through inhibition of tyrosine phosphorylation of CD28, thus preventing recruitment of PI3K to the receptor (Akdis et al., 2000; Joss et al., 2000). The anti-inflammatory effects of IL-10 are mediated by down-regulation of production of proinflammatory cytokines, and inhibition of cellular responses to mediators of inflammation. In monocytes, IL-10 inhibits the production of IL-1 and TNFa,

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and down-regulates the expression of IL-1RI and RII (Dickensheets and Donnelly, 1997). In addition, IL-10 inhibits COX2 expression in LPS-stimulated monocytes, and accelerates degradation of COX2 mRNA, thus controlling the inflammatory response at yet another level (Niiro et al., 1995). In addition to inhibition of proliferation and inflammatory response, IL-10 has been shown to play a role in inhibition of leukocyte recruitment and homing. Treatment with IL-10 down-regulates CCR7 mRNA expression, resulting in inhibition of dendritic cell chemotaxis toward macrophage inflammatory protein 3B (Takayama et al., 2001). Blocking IL-10 receptor restores CCR expression and cellular migration.

AREAS OF ACTIVE RESEARCH AND SUMMARY As for most cytokines, IL-10 is produced by many different cell types under a very wide variety of stimuli. In addition, the IL-10 receptor complex is expressed by many different immune and parenchymal cell types, and signal transduction is regulated by multiple other signaling pathways. As a result, IL-10 is responsible for many different pathophysiological events, and it is not precisely clear what are the true and dominant roles for IL-10 in regulating disease states. This is particularly true for EAE and IDDM, as reviewed above. Thus, a major area that remains under active investigation is to delineate precisely the immunomodulatory roles of IL-10 in important disease states, particularly autoimmune disease, transplantation, infection and ischemia-reperfusion. The goal of such research will be to define robust methods to manipulate IL-10 and/or its signaling pathways to control reliably inflammatory and immune responses. Put in another way, the research is directed toward separating out the immunosuppressive from the immunostimulatory activities of IL-10, so that immune manipulations can be made more precisely. In this regard, the biological spectrum of activity of vIL-10 versus cIL-10 suggests that it may already be possible to channel the activity of IL-10 in the desired direction. The description of new members and orphan members of the type II cytokine ligand and receptor

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families suggests that there will be much more to learn about the regulation of receptor–ligand interactions and signaling pathways. Thus, it is possible and even likely that agonist and antagonist ligands remain to be discovered and characterized. It is also likely that additional components or shared receptor subunits of the IL-10R complex will be shown to influence the complexity of signal transduction. Indeed, the demonstration that IL-10 and IL-22 share the IL-10R2 subunit suggests an additional level of regulation through alternative heterodimers on the cell surface.

Despite the many varied effects of IL-10, its importance is undiminished and lies in the fact that it is one of the few cytokines that predominantly assumes anti-inflammatory and immunosuppressive functions in many different settings. Thus, IL-10 has been and remains a target for both basic and clinical studies aimed at understanding, controlling and limiting mechanisms of inflammation. The summary in Tables 25.2 and 25.3 reveals that for all its multifarious effects, IL-10 is predominantly an immunosuppressive molecule.

TABLE 25.2 Cellular functions of IL-10 Cell type

TH

Function Immunosuppressive Anti-inflammatory

Stimulation Proliferation

Decreased cytokine synthesis (IFNc) Anergy, immune deviation, generation of suppressor type cells, inhibits TH1

Stimulates TH2

Tc

Stimulates maturation and proliferation

Thymocytes

Proliferation

NK

Decreased cytokine synthesis (IFNc)

Macrophage, monocytes

Decreased APC function Decreased cytokine synthesis (IL-1a, IL-1b, IL-6, IL-8, IL-12, VEGF, MMP-9, TNF a, GM-CSF, NO, H2O2) Decreased receptor expression (CD80, CD86, MHCII, IL-1RI, IL-1RII)

DC

Decreased cytokine synthesis Decreased receoptor expression (CD80, CD86, MHC II, MHC I) Decreased APC function

Vascular endothelial cell

Decreased receptor expression (ICAM-1)

Increased receptor expression (CD62E)

B cells

T dependent responses

T independent responses, isotype switching, growth and differentiation, increased MHC II

Mast cell

Increased proliferation Increased cytotoxicity

Stimulates proliferation

Neutrophils

Decreased cytokine secretion

Oligodendrocytes

Decreased cytokine synthesis

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TABLE 25.3 Pathophysiologic function of IL-10 Disease state

Function Anti-inflammatory Immunosuppressive

Cytokine syndrome Endotoxemia Rheumatoid arthritis Thyroiditis Collagen arthritis Herpetic keratitis IDDM EAE Tumor immunity Allograft immunity GVHD Colitis SLE Angiogenesis

         (vIL-10)  (vIL-10)

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26 The IL-10 family [Interleukins -19, -20, -22, -24 and -26] Holger Hackstein1,2, Grant Gallagher3, Sergei Kotenko3 and Angus W. Thomson1 1

University of Pittsburgh, PA, USA; 2Justus-Liebig University Giessen, Germany; and 3University of Medicine and Dentistry of New Jersey, NJ, USA

There are no facts, only interpretations. Frederich Nitzsche

INTRODUCTION Many cytokines exist within structurally related families. This is well-recognized in the case of the tumor necrosis factor (TNF) family of ligands, which comprises three adjacent, closely related genes within the MHC region on chromosome 6 (Nedospasov et al., 1986) and several other genes on a variety of chromosomes, which continue to be uncovered (Suda et al., 1993; Hahne et al., 1998). More recently, the T cellderived cytokine IL-17 was shown to be the prototype for a new family of cytokines which so far comprises IL-17A to IL-17E (Li et al., 2000; Aggarwal and Gurney 2002); a further example is to be found in the IL-1 family. These families are defined according to structural similarities in the encoded proteins. While some members of some families share certain functions (for example in the IL-1 family), frequently they do not. The recently described cytokines discussed in this chapter are largely of poorly defined function, but it

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

appears that the IL-10 homologues are not functionally similar. At the time of writing, there were six known members of the IL-10 family, IL-10 itself and the five homologues: IL-19, IL-20, IL-22, IL-24 and IL-26 (Figure 26.1). One of them, IL-24 has been in the public domain for some time known as MDA-7, but never discussed as being similar to IL-10, while the others are more recent discoveries. Two of these (IL-19 and IL-20) were discovered as a result of purposeful searching for homologues of IL-10, while the remainder were discovered by other means and subsequently found to be IL-10 homologues. According to structural similarity then, IL-19 is a novel member of the IL-10 protein family, and was first described in 2000 (Gallagher et al., 2000). Other members of this cytokine family are IL-20 (Blumberg et al., 2001), IL-22 (formerly IL-TIF) (Dumoutier et al., 2000a), melanoma differentiation associated gene 7 (MDA-7; Jiang et al., 1995), now designated IL-24 and AK-155

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THE IL - 10 FAMILY

HulL19 HulL20 HulL24 HulL22 HulL26 HulL10

---------------------------MKLQCVSLWLLGTILILCSVDNHGLRR-----C ---------------------------MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSC MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQ -----------------MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLD ----------------------------MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSC ----------------------------MHSSALLCCLVLLTGVRASPGQGTQSENSCTH

HulL19 HulL20 HulL24 HulL22 HulL26 HulL10

LISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTL-ETLQIIKPLDVCCVTKNLLAFYVD VIATNLQEIRNGFSDIRGSVQAKDGNIDIRILRRT-ESLQDTKPANRCCLLRHLLRLYLD VKGVVPQKLWEAFWAVKDTMQAQDNNTSCRLLQQ--EGLQNVSDAESCYLVHTLLEFYLK KSNFQQPYITNRTFMLAKEASLADNNTDVRLIGE--KLFHGVSMSERCYLMKQVLNFTLE YPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLL-KKKTKKQFMKNCQFQEQLLSFFME FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLE

HulL19 HulL20 HulL24 HulL22 HulL26 HulL10

RVFKDHQ--EPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYD RVFKNYQ--TPDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMKKYSQILSHFE TVFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFK EVLFPQS---------DRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTV DVFGQLQ----------LQGCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIF EVMPQAE------NQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAF

HulL19 HulL20 HulL24 HulL22 HulL26 HulL10

QLEVHAAAIKSLGELDVFLAWINKNHEVMSSA KLEPQAAVVKALGELDILLQWMEETE-----QLDVEAALTKALGEVDILLTWMQKFYKL---KKLGESGEIKAIGELDLLFMSLRNACI----YRIGNKGIYKAISELDILLSWIKKLLESSQ-NKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

FIGURE 26.1 Alignment of peptide sequences of IL-10 family members. Peptide sequences for IL-10, IL-19, IL-20, IL-22 and IL-26 were aligned using the ‘MAGI’ multiple sequence alignment software at the Medical Research Council’s Human Genome Mapping Project resource website (http://www.hgmp.mrc.ac.uk/Registered/Webapp/magi/) and displayed using the ‘boxshade’ option. White letters on black are identical, black letters on gray are similar, black letters on white are dissimilar. It will be seen that the six members of the IL-10 family are particularly similar towards their C-termini, but that distinct areas of similarity exist in other parts of the molecules.

(Knappe et al., 2000), now designated IL26. Human IL-10, IL-19, IL-20 and IL-24 form a gene cluster on chromosome 1q32 (Blumberg et al., 2001), whereas IL-24 and IL-22 are located on human chromosome 12q15 (Knappe et al., 2000; Dumoutier et al., 2000b), close to the interferon-gamma gene. The characteristic 6-helical structure of IL-10 seems to be conserved throughout all the homologues, but the F-helix is the region of greatest similarity, being highly conserved between all six proteins. However, a consideration of the evolutionary relationship between these molecules (Figures 26.1 and 26.2) demonstrates that there are two distinct branches to this family. One contains IL-10 itself, IL-22 and IL-26, while the other contains IL-19, IL-20 and IL-24 (interestingly, this does not coincide exactly with the chromosomal location of these genes). As will be discussed later, this is reflected in receptor-chain sharing between members of either the IL10 sub-family or the IL19 subfamily. Note however, that this is true only

with the caveat that not all receptors are defined, nor do all the six ligands have defined receptors – the receptor for IL26/AK155 is completely undefined at the time of writing while IL-19, 20 and 24 have a poorly understood sharing of two pairs of receptors chains (Dumoutier et al., 2001a; Wang et al., 2001).

INTERLEUKIN-19 The gene for human IL-19 was identified by screening publicly available expressed sequence tag (EST) databases for gene fragments which encoded peptides with homology to IL-10 (Gallagher et al., 2000). However, it is of interest that screening these databases with the peptide sequence of human IL-10 or fragments thereof, did not in fact reveal IL-19, it was only when the MDA-7 (now IL-24) sequence was used that the presence of IL-19 became apparent. At the time, it was not widely recognized that MDA-7 was a homo-

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INTERLEUKIN - 19

629

HulL19 HulL20 HulL24

---------------------------MKLQCVSLWLLGTILILCSVDNHGLRR-----C ---------------------------MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSC MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQ

HulL19 HulL20 HulL24

LISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDR VIATNLQEIRNGFSDIRGSVQAKDGNIDIRILRRTESLQDTKPANRCCLLRHLLRLYLDR VKGVVPQKLWEAFWAVKDTMQAQDNNTSCRLLQQ-EGLQNVSDAESCYLVHTLLEFYLKT

HulL19 HulL20 HulL24

VFKDHQ--EPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYDQ VFKNYQ--TPDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMKKYSQILSHFEK VFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQ

HulL19 HulL20 HulL24

LEVHAAAIKSLGELDVFLAWINKEHEVMSSA LEPQAAVVKALGELDILLQWMEETE-----LDVEAALTKALGEVDILLTWMQKFYKL---IL-24 IL-19 IL-20 IL-26 IL-10 IL-22

HulL26 HulL10 HulL22

---------MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAW ---------MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRV MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLA

HulL26 HulL10 HulL22

LKATIPEDRIKNIRLLKKKTKK-QFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFV-KTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNS KEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVV-

HulL26 HulL10 HulL22

--EDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKL LGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAY --PFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA

HulL26 HulL10 HulL22

LESSQ-MTMKIRN CI-----

FIGURE 26.2 Two distinct subfamilies of IL-10 homologues. The MAGI software was used to generate a nearest neighbours tree, revealing two distinct subfamilies as described in the text. It will be seen that the two subfamilies have considerably greater similarity within themselves than is suggested by examining all six homologues together. logue of IL-10 despite its being discussed as such at the European Immunology meeting in 1995 and, because of the vast number of IL-10 molecules represented in the databases from non-human species, human molecules with lower homology to human IL-10 (i.e. the homologues), than IL-10 from other animals, were never revealed. With the benefit of hindsight, this may reflect the fact of the two subfam-

ilies of IL-10 homologues and it is of interest that viral homologues have been identified in each subfamily; the long-recognized EBV-IL-10 (Vieira et al., 1991) whose amino acid sequence is virtually identical to that of human IL-10 and the more recently discovered cmvIL-10, whose amino acid sequence is quite distinct from that of human IL-10, but which nonetheless can signal through the IL-10 receptor (Kotenko

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et al., 2000) and on the other side, a YABA-like virus has a homologue of IL-24 (Lee et al., 2001). Cloning of the full-length cDNA yielded a predicted protein of 179 amino acids (aa), including a signal peptide of 18 aa. Besides an aa identity of 21%, IL-19 shares several important structural motifs with IL-10, such as the four cysteine residues necessary to fold the IL-10 monomer. Further homologies with IL-10 are apparent within the areas forming the cytokine helices or the hydrophobic core. In contrast, less homology to IL-10 was observed in the regions predicted to interact with the alpha-chain of the IL-10 receptor. Thus, it is probable that IL-19 is secreted in a homodimeric form like IL-10, but it is unlikely that this cytokine interacts with the IL-10 receptor. The specific IL-19 receptor remains to be identified, although it is known to cross-react with the IL-20 receptor (Dumoutier et al., 2001a). Transient expression of the IL-19 peptide tagged with the FLAG epitope in COS-1 cells resulted in the presence of multiple bands in the 35–40 kDa region by Western blot analysis of the conditioned medium, indicating that the secreted protein was glycosylated, perhaps variably so. Enzymatic deglycosylation of secreted IL-19 revealed the presence of a single 21 kDasized protein that is in accordance with the predicted size of IL-19.

IL-19 gene structure Similar to IL-10, the human IL-19 gene consists of five exons and four introns. Table 26.1 summarizes the gene structure of IL-19 and compares it with IL-10 and the other homologues. By sequencing different cDNA clones, at least two IL-19 mRNA species that differ in their 5 sequences have been observed (Gallagher et al., 2000). The longer 5 sequence contains a second translation start site that is in-frame with the rest of the IL-19 mRNA and encodes for an elongated leader sequence. Transfection of COS-1 cells with the longer 5 sequence strongly reduces the amount of secreted protein, indicating that the elongated leader sequence influences IL-19 secretion. It should be noted that an intron is present immediately upstream of the major coding ATG and that the promoter is not immediately apparent. However, the two sequences obtained from 5UTR of the long and short form of IL-19 mRNA suggest that they are a considerable distance upstream of the primary ATG, at least tens of kilobases.

IL-19 expression IL-19 mRNA expression was detected in human peripheral blood mononuclear cells and purified

TABLE 26.1 Comparison of human IL-19, IL-20, IL-22, IL-24, IL-26 and IL-10 genes Feature

IL-19a,b

IL-20

IL-22

IL-24

IL-26

IL-10

Gene location (chromosome)

1q32

1q32

12p15

1q32

12p15

1q32

Gene size (excluding 3UTR)

5943 bp2

3747 bp

3UTR size

354 bp

1033 bp

Exon-intron number

5–4

5–4

Exon sizes (exons 1–5) mRNA instability motifs a b

144, 66, 153, 75, 96 bp

159, 66, 153, 75, 96 bp

186, 66, 144, 66, 78 bp

196, 63, 159, 75, 84 bp

1

186, 66, 144, 66, 78 bp

165, 60, 153, 66, 93 bp 5

According to Gallagher et al., (2000). Genbank Accession No. AF276915. THE CYTOKINES AND CHEMOKINES

INTERLEUKIN - 20

monocytes after LPS stimulation (Gallagher et al., 2000). With respect to monocytes, IL-19 mRNA can be further induced by stimulation with GM-CSF, but not with IFN-b, IFN-c, IL-4 or IL-13. However, it was noteworthy that pre-priming monocytes with IFN-c, prevented the induction of IL-19 by LPS while prepriming with IL-4 enhanced LPS-induced IL-19 expression. The observation that IL-19 mRNA expression is delayed relative to IL-10 (4 versus 2 h in LPSstimulated monocytes) gave rise to the suggestion that it may function primarily as a feedback inhibitor of proinflammatory cytokines (Gallagher et al., 2000). Recently, expression of IL-19 has been demonstrated in a wide panel of human TH1 and TH2 clones (Gallagher et al., unpublished observations). However, the true function of IL-19 remains to be defined.

INTERLEUKIN-20 Background Interleukin-20 (IL-20) first described by Blumberg et al. (2001), constitutes a novel IL-10-related cytokine. IL-20 was discovered by utilizing an algorithm designed to identify common structural motifs of helical cytokines in EST databases. Experiments with recombinant IL-20 led to the identification and characterization of a heterodimeric IL-20 receptor that is structurally related to the IL-10 receptor (Blumberg et al., 2001). IL-20 is considered to be of critical importance in skin inflammation (Rich and Kupper, 2001). It promotes keratinocyte proliferation and its receptor subunits are somewhat up-regulated in the skin of patients suffering from psoriasis (Blumberg et al., 2001).

IL-20 protein The IL-20 protein is encoded by a gene located on human chromosome 1q32. The murine IL-20 gene lies on chromosome 1. Based on amino acid sequence homology, IL-19 (40%), MDA-7 (30%) and IL-10 (20%) show the highest similarity to IL-20 (Figures 27.1 and 27.2) (Blumberg et al., 2001). The highly significant structural homology and spatial proximity of these cytokines suggests that they have evolved through duplication of a common ancestral gene,

631

although further sub-evolution has probably occurred (above). However, the question of why IL-10 itself is more closely related to the homologues on chromosome 12 remains unresolved in this context. Both human and mouse IL-20 genes encode a protein of 176 aa and share 76% aa sequence homology (Genbank Accession Nos AF 224266 and AF224267 for human and mouse IL-20, respectively) (Blumberg et al., 2001).

IL-20 receptor and signaling Based on its homology with IL-10, Blumberg et al. (2001) hypothesized that IL-20 interacts with a heterodimeric class II cytokine receptor. Binding assays with biotinylated and I125-labeled IL-20 revealed that IL-20 bound only to a combination of two orphan class II cytokine receptor subunits: Zcytor7 (IL-20R1, Genbank Accession No. AF184971) and DIRS1 (IL-20R2). No binding was observed to any tested single receptor subunit (IL-10Ra, IL-10Rb, IFN-aR1, IFN-aR2, IFN-cR1, zcytor7, zcytor11, DIRS1) was observed. Competition experiments with unlabeled IL-20 and the unrelated cytokine IL-21 confirmed the specificity of the interaction between IL-20 and its heterodimeric receptor. Analysis of various human tissues by RT-PCR, revealed high amounts of IL-20R1 and IL-20R2 mRNA in skin and testis. In general, IL-20R1 (Zcytor7) was detectable in many different tissues, whereas the expression of IL-20R2 (DIRS1) was several magnitudes lower or undetectable (Blumberg et al., 2001). Luciferase reporter constructs with STAT-responsive elements demonstrated that receptor pairing was not only necessary for IL-20 binding, but also for IL-20mediated activation of STAT-luciferase. Utilization of a nuclear translocation assay further revealed that IL-20 stimulation resulted in nuclear translocation of STAT3, but not STAT1, in the human HaCaT keratinocyte cell line (Blumberg et al., 2001).

Biological functions of IL-20 The biological functions of IL-20 were investigated by means of transgenic mice, overexpressing IL-20 under control of different tissue-specific promotors (Blumberg et al., 2001). IL-20 transgenic animals died within a few days of birth. They were phenotypically

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smaller, lacked visible adipose tissue, and showed delayed ear and toe development, as well as swollen extremities, when compared with non-transgenic controls. Most prominently, the skin architecture of IL-20 transgenic animals was altered, characterized by a thickened epidermis, hyperkeratosis and proliferation in the suprabasal layer of the epidermis. The skin alterations were related to circulating IL-20, since they were also observed in IL-20 transgenic animals bearing a liver-specific albumin promotor, although the authors did not report the effect of administering IL-20 protein directly.

Clinical relevance of IL-20 The skin alterations seen in IL-20 transgenic animals resemble many characteristics of human psoriasis. However, in contrast to psoriasis, no immune infiltrates were seen in IL-20 transgenic mice and similar skin abnormalities have been reported in other transgenic mouse models – TGF-a, IFN-c and keratinocyte growth factor (Vassar and Fuchs, 1991; Guo et al., 1993; Carroll et al., 1997). Further evidence for the importance of the IL-20 pathway in the pathogenesis of psoriasis comes from experiments where the expression of the two IL-20R subunits has been analyzed in patients with psoriasis. Compared with healthy controls, IL-20R1 and IL-20R2 mRNA expression were found to be up-regulated in psoriatic skin (Blumberg et al., 2001). Interestingly, both IL-20R subunits were also detectable in immune infiltrates (mononuclear cells) and endothelial cells of some of the affected patients, indicating that IL-20 does not affect keratinocytes exclusively. Recent evidence has suggested that IL-10 may represent a viable new treatment for psoriasis (Asadullah et al., 2001a) and it has been demonstrated that patients with a family history of psoriasis show a genetic association with specific alleles in the IL-10 gene (Asadullah et al., 2001b). It is of interest to speculate that IL-10 may antagonize the activity of IL-20 and that the association with IL-10 markers seen in familial psoriasis actually represents linkage disequilibrium into the nearby IL-20 locus.

INTERLEUKIN-22 Background In 2000, Dumoutier et al. described a novel cytokine induced by IL-9 in murine T cells and named this molecule IL-10-related T cell-derived inducible factor (IL-TIF) because of its structural similarity to IL-10 (Dumoutier et al., 2000a). In the same year, the human counterpart of IL-TIF was identified and termed IL-22 and its receptor was seen to be made up from the IL-10 receptor b chain (IL-10Rb) and the orphan CRF2-9 (cytokine receptor family 2-9) chain (Dumoutier et al., 2000c; Xie et al., 2000; Kotenko et al., 2001a). In 2001, almost contemporaneously, four groups identified a novel, soluble human class II cytokine receptor that antagonizes IL-22 (Dumoutier et al., 2001b; Gruenberg et al., 2001; Kotenko et al., 2001b; Xu et al., 2001). This molecule was designated IL-22 receptor-a 2 (IL-22RA), or IL-22 binding protein (IL22BP).

IL-22 gene Mouse IL-22 (also called IL-TIF) is encoded by a gene on chromosome 10 in proximity to the IFN-c gene (Dumoutier et al., 2000b). The gene with a size of approximately 6 kb consists of six exons and encodes a 179 aa long protein, with a glycosylated molecular weight of approximately 25 kDa (Dumoutier et al., 2000a, 2000b). It is a single gene in BALB/c and DBA/2 mice and duplicated in other strains (C57BL/6, FVB, 129) (Dumoutier et al., 2000b). The two genes, IL-TIFa and IL-TIFb are almost identical and show 96% nucleotide homology with respect to the coding exons. The major difference between IL-TIFa and IL-TIFb is the deletion of a 658 bp fragment in the IL-TIFb gene including the first noncoding exon and the proximal putative promotor region (Dumoutier et al., 2000b). Since this fragment alone was found to transfer IL-9 responsiveness in a luciferase reporter gene assay and no expression of the IL-TIFb has been observed, it was concluded that this gene was either not expressed or was differently regulated from the ILTIFa gene (Dumoutier et al., 2000b). Human IL-22 is encoded by a gene mapped to chromosome 12q15, only 90 kb from the IFN-c gene (Dumoutier et al., 2000b). Like mouse IL-22, it consists of one noncod-

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INTERLEUKIN - 22

mIL-TIF MAVLQKSMSFSLMGTLAASCLLLIALWAQEANALPVNTRC ** **** * ****** ***** ** * * * * hIL-TIF MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHC

40

mIL-TIF KLEVSNFQQPYIVNRTFMLAKEASLADNNTDVRLIGEKLF * ******** ************************** hIL-TIF RLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLF

80

mIL-TIF RGVSAKDQCYLMKQVLNFTLEDVLLPQSDRFQPYMQEVVP *** ************* ** *************** hIL-TIF HGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVP

120

mIL-TIF FLTKLSNQLSSCHISGDDQNIQKNVRRLKETVKKLGESGE ** *** ** *** *** ** ** ** ********** hIL-TIF FLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGE

160

mIL-TIF IKAIGELDLLFMSLRNACV ****************** hIL-TIF IKAIGELDLLFMSLRNACI

179 179

ing and five coding exons and encodes a 179 aa long protein that shares 79% aa identity with the murine analogue (Figure 26.3) (Dumoutier et al., 2000b, 2000c; Xie et al., 2000). Both human and mouse IL-22 genes encode proteins which show 20% aa sequence identity with IL-10. Constitutive expression of murine IL-22 was detected by RT-PCR in thymus and brain. Murine IL-22 can be induced in T cells and mast cells by IL-9 and in various organs in vivo (gut, liver, kidney, spleen, lung, stomach, heart, thymus) after LPS injection (Dumoutier et al., 2000a, 2000c).

Functional IL-22 receptor complex As described above, the functional IL-22R complex consists of the IL-10Rb chain (also called IL-10R2, CRF2-4) and the (previously) orphan class II cytokine receptor CRF2-9 (IL-22R1) (Figure 26.4). CRF2-9 is also called ZcytoR11 in patent databases (Lok et al., 1999). The IL-10R2 chain was the first class II cytokine receptor chain shared by at least two different cytokine receptor complexes and was therefore proposed to be termed receptor 2 common chain (R2c) (Kotenko et al., 2001a), although IL-22R1 is now also known to share this property – see above. It was proposed that CRF2-9 be named IL-22R (Xie et al., 2000). Its cDNA encodes for a 574 aa long protein with significant homology to the IL-10R1 chain (Xie et al., 2000; Kotenko et al., 2001a). Cross-linking studies showed that either chain alone, CRF2-9 and IL-10R2 chain could bind IL-22 independently (Kotenko et al., 2001a), although IL-10R2 cannot bind IL-10 in the absence of IL10-R1. Expression of both chains is nec-

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FIGURE 26.3 Alignment of murine IL-TIF and human IL-TIF protein sequences. Conserved residues are boxed. Reprinted from Dumoutier et al. (1997). Interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocytestimulating factor. Proc. Natl Acad. Sci. USA 97, pp 10144–10149. Copyright (2000) with permission from National Academy of Sciences USA.

essary to form the functional IL-22R complex and to induce STAT activation, as shown in transfected hamster cells and IL-22 responsive cell lines (Xie et al., 2000; Kotenko et al., 2001a). IL-22 was found to promote STAT1, 3 and 5 activation in cell lines expressing the functional IL-22 receptor complex (Xie et al., 2000). Since the IL-10R2 chain is expressed ubiquitously, expression of the CRF2-9 is critical to determine IL-22 responsiveness (Kotenko et al., 2001a). High level expression of CRF2-9/IL-22R1 mRNA has been found in normal liver and kidney tissue and in cell lines (e.g. melanoma G-361, Burkitt’s lymphoma Raji, renal cell carcinoma TK-10). Further evidence for the indispensable role of the IL-10R2 chain as a subunit of the functional IL-22R complex comes from experiments in which IL-22 responsiveness of HepG2 human hepatoma cells was blocked in the presence of anti-IL-10R2 chain antibodies (Dumoutier et al., 2000c).

Biological effects of IL-22 IL-22 promotes acute phase protein production (haptoglobin, serum amyloid A, a1-antichymotrypsin) in HepG2 human hepatoma cells in vitro and in liver cells in vivo, suggesting an involvement in livermediated inflammatory responses (Dumoutier et al., 2000c). In T helper 2 cells it was found to inhibit IL-4 production (Xie et al., 2000). The constitutive expression of IL-22 in the brain and the IL-22 responsiveness of the neuronal cell line PC12 (rat pheochromocytoma) is indicative of a role of IL-22 in neurological processes (Dumoutier et al., 2000a).

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THE IL - 10 FAMILY

TYk2

IL-22 IL-22

Tyk2 Jak?

IL-22 IL-22

IL-22 IL-22

R2c

Stat3

IL-22 IL-22

IL-22R1

Jak? TYk2

R2c

Stat3

IL-22R1

Jak?

IL-22BP

Stat3 Stat5

StatXStat1,3 or 5

Stat1

StatX StatX

FIGURE 26.4 Model of the IL-22R complex and signal transduction. The functional IL-22 (IL-10-related T cell-derived inducible factor) receptor complex consists of two receptor chains, the unique IL-22R1 chain and the IL-10R2 (R2c) chain, which is a common chain for the IL-10R complex and the IL-22R complex. Both chains can independently bind IL-22, but both chains must be present in the receptor complex to induce the signal transduction events. The IL-22 activity can be negatively regulated by the expression of the R2c chain alone because IL-22 binding to the R2c does not lead to signaling, preventing shedding of IL-22 into the circulation (local suppression). In addition, the secretion of the soluble IL-22BP into the circulation can provide systemic inhibition of IL-22 action. Reprinted from Kotenko et al. (2001). Identification, cloning and characterization of a novel soluble receptor that binds IL-22 and neutralizes its activity. J. Immunol. 166, 7096–7103. Copyright (2001) with permission from The American Association of Immunologists.

IL-22 binding protein As mentioned above, IL-22 binding protein (IL-22BP; also called IL-22 receptor-a 2, CRF2-10) is a novel soluble class II cytokine receptor encoded by a gene positioned on human chromosome 6q23.3-24.2, approximately 24 kb and 100 kb apart from the IFNcR1 and the IL-20Ra gene, respectively (Dumoutier et al., 2001b; Gruenberg et al., 2001; Kotenko et al., 2001b; Xu et al., 2001). The IL-22 BP gene is organized in six exons and encodes a 231 aa long protein with-

out transmembrane or intracellular domains. Two splice variants with either an additional exon (exon 4a) or lacking exon 5 resulting in a longer, or a prematurely terminated short protein, respectively, have been described (Gruenberg et al., 2001; Kotenko et al., 2001b). IL-22BP shares 34% aa homology with the extracellular domain of the IL-22R (also called CRF2-9). IL-22BP binds IL-22 and prevents interaction of IL-22 with the functional IL-22R cell surface complex (Figure 27.4) (Dumoutier et al., 2001b; Kotenko et al., 2001b; Xu et al., 2001). As a consequence, IL-22 is unable to induce STAT activation and acute phase reactant production in responsive cells in the presence of IL-22BP. There is currently no evidence that IL-22BP can also enhance IL-22 activity by functioning as a cytokine carrier that releases the molecule for interaction with its membrane-bound receptor or inhibits proteolytic degradation. Instead, in vitro experiments suggest that the IL-22/IL-22BP complex is stable over several days and little to no dissociation occurs (Kotenko et al., 2001b). IL-22BP is expressed in various tissues with the highest levels being detected in placenta, breast tissue, colon, lung, skin and spleen (Dumoutier et al., 2001b; Gruenberg et al., 2001; Kotenko et al., 2001b; Xu et al., 2001). The major cell types expressing IL-22BP are monocytes, activated B cells and epithelial cells (Xu et al., 2001).

INTERLEUKIN-24 (MDA-7) As previously mentioned, IL-24 has been known for some time, as MDA-7. The first member of the IL-10 homologue family, IL-24 was cloned by subtraction hybridization in 1995 as a protein whose expression is elevated in terminally differentiated human melanoma cells (Jiang et al., 1995). The protein was designated MDA-7, melanoma differentiation associated gene 7. Although MDA-7 protein is expressed in normal melanocytes, its expression is gradually reduced with melanoma progression and is undetectable at the metastatic stage (Ekmekcioglu et al., 2001; Huang et al., 2001). Nevertheless, the expression of MDA-7 is increased in human melanomas after treatment with IFN-b and the protein kinase C activator mezerein (Jiang et al., 1995), resulting in irreversible lost of growth potential and terminal differentiation (Su et al., 1998) The function of MDA-7

THE CYTOKINES AND CHEMOKINES

INTERLEUKIN - 26 ( AK - 155)

has been linked to selective suppression of tumor growth (Jiang et al., 1996; Su et al., 1998; Madireddi et al., 2000; Saeki et al., 2000; Ekmekcioglu et al., 2001; Huang et al., 2001; Mhashilkar et al., 2001), through a mechanism involving the induction of apoptosis in cancer cells, which appeared to be p53 and retinoblastoma protein (RB) independent and correlated with up-regulation of BAX protein expression (Jiang et al., 1996; Su et al., 1998). Also, G2/M cell cycle arrest inhibiting entry into S phase of the cell cycle was observed in cancer cells overexpressing MDA-7 (Ekmekcioglu et al., 2001; Mhashilkar et al., 2001). MDA-7 was demonstrated to be a secreted glycosylated protein which appears on SDS-PAGE as several bands in the region of 35–40 kDa (Wang and Razzaque 1993; Dumoutier et al., 2001a; Zhang et al., 2000). Moreover, MDA-7 was reported to be expressed at high level in several human colon cancer specimens tested (Zhang et al., 2000). Its identity as a cytokine was confirmed by finding of its receptor, first as an entity expressed in ras-transformed cells (Zhang et al., 2000) and then as specific membrane-bound proteins able to induce signaling after binding of MDA-7 (Dumoutier et al., 2001a; Wang et al., 2001, see below for details). Based on its cytokine-like properties, MDA-7 was designated IL-24. Identification and characterization of rat and mouse IL-24 (MDA-7) orthologs suggested that the function of IL-24/MDA-7 is even more complex. The expression of the rat IL-24 ortholog was linked to wound healing (Soo et al., 1999; the IL-24 protein was designated C49A) and to ras transformation (Wang et al., 2001) the IL-24 protein was designated Mob-5), and the mouse IL-24 ortholog (Schaefer et al., 2001; the IL-24 protein was designated FISP, IL-4-induced secreted protein) was demonstrated to be specifically produced by activated TH2 cells. Rat IL-24 (C49A) cDNA was cloned by differential display as a gene expressed at elevated levels by up to 12-fold during wound healing (Soo et al., 1999). The up-regulation was rapid reaching maximum level within 12 to 24 h with subsequent gradual decline to a baseline around the day 14. The expression of C49A protein was localized primarily to spindle-shaped fibroblast-like cells at the wound edge and base. It was also reported that increased proliferation of normal human skin fibroblasts was associated with increased level of endogenous IL-24 expression (Soo

635

et al., 1999). In addition, expression of rat IL-24 (mob5) was induced by expression of oncogenic Ha-ras in fibroblasts or Ki-ras in intestinal epithelial cells (Wang et al., 2001), and secreted, migrating on the SDS-PAGE as a single band of about 23 kDa. It was also demonstrated that in addition to Mob-5 itself, its putative receptor is also oncogenic ras specific target, because Mob-5 binds to the cell surface of ras-transformed cells, but not of parental untransformed cells (Wang et al., 2001). Thus, this coordinated regulation of a ligand-receptor matching pair by ras oncogene may create an autocrine activation loop in ras-transformed cells. Constitutive signaling though this receptor could be one of the mechanisms mediating rasinduced transformation. A possible function within the immune system is also suggested by properties of mouse IL-24 analog designated FISP (Schaefer et al., 2001). FISP was identified by representational difference analysis method, which was employed to isolate genes expressed specifically by TH2 cells. Northern blot analysis demonstrated the expression of FISP mRNA in either total mouse splenocytes or CD4-enriched T cells cultured under TH2 (anti-CD3, IL-2, IL-4, anti-IL-12) differentiation conditions. The expression of FISP mRNA gradually increased from day 3, peaking at day 5. Splenocytes or CD4-enriched T cells incubated under TH1 (anti-CD3, IL-2, anti-IL-4, IL-12) differentiation conditions failed to produce a detectable amount of FISP transcripts. Although it is clear that human IL-24 (MDA-7), mouse FISP, and rat C49A/Mob-5 represent speciesspecific products of the same evolutionary conserved gene, it is possible that the function of these proteins diverged between species. They all are cytokine-like secreted molecules highly homologous to each other, but their known up-to-date activities do not overlap. In support of this possibility mouse receptors for IL-24 have unexpectedly low homology in their intracellular domains (see below). Thus, overlapping but distinct signal transduction pathways may be activated by IL-24 in different species.

INTERLEUKIN-26 (AK-155) Interleukin-26, formally known as AK-155, is encoded on chromosome 12, between the interferon gamma

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gene and that for IL-22. Its function is completely undefined, as is the nature of its receptor. IL-26 was originally discovered during a project which was examining the effect of infecting human T cells with the virus Herpesvirus saimiri (Knappe et al., 2000). H. saimiri is a transforming virus of New World monkeys, which has the interesting property of being able to ‘immortalize’ human T cells. Subtractive hybridization experiments between virally transformed human T cells and their non-infective parents let to the cloning of several mRNAs originating from the transformed cells and one of these encoded IL-26. IL-26 is a 171 amino acid protein, with 24.7% identity and 47% similarity to human IL-10. It compares similarly to IL-22 and these three proteins are in many ways distinct from the other three, IL-19, IL-20 and IL-24. The cysteine residues important for the folding of IL-10 are conserved in IL-26. In addition, IL-26 is known to be secreted from cells as a homodimer, similarly to the required folding pattern for IL-10. The gene has a similar intron/exon structure to IL-10 and the other homologues, with the exception that intron 3 is particularly long, at 23 kb. Expression studies reported in the original paper demonstrated that IL-26 was present in a range of transformed human T cell lines such as Jurkat, Molt-3 and Hut-102, as well as PHA blasts. IL-26 is poorly expressed in B cells or monocyte lines and absent from the small number of carcinoma lines examined. Very interestingly, the authors reported that IL-26 was present in the mRNA of resting, freshly isolated human peripheral blood T cells, in all of 10 tested individuals. Recently, Goris et al. (2001) provided identification and characterization of two microsatellite polymorphisms at the AK155 locus, i.e. D12S2511 and D12S2510. The first is located in the third intron and the second in the 3 region of the gene and various alleles were shown to be in linkage disequilibrium, working together as haplotypes.

RECEPTOR REDUNDANCY AMONGST IL-10 FAMILY MEMBERS At the time of writing, six ligands of the IL-10 family have been defined. Following the prototype of IL-10, these all appear to bind to two-chain receptors,

although it should be emphasized that the receptor for IL-26/AK155 is presently undefined. The IL-10 receptor is made up of the IL10-R1 chain and the IL10-R2 chain, crf2/4 (Kotenko et al., 1997) and if each ligand were to have unique receptors, 12 chains would be required. However, as found with many cytokine families it appears that a significant redundancy exists in the usage of these receptor chains, with individual specificities being derived through a process of ‘mixing and matching’. Presently, five receptor chains have been identified: IL10-R1, IL10-R2, IL20-R1, IL20-R2, IL22-R1, almost certainly insufficient to construct six discrete two-chain receptors. Although a variety of names exist for these in the literature, we shall use this nomenclature here for simplicity’s sake, secure in the knowledge that more interested readers will investigate things more deeply for themselves. Receptor chain sharing (note: not receptor sharing, which came later) was demonstrated in the IL-10 family with the definition of the IL22 receptor (Xie et al., 2000), which combines a unique chain, IL22-R1, with the IL10-R2 receptor chain. Ligand-binding studies with IL-19, IL-20 and IL-24 have shown that these cytokines, too, utilize common receptor chains although interestingly they can also share complete receptors, too. The initial receptor defined on this side of the IL-10 family was that for IL-20, comprising the IL20-R1 and IL20R2 chains. Recently, it was shown that in fact, IL-19, IL-20 and IL-24 can all bind to and signal through this pair of molecules (Dumoutier et al., 2001a), a result which is somewhat counter-intuitive. Some clarification of this came recently when it became apparent that IL20 and IL-24 can also signal through a second receptor pair, IL22-R1 and IL20-R2 (Dumoutier et al., 2001a; Wang et al., 2001). Cells transfected with either IL22-R1  IL20-R2 or IL20-R1  IL20-R2 bound IL-24 with similar saturation kinetics and both receptors gave rise to STAT activation. Nonetheless, these two studies do appear to reveal a complex pattern of receptor chain usage and ‘mix-matching’ by these three ligands. Further examination of the possible combinations of these receptor chains may yet reveal additional combinations which bind only one ligand. Perhaps these questions and those relating to the functions of the IL-10 homologues themselves will be resolved in time for the next edition of The Cytokine Handbook!

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Rich, B.E. and Kupper, T.S. (2001). Cytokines: IL-20 – a new effector in skin inflammation. Curr. Biol. 11, R531–R534. Saeki, T., Mhashilkar, A., Chada, S. et al. (2000). Tumorsuppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7, 2051–2057. Schaefer, G., Venkataraman, C. and Schindler, U. (2001). Cutting edge: FISP (IL-4-induced secreted protein), a novel cytokine-like molecule secreted by Th2 cells. J. Immunol. 166, 5859–5863. Soo, C., Shaw, W.W., Freymiller, E. et al. (1999). Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma differentiation associated gene, mda-7. J. Cell. Biochem. 74, 1–10. Su, Z.Z., Madireddi, M.T., Lin, J.J. et al. (1998). The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Natl Acad. Sci. USA 95, 14400–14405. Suda, T., Takahashi, T., Golstein, P. and Nagata. S. (1993). Molecular cloning and expression of the Fas ligand, a novel member of the tumour necrosis factor family. Cell 75, 1169–1178. Vassar, R. and Fuchs, E. (1991). Transgenic mice provide new insights into the role of TGF-alpha during epidermal development and differentiation. Genes Dev. 5, 714–727.

Vieira, P., de Waal-Malefyt, R., Dang, M.N. et al. (1991). Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein–Barr virus open reading frame BCRFI. Proc. Natl Acad. Sci. USA 88, 1172–1176. Wang, J. and Razzaque, A. (1993). Mapping and DNA sequence analysis of the cytomegalovirus transforming domain III (mtrIII). Virus Res. 30, 221–238. Wang, M., Tan, Z., Zhang, R. et al. (2001). Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J. Biol. Chem. 12, 12. Wang, M., Tan, Z., Zhang, R. et al. (2002). Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J. Biol. Chem. 277, 7341–7347. Xie, M.H., Aggarwal, S., Ho, W.H. et al. (2000). Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R. J. Biol. Chem. 275, 31335–31339. Xu, W., Presnell, S.R., Parrish-Novak, J. et al. (2001). A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist. Proc. Natl Acad. Sci. USA 98, 9511–9516. Zhang, R., Tan, Z. and Liang, P. (2000). Identification of a novel ligand-receptor pair constitutively activated by ras oncogenes. J. Biol. Chem. 275, 24436–24443.

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27 Interleukin-1 Family [IL-1F1, F2] Charles A. Dinarello University of Colorado Health Sciences Center, Denver, CO, USA

The price of greatness is responsibility Sir Winston Churchill

INTRODUCTION Unlike IL-2 and cytokines that affect primarily lymphocyte function and lymphocyte expansion, IL-1 and its related family members are primarily proinflammatory cytokines by their ability to stimulate the expression of genes associated with inflammation and autoimmune diseases. The most salient and relevant properties of IL-1 in inflammation are the initiation of cyclooxygenase type 2 (COX-2), type 2 phospholipase A and inducible nitric oxide synthase (iNOS). This accounts for the large amount of prostaglandin-E2 (PGE2), platelet activating factor and nitric oxide (NO) produced by cells exposed to IL-1 or in animals or humans injected with IL-1. Another important proinflammatory property of IL-1 is its ability to increase the expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), on mesenchymal cells and vascular-cell adhesion molecule-1 (VCAM-1) on endothelial cells.

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

This latter property promotes the infiltration of inflammatory and immunocompetent cells into the extravascular space. IL-1 is also an angiogenic factor by increasing the expression of vascular endothelial growth factor; IL-1 thus plays a role in tumor metastasis and blood vessel supply. The strongest case for the importance of IL-1 in disease processes comes from the administration of the IL-1 receptor antagonist, a member of the IL-1 family that prevents IL-1 activity. Although IL-1 receptor antagonist is discussed in detail elsewhere in this volume, injections of the IL-1 receptor antagonist (IL-1Ra) into humans with rheumatoid arthritis has resulted in a reduction in the inflammatory and joint destructive nature of their disease (Bresnihan et al., 1998; Bresnihan et al., 2000; Cunnane et al., 2001). However, in addition to proinflammatory properties, IL-1 is also an adjuvant during antibody production and acts on bone marrow stem cells for differentiation in the myeloid series. In fact, IL-1 was administered to humans in order to

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reduce the nadir of white blood cells and platelets of patients undergoing bone marrow transplantation (Smith et al., 1993). A great deal has been learned from studies using either the administration of IL-1Ra or in mice deficient in either IL-1 or IL-1 receptors. For example, in mice lacking IL-1 receptor type I, there is a failure to develop proliferative lesions of vascular smooth muscle cells in mechanically injured arteries (Rectenwald et al., 2000). Mice deficient in tumor necrosis factor-a (TNFa) also exhibit decreased neointimal hyperplasia but in these mice, there is no expression of IL-1a, suggesting that the effect in TNFa-deficient mice is due to a lack of IL-1a expression. This conclusion is supported by the observation that TNFa is expressed in mice deficient in IL-1 receptors, but there is reduced intimal hyperplasia. These and similar experiments are consistent with the concept that some effects of TNFa are mediated by IL-1. Early studies on the effects of TNFa revealed that TNFa induces IL-1 (Dinarello et al., 1986). It is also possible that the effect of blocking TNFa in patients with rheumatoid arthritis is due to a reduction in IL-1 production and/or activity. For example, in rheumatoid arthritis patients injected with anti-TNFa monoclonal antibodies, there is a rapid reduction in circulating IL-1b levels. Mice overexpressing TNFa develop a spontaneous rheumatoid arthritis-like joint disease; however, if treated early in their disease process with anti-IL-1 receptor antibody, there is no development of arthritis (Probert et al., 1995). IL-1a, IL-1b and IL-18 are unique in the cytokine families. Each is initially synthesized as precursor molecules without a signal peptide. After processing by the removal of N-terminal amino acids by specific proteases, the resulting peptides are called ‘mature’ forms. The 31 kDa precursor form of IL-1b is biologically inactive and requires cleavage by specific intracellular cysteine protease called IL-1b converting enzyme (ICE). ICE is also termed caspase-1, the first member of a large family of intracellular cysteine proteases with important roles in programmed cell death. However, there is little evidence that ICE (caspase-1) participates in programmed cell death (Watanabe et al., 1998). Rather, ICE seems to be primarily used by the cell to cleave the IL-1b and IL-18

precursors. As a result of the cleavage, the mature form of IL-1b is a 17.5 kDa molecule and of IL-18 is an 18 kDa peptide. Although ICE is primarily responsible for cleavage of the precursor intracellularly, other proteases such as proteinase-3 can process the IL-1b precursor extracellularly into an active cytokine (Coeshott et al., 1999). In terms of the role of IL-1 in human disease, specific blockade of the IL-1 receptor type I (the ligand binding chain of the heterodimeric IL-1 receptor signaling complex) with the naturally occurring IL-1 receptor antagonist (IL-1Ra) in patients with rheumatoid arthritis has resulted in reduced disease activity and reduced joint destruction (Bresnihan et al., 1998; Jiang et al., 2000; Cunnane et al., 2001). To date IL-1Ra has been approved for use in the United States, Canada and Europe for the treatment of rheumatoid arthritis and several thousand patients receive daily treatment; the results support the essential inflammatory and tissue remodeling functions of IL-1. However, IL-1 and IL-18 (a new member of the IL-1 family) are truly pleiotropic cytokines and affect the innate, as well as the acquired immune systems. Mice deficient in the IL-1 receptor type I, IL-1a, IL-1b or double-deficient in IL-1a and IL-1b exhibit no phenotype different from the same strain wild-type mice. A similar observation has been made with mice deficient in IL-18 or the IL-18 receptor. Thus, IL-1and IL-18-deficient mice live in routine, microbially unprotected animal facilities. From these observations, one can conclude that these three agonist members of the IL-1 family, which play important roles in disease, are not essential for normal embryonic development, post-natal growth, homeostasis, reproduction or resistance to routine microbial flora. These mice also do not exhibit evidence of spontaneous carcinogenesis and their life-span appears normal. Lymphoid organ architecture is also normal. Nevertheless, in the context of an inducible disease, a deficiency in any one of these three members of the IL-1 superfamily reveals a role in disease severity. In contrast, as described below, mice deficient in IL-1Ra do not exhibit normal reproduction, have stunted growth and in selected strains develop spontaneous diseases, such as rheumatoid arthritis-like polyarthropathy and a fatal arteritis (Horai et al., 2000; Nicklin et al., 2000).

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HISTORICAL BACKGROUND The history of IL-1 begins with studies on the pathogenesis of fever. These were studies performed on the fever-producing properties of proteins released from rabbit peritoneal exudate cells by Menkin and Beeson in 1943–1948, and were followed by contributions of several investigators, who were primarily interested in the link between fever and infection/inflammation. In 1972, Waksman and Gery made an important contribution with the discovery that soluble factors augmented lymphocyte proliferation in response to antigenic or mitogenic stimuli. Kamschmidt also contributed to the ‘discovery phase’ of IL-1 in describing macrophage products that induced the synthesis of acute phase proteins. The basis for the term ‘interleukin’ was to streamline the growing number of biological properties attributed to soluble factors from macrophages and lymphocytes. IL-1 was the name given to the macrophage product, whereas IL-2 was used to define the lymphocyte product. At the time of the assignment of these names, there was no amino acid sequence analysis known and the terms were used to define biological properties. In the field of rheumatoid arthritis, Krane and Dayer described IL-1 as an inducer of collagenases and Saklatvala described IL-1 for its property to destroy cartilage. The large number of diverse multiple biological activities attributed to a single molecule engendered considerable skepticism in the scientific community, but with the cloning of IL-1 in 1984 (Auron et al., 1984; Lomedico et al., 1984), the use of recombinant IL-1 established that IL-1 was indeed a pleiotropic cytokine mediating inflammatory, as well as immunological responses. With the use of targeted gene disruption, a more precise role for IL-1 in immune responses has been possible. For example, immunization with sheep red blood cells fails to elicit an antibody response in IL-1b-deficient mice and hypersensitivity responses to antigens are suppressed in IL-1b-deficient mice.

THE IL-1 LIGAND SUPERFAMILY The intron–exon organization of the IL-1 genes suggests duplications of a common gene some 350 mil-

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lion years ago. Before this common IL-1 gene, there may have been another ancestral gene from which fibroblast growth factors (FGF) such as acidic and basic FGF also evolved, since IL-1 and FGFs share significant amino acid homologies, and similar to IL-1, form an all-beta-pleated sheet tertiary structure. To date, 10 individual members of the IL-1 gene superfamily have been described. Of these, four gene products have been thoroughly studied. The other six members have been shown to exit in various human tissues, but their role in health or disease is presently unknown. The four primary members of the IL-1 gene superfamily are IL-1a, IL-1b, IL-18 and IL-1 receptor antagonist (IL-1Ra). IL-1a, IL-1b and IL-18 are each agonists; IL-1Ra, on the other hand, is the specific receptor antagonist for IL-1a and IL-1b, but not for IL-18. When IL-1Ra occupies the IL-1 receptor, bona fide IL-1 cannot bind to the receptor and there is no biological response to IL-1. The existence of a highly specific and naturally occurring receptor antagonist in cytokine biology appears to be unique to the IL-1 family. Recombinant IL-1Ra is approved in the United States and Europe to treat patients with rheumatoid arthritis by reducing inflammation and joint destruction (Bresnihan et al., 1998; Jiang et al., 2000). Similar to the use of anti-TNFa monoclonal antibodies or soluble TNF receptors, the beneficial effects of these anti-cytokine strategies is limited to amelioration of disease activity without affecting the dysfunctional autoimmune nature of rheumatoid arthritis. Members of the IL-1 superfamily have been assigned a new nomenclature using the expression IL-1F reflecting their being part of a ‘family’ of related ligands. Table 27.1 lists the current members of the IL-1 superfamily. In this chapter, the terms IL-1a, IL-1b and IL-18, as well as IL-1Ra will be retained. Most members of the IL-1 superfamily are located on the long arm of chromosome 2. The intron–exon organization of the new members is also similar to that of the primary four members of the IL-1 superfamily. The six new members are closely related to IL-1b and IL-1Ra. From the intron–exon organization, some members represent gene duplications. In the case of IL-1F5, and possibly other newly described members, the duplication of the IL-1Ra gene has taken place (Mulero et al., 1999). IL-1F7 and IL-1F9 are also closely related to IL-1Ra (Busfield et al., 2000). IL-1F5 shares 47% amino acid identity with IL-1Ra

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TABLE 27.1 IL-1 superfamily members New name

Former name(s)

Property

IL-1F1 IL-1F2 IL-1F3 IL-1F4 IL-1F5

IL-1a IL-1b IL-1Ra IL-18; IFNc inducing factor IL-1Hy1, FIL1d, IL-1H3, IL-1RP3, IL-1L1, IL-1d FIL-1e, IL-1e FIL-1f, IL-1H4, IL-1RP1 FIL-1h, IL-1H2 IL-1H1, IL-1RP2 IL-1Hy2, FKSG75

Agonist Agonist Antagonist Agonist Unknown

IL-1F6 IL-1F7 IL-1F8 IL-1F9 IL-1F10

Unknown Unknown Unknown Unknown Unknown

and is expressed in human monocytes activated by endotoxins. From the gene sequence, the predicted amino acids sequence of IL-1F5 does not have a leader peptide for secretion, which is in sharp contrast to the IL-1Ra (IL-1F3). IL-1F5 failed to exhibit agonist activity using induction of IL-6 from fibroblasts, a well-described biological property of IL-1a and IL-1b (Barton et al., 2000). Furthermore, IL-1F5 did not block the IL-1 or IL-1b-induced IL-6 or IL-18induced production of IFNc (Barton et al., 2000). Therefore, IL-1F5 possesses neither IL-1- or IL-18-like agonist activities nor the property to act as a receptor antagonist for IL-1, despite it close amino acid identity to IL-1Ra. Although IL-1F7 (formerly IL-1f, IL-1H4, IL-1H and IL-1RP1) is structurally related to IL-1Ra (36%), this IL-1 superfamily member binds to the IL-18 receptor chain and therefore has attracted attention as being related to IL-18 (Pan et al., 2001). IL-1F7 has no leader peptide and the recombinant form has been expressed with a N-terminus from a predicted caspase-1 site (Kumar et al., 2000). There are two forms of IL-1F7, a full-length peptide and a splice variant with an internal 40 amino acids deletion (Pan et al., 2001). The binding of IL-1F7 to the soluble IL-18R a-chain has also been observed. However, compared with IL-18, recombinant IL-1F7 does not induce IFNc from whole human blood cultures, in peripheral blood mononuclear cells (PBMC) or various cell lines. Therefore, it is unlikely that IL-1F7 is a true agonist for the IL-18 receptor. Whether IL-1F7 is a receptor antagonist for IL-18 remains to be determined. IL-1F9 is constitutively expressed primarily in the placenta and the squamous epithelium of the esoph-

agus. The three-dimensional folding of IL-1F9 is similar to that of IL-1Ra; therefore, IL-1F9 appears to be a possible IL-1 receptor antagonist rather than an agonist. IL-1F10 shares 37% amino acid identity with the IL-1Ra and a similar three-dimensional structure (Lin et al., 2001). This cytokine is secreted from cells and is expressed in human skin, spleen and tonsil. To date, recombinant IL-1F10 has been shown to bind to the recombinant soluble IL-1 receptor type I, but it is unclear whether IL-1F10 binds to cell surface IL-1 receptors. Although these data suggest that IL-1F10 is likely to be a receptor antagonist, compared with IL-1Ra, its role in health and disease remains unclear. In general, the function(s) of the newly described members of the IL-1 superfamily (IL-1F5-10) is presently unclear. It is unlikely that any possess proinflammatory properties, since recombinant forms have not revealed detectable effects in primary cells similar to those for IL-1a, IL-1b or IL-18. Since most share significant amino acid identities with IL-1Ra and since the intron–exon organization appears to reveal gene duplication of the IL-1Ra gene, these IL-1 superfamily members may be receptor antagonists. Whether these IL-1Ra-like homologues can block IL-18 is also presently unclear. Because deletion of only the IL-1Ra gene has resulted in a significant disease-producing phenotype in mice (see below), one can assume that the genes coding for the IL-1Ra homologues (IL-1F5-10) do not play a significant role in health. At present, the effect of deletion of IL-1F5-10 in mice is unknown.

Structures of the IL-1 family The three-dimensional structure of the IL-1a is similar to that of IL-1b and IL-18 in that each cytokine forms an open-ended barrel comprised of all betapleated strands. Crystal structural analysis of the mature form of IL-1a is similar to that of IL-1b. IL-1a has two sites of binding to IL-1 receptor. There is a primary binding site located at the open top of its barrel, which is similar, but not identical to that of IL-1b. IL-1Ra is structurally related to IL-1b rather than IL-1a. The unique structure of IL-1Ra that allows binding to the IL-1 receptor but without triggering signal transduction is due to the lack of a second binding site on the backside of the molecule (Evans et al., 1994). There are no data on the structure of

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IL-18 crystals. However, specific mutations in human IL-18 have revealed the importance of glutamic acid in position 35 and of lysine in position 89 for biological activity and binding to the IL-18 binding protein (Kim et al., 2001a).

Cells producing IL-1 family members The cells producing IL-1F5-10 are described above. The primary sources of IL-1b are the blood monocyte, tissue macrophages and dendritic cells. Blymphocytes and NK cells are also sources. Keratinocytes produce IL-1b under inflammatory conditions, but there is no constitutive expression of IL-1b in these cells unlike that of keratinocyte IL-1a. Fibrobasts and epithelial cells generally do not produce IL-1b. In health, the circulating human blood monocyte or bone marrow aspirate do not constitutively express IL-1b. However, there seems to be constitutive expression of IL-1b in the human hypothalamus (Breder et al., 1988). Nearly all microbial products induce IL-1b via the Toll-like receptor (TLR) family of receptors. TLR-4 is used by endotoxins to induce several cytokines and the induction of IL-1b is particularly sensititive to low (1–10 pg ml1) concentrations of endotoxins. Depending on the stimulant, IL-1b mRNA levels rise rapidly within 15 min but begin to decline after 4 h. This decrease is likely due to the synthesis of a transcriptional repressor and/or a decrease in mRNA half-life (Fenton et al., 1987). However, using IL-1 itself as a stimulant of its own gene expression, IL-1b mRNA levels are sustained for over 24 h compared with microbial stimulants. Raising intracellular cAMP levels with histamine enhances IL-1a-induced IL-1b gene expression and protein synthesis (Vannier and Dinarello, 1993). In human peripheral blood mononuclear cells (PBMC), retinoic acid induces IL-1b gene expression but the primary precursor transcripts fail to yield mature mRNA (Jarrous and Kaempfer, 1994). Inhibition of translation by cycloheximide results in enhanced splicing of exons, excision of introns and increased levels of mature mRNA (superinduction) by two orders of magnitude. Thus, synthesis of mature IL-1b mRNA requires an activation step to overcome an apparently intrinsic inhibition to process precursor mRNA. Stimulants such as the complement component C5a, hypoxia, adherence to surfaces, or clotting of blood

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induce the synthesis of large amounts of IL-1b mRNA in monocytic cells without significant translation into the IL-1b protein (Schindler et al., 1990). This dissociation between transcription and translation is characteristic of IL-1b, but also of TNFa. It appears that the above stimuli are not sufficient to provide a signal for translation despite a vigorous signal for transcription. Without translation, most of the IL-1b mRNA is degraded. Although the IL-1b mRNA assembles into large polyribosomes, there is little significant elongation of the peptide (Kaspar and Gehrke, 1994). However, adding bacterial endotoxin or IL-1 itself to cells with high levels of steady state IL-1b mRNA results in augmented translation in somewhat the same manner as the removal of cycloheximide following superinduction. One explanation is that stabilization of the AU-rich 3 untranslated region takes place in cells stimulated with LPS. These AU-rich sequences are known to suppress normal hemoglobin synthesis. The stabilization of mRNA by microbial products may explain why low concentrations of LPS or a few bacteria or Borrelia organisms per cell induce the translation of large amounts of IL-1b (Miller et al., 1992).

Transcriptional regulation The promoter of IL-1a does not contain a clear TATA box, a typical motif of inducible genes. Inducible gene expression for IL-1a involves both a 4.2 kb upstream and a proximal promoter region of 200 bp. A construct containing sequences 1437 to 19 does not allow for stimulation of specific expression, but an additional 731 bp spanning exon I, intron I and a segment of exon II controls a 20-fold increase in stimulation over background levels in murine macrophagic cells. Interestingly, using the same construct in human leukemic cells, only a two-fold increase was observed. These additional 731 bp contain nuclear factor (NF) IL-6 and NFjB within intron I. Unlike the promoter of IL-1a, the promoter region for IL-1b contains a clear TATA box. The half-life of IL-1b mRNA depends upon the cell type and the conditions of stimulation. The most studied cells are freshly obtained human blood monocytes and macrophage cell lines derived from myelomonocytic leukemias. Endotoxin triggers transient transcription and steady state levels of IL-1b mRNA which accumulate for 4 h followed by a rapid fall due to synthesis

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of a transcriptional repressor (Fenton et al., 1988). Unlike most cytokine promoters, IL-1b regulatory regions can be found distributed over several thousand base pairs upstream and a few base pairs downstream from the transcriptional start site. The topic of IL-1b gene regulation has been reviewed in detail (Auron and Webb, 1994). The IL-1b promoter required for transcription has two independent enhancer regions (2782 to 2729) and (2896 to 2846) which appear to act cooperatively. The latter contains a cAMP response element, whereas the former is a composite cAMP response element-NFIL-6 which is responsive to LPS. The 80 bp fragment (2782 to 2729) is required for transcription and contains, in addition to a cAMP response element, an NFjB-like site. Activating protein-1 (AP-1) sites also participate in endotoxin-induced IL-1b gene expression. Proximal promoter elements between 131 and 14 have also been identified and sequences in this region contain the binding sites for the novel nuclear factor NFbA, which appears to be similar to nuclear factors termed NFb1 and NFb2. This proximal promoter is required for maximal IL-1b gene expression. Importantly, the nucleotide binding sequences of NFbA were found to be identical to those of the transcription factor Spi-1/PU.1 (Fenton et al., 1994), a well-established NF in cells have myeloid and monocyte lineage. The requirement for Spi-1/PU.1 for IL-1b gene expression imparts tissue specificity since not all cells constitutively express this NF. Human blood monocytes, which constitutively express Spi-1/PU.1, are exquisitely sensitive to gene expression of IL-1b by 1–10 pg/ml of LPS. Interestingly, the IL-1Ra promoter contains the proximal Spi-1/PU.1 site, which is also highly sensitive to LPS. There is no constitutive gene expression for IL-1b in freshly obtained human PBMC from healthy donors using over 40 cycles of PCR (Shapiro and Dinarello, 1997). However, the same PBMC express constitutive mRNA for IL-18. Constitutive expression was also observed using Western blot analysis for precursor IL-18 in lysates from the same PBMC. Yet, there was no proIL-1b in the same cells. Constitutive IL-18 gene expression and the presence of precursor IL-18 protein were also observed in freshly obtained murine splenocytes (Puren et al., 1999). In these splenocytes, there was no constitutive expression of the IL-1b gene or protein. The promoter regions for IL-1b and IL-18

gene expression have been studied and may provide an insight into these observations. The promoter for IL-18 is TATA-less and IL-18 promoter activity upstream of exon 2 acts constitutively (Tone et al., 1997). The additional finding that the 3 untranslated region of human IL-18 lacks the AUUUA destabilization sequence is also consistent with these observations. This would allow for more sustained levels of the polyadenylated species and translation into protein. Other than to distinguish differences between IL-1b and IL-18 in the same cells, the clinical significance of constitutive gene and protein expression for IL-18 in mononuclear cells remains unclear, but certainly would focus on regulation being at the level of processing the precursor and secretion of the mature form(s). Osteoclasts also produce IL-18 (Martin et al., 1998) and regulation of bone density may be a property of IL-18 as it is for IL-1b. IL-1a is also synthesized as a precursor molecule without a signal peptide; unlike IL-1b, the IL-1a precursor is biologically active. Processing of the IL-1a precursor yields a mature molecule of 17.5 kDa. Calpain, a calcium activated cysteine protease associated with the plasma membrane, is responsible for cleavage of the IL-1a precursor into a mature molecule. It is unclear if this process of calpain cleavage of the IL-1a is functional under physiological conditions since IL-1a is rarely measured in the circulation. Even under conditions of cell stimulation, human blood monocytes do not process or readily secrete mature IL-1a. The 31 kDa IL-1a precursor is synthesized in association with cytoskeletal structures (microtubules), unlike most proteins, which are translated in the endoplasmic reticulum. It is unknown whether the IL-1a precursor is active intracellularly and there is no appreciable accumulation of IL-1a in any specific organelle. Immunohistochemical studies of IL-1a in endotoxin-stimulated human blood monocytes revealed a diffuse staining pattern. By comparison, in the same cell, IL-1Ra, which has a signal peptide, is localized to the Golgi complex (Andersson et al., 1992). In contrast to IL-1b, IL-1a is not commonly found in the circulation or in body fluids except during severe disease, in which case the cytokine may be released from dying cells. IL-18 is also initially synthesized as an inactive precursor (24 000) and requires ICE cleavage for processing into a mature molecule of 18 000. ICE-deficient

THE CYTOKINES AND CHEMOKINES

THE IL - 1 LIGAND SUPERFAMILY

mice have been helpful in revealing non-ICEmediated pathways of IL-18 processing. Following endotoxin, ICE-deficient mice do not exhibit circulating IFNc as endotoxin-induced IFNc is IL-18dependent (Gu et al., 1995). IL-12-induced IFNc is also ICE-dependent (Fantuzzi et al., 1999), again suggesting that microbial toxins (via Toll-like receptors) require IL-18 for IFNc production. In general, processing of the IL-18 precursor is ICE-dependent, but exceptions exist. Fas ligand stimulation results in release of biologically active IL-18 in ICE-deficient murine macrophages (Tsutsui et al., 1999). Similar to IL-1b processing, proteinase-3 appears to activate processing to mature IL-18. In contrast to the agonists IL-1a, IL-1b and IL-18, IL-1Ra has two prominent forms due to alternative mRNA splicing events. The IL-1Ra gene codes for a form with a strong signal peptide but this signal peptide can be deleted and the resulting ligand lacking a signal peptide remains intracellularly. Although the blood monocyte and tissue macrophages are the primary sources of IL-1b, in health, these cells do not constitutively express IL-1b. Expression of IL-1b from blood monocytes in health are due to the activation of the IL-1b transcriptional process by surface contact (Schindler et al., 1990). However, several malignant tumors express IL-1b as part of their neoplastic nature, particularly acute myelogenous leukemia, multiple myeloma and juvenile myelogenous leukemia, each of which exhibit constitutive expression of IL-1b.

IL-1b converting enzyme ICE (caspase-1) is constitutively expressed in various cells as a primary transcript of 45 kDa (inactive precursor) requiring two internal cleavages before becoming the enzymatically active heterodimer comprised of a 10- and 20-kDa chain. The active site cysteine is located on the 20-kDa chain. ICE itself contributes to autoprocessing of the ICE precursor by undergoing oligomerization with itself or homologs of ICE. In the presence of specific inhibitors of ICE, the generation and secretion of mature IL-1b is reduced and precursor IL-1b accumulates mostly inside, but the precursor is also found outside the cell. This latter finding supports the concept that precursor IL-1b can be released from a cell independent of processing by

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ICE. Due to alternate RNA splicing, there are five isoforms of human ICE (ICEa, b, c, d and e); ICEa, cleaves the ICE precursor and the IL-1b precursor. It is presumed that ICEb and c also process precursor ICEe. ICE is a truncated form of ICE which may inhibit ICE activity by binding to the p20 chain of ICE to form an inactive ICE complex. In addition to ICE, the IL-1b precursor is cleaved by elastase, chymotrypsin, a mast cell chymase, proteinase-3, granzyme A, and a variety of proteases commonly found in inflammatory fluids. Some matrix metalloproteases (MMPs) commonly found in joint fluids from patients with rheumatoid arthritis also cleave the precursor of IL-1b into biologically active IL-1b. These include gelatinase-B, MMP-2, MMP-3 (stromelysin-1) and MMP-9. These alternative, extracellular proteases may account for the observation that mice deficient in ICE can exhibit a full inflammatory response to subcutaneous turpentine, an IL-1b-dependent mode. The secretion of mature IL-1b is facilitated by a fall in the intracellular levels of potassium which takes place when a cell is exposed to high levels of ATP (Perregaux et al., 1992). Treatment of stimulated macrophages with millimolar concentrations of ATP also result in the processing and release of IL-1b. The effect of ATP or nigericin is due to a net decrease in the intracellular levels of potassium. Increasing the extracellular level of potassium also results in the inhibition of caspases by preventing the formation of a large intracellular complex associated with activation of caspases (Thompson et al., 2001). Figure 27.1 illustrates the cleavage of the IL-1b, as well as the IL-18 precursors by ICE.

The P2X-7 receptor and secretion of IL-1b Since ATP results in the rapid release of mature IL-1b within minutes, a receptor-mediated event has been proposed. For ATP, a purinergic receptor (since adenosine is a purine) is found on monocytes and macrophages and designated P2X-7. When triggered by millimolar concentrations of ATP, reversible pores form in the plasma membrane and due to ion fluxes, the electrical potential of the membrane is transiently lost. In the presence of LPS, this activation by ATP triggers the release of mature IL-1b (Ferrari et al., 1997). A monoclonal antibody to the P2X-7 receptor prevents

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INTERLEUKIN - 1 FAMILY

Soluble IL-1R II

Soluble IL-1R II

+

(neutralization)

IL-1β

IL-1R AcP

IL-1R II IL-1R I

IL-1R AcP

Approximation of cytoplasmic Toll-domains

No Signal

Nucleus MyD88

IRAKs Recruitment of kinases

IKKα IKKβ

No Signal NFkB

TRAF-6 IkB

MAP kinases

FIGURE 27.1 Interleukin-1b converting enzyme (ICE, caspase-1) cleaves the IL-18 and IL-1b precursors at the aspartic acid in P1 position (see text). the release of mature IL-1b from activated macrophages (Buell et al., 1998). Importantly, the secretion of IL-1b via activation of the P2X-7 receptor by TP is independent of ICE, since highly specific inhibitors of ICE prevent processing of the IL-1b precursor, but have no effect on release of the IL-1b precursor or the release of lactic dehydrogenase in cells stimulated with ATP (Perregaux and Gabel, 1998). Triggering of the P2X-7 receptor is specific for the release of mature IL-1b and also IL-18, but does not result in the release of TNFa. ATP or nigericin also stimulate the release of IL-1 and IL-18 in LPS-stimulated whole blood cultures (Perregaux et al., 2000). Convincing evidence for a role of the P2X-7R in the post-translational processing and secretion of IL-1b is found in mice deficient in this receptor. Similar to wild-type macrophages, P2X7R-deficient macrophages synthesize PGE2 and the IL-1b precursor in response to endotoxins. However, when activated by ATP, P2X-7R-deficient macrophages do not process or release IL-1b (Solle et al., 2001). In vivo, wild-type and P2X-7R-deficient release the same amounts of IL-6 into the peritoneal cavity, but there is no release of mature IL-1b from P2X-7Rdeficient mice.

first, precursor IL-1a is synthesized and remains inside the cell where it can bind to the nucleus; second, intracellular precursor IL-1a complexes to an intracellular pool of IL-1RI before exerting an effect as a ligand/receptor complex; and third, either precursor IL-1a or mature IL-1a bound to surface IL-1RI is internalized with subsequent translocation to the nucleus (similar to steroid receptors). Each mechanism has supporting experimental data. Some investigators have considered that intracellular precursor IL-1a regulates normal cellular differentiation, particularly in epithelial and ectodermal cells. In the case of keratinocytes, constitutive production of large amounts of precursor IL-1a is found in healthy human skin. In support of the concept that precursor IL-1a functions as an intracellular messenger in certain cells, an antisense oligonucleotide to IL-1a reduces senescence in endothelial cells (Maier et al., 1990, 1994). In the murine TH2 cell line, IL-1a was proposed as an essential autocrine and paracrine growth factor using an antisense IL-1a oligonucleotide or anti-IL-1a antibodies. Thymic epithelium produces IL-1a and a requirement for IL-1a has been demonstrated in the expression of CD25 (IL-2 receptor a chain) and maturation of thymocytes. However, these data must be viewed with the report that in mice deficient for IL-1a, there are no demonstrable defects in growth and development, including skin, fur, epithelium and gastrointestinal function (Horai et al., 1998). The large amounts of precursor IL-1a in normal skin keratinocytes is thought to affect terminal differentiation. If there is a role for intracellular precursor IL-1a in normal cell function, this should be carefully regulated. The presence of large amounts of an intracellular form of the IL-1Ra (icIL-1Ra) (Hammerberg et al., 1992) produced in the same cells expressing precursor IL-1a is thought to compete with the intracellular pool of precursor IL-1a for nuclear binding sites. The IL-1a-deficient mouse does not support this concept (Horai et al., 1998).

Membrane IL-1a

IL-1a IL-1a as an autocrine growth factor The concept that IL-1a can be an autocrine growth factor takes into account three distinct observations:

Precursor IL-1a can be found on the surface of several cells, particularly on monocytes and B lymphocytes, where it is referred to as membrane IL-1a (Kurt-Jones et al., 1985). Membrane IL-1a is biologically active; its biological activities are neutralized by anti-IL-1a but

THE CYTOKINES AND CHEMOKINES

EFFECTS IN IL - 1 KNOCKOUTS

not by anti-IL-1b. Membrane IL-1a appears to be anchored to the cell membrane via a lectin interaction involving mannose residues. A mannose-like receptor appears to bind membrane IL-1a (Brody and Durum, 1989). The role of membrane IL-1a in disease remains unclear. In vitro, the amount of IL-1Ra needed to block membrane IL-1a was 10- to 50-fold greater than the amount required to block mature IL-1b (Kaplanski et al., 1994).

Autoantibodies to IL-1a Neutralizing autoantibodies directed against IL-1a may function as natural buffers for IL-1a. Autoantibodies to IL-1a have been detected in healthy subjects, as well as in patients with various autoimmune diseases. Autoantibodies to IL-1a are neutralizing IgG antibodies that bind natural precursor form of IL-1a, as well as 17 kDa recombinant IL-1a (Bendtzen, 1990). The incidence of these antibodies is increased in patients with autoimmune diseases. For example, in 318 patients with chronic arthritis, anti-IL-1a, but not anti-IL-1b or anti-TNFa, IgG antibodies were detected in 18.9% of arthritis patients, but in 9% of healthy subjects. Anti-IL-1a was present more commonly and at a higher level in patients with nondestructive arthritis. An inverse correlation has been observed between the levels of anti-IL-1a antibodies and the clinical disease activity.

EFFECTS IN IL-1 KNOCKOUTS The IL-1b-deficient mouse. The IL-1b deficient mouse is without abnormal findings after 6 years of continuous breeding. However, upon challenge, IL-1b deficient mice exhibit specific differences from their wild-type controls. The most dramatic is the response to local inflammation followed by a subcutaneous injection of turpentine (50–100 ll). Within the first 24 h, IL-1b-deficient mice injected with turpentine do not manifest an acute phase response, do not develop anorexia, have no circulating IL-6 and no fever (Zheng et al., 1995, Fantuzzi et al., 1997a). These findings are consistent with those reported in the same model using anti-IL-1R type I

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antibodies in wild-type mice (Zheng et al., 1995). IL-1b-deficient mice also have reduced inflammation following zymosan-induced peritonitis (Fantuzzi et al., 1997b). Additional studies have also found that IL-1bdeficient mice have elevated febrile responses to IL-1b and IL-1a (Alheim et al., 1997). In contrast, IL-1b-deficient mice have nearly the same responses to LPS as do wild-type mice (Fantuzzi et al., 1996) with one notable exception. IL-1bdeficient mice injected with LPS have little or no expression of leptin mRNA or protein (Faggioni et al., 1998). In IL-1b pregnant mice, there is a normal response to LPS-induced pre-mature delivery. However, in these mice, there is decreased uterine cytokines following LPS (Reznikov et al., 2000). The reduction in LPS-induced cytokines is not found in non-pregnant IL-1b-deficient mice suggesting that the combination of the hormonal changes in pregnancy and the state of IL-1b deficiency act together to reduce the responsiveness to LPS. The mechanism for the reduced cytokine production in pregnant IL-1bdeficient mice appears to be due to a reduction in the constitutive level of the p65 component of NFjB. No differences were noted in plasma elevations of glucocorticoid steroids between IL-1b-deficient and wild-type mice following injection of LPS, indicating that IL-1b is not required for activation of the HPA axis during endotoxemia. The data demonstrate that in the mouse, IL-1b is critical for the induction of fever during local inflammation. Another characterized body temperature, activity and feeding live influenza virus in IL-1b-deficient mice. Body temperature and activity were lower in IL-1b-deficient mice (Kozak et al., 1998). The anorexic effects of influenza infection was similar in both groups of mice. The mice deficient in IL-1b exhibited a higher mortality to influenza infection than the wild-type mice.

Studies in IL-1a-deficient mice Mice deficient in IL-1a are born healthy and develop normally. Following subcutaneous injection of turpentine, which induces a local inflammatory response, wild-type and IL-1a-deficient mice develop fever, whereas IL-1b-deficient mice do not (Horai et al., 1998). The induction of glucocorticoids after turpentine injection was suppressed in IL-1b- but not in IL-1a-deficient mice. Expression of IL-1b mRNA in

THE CYTOKINES AND CHEMOKINES

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the brain decreased 1.5-fold in IL-1a-deficient mice, whereas expression of IL-1a mRNA decreased more than 30-fold in IL-1b-deficient mice. These data suggest that IL-1b exerts greater control over production of IL-1a than does IL-1a over the production of IL-1b. In ICE-deficient mice, IL-1a production is also reduced (Kuida et al., 1995), suggesting that production of IL-1a is under the control of IL-1b.

Differences between IL-1a-and IL-1b-deficient mice Studies on the effects of selective deficiency in IL-1b in mice are summarized in Table 27.2. These differences are to be compared with the same models in mice deficient in IL-1a. For example, mice deficient in IL-1a develop a normal immune response to immunization with sheep red blood cells, whereas mice deficient in IL-1b do not produce anti-sheep red blood cell antibodies, a T-dependent response (Nakae et al., 2001). However, antibody production by Tindependent antigens was normal in mice deficient in both IL-1a and IL-1, as was the proliferative response to anti-CD3. In mice deficient in IL-1Ra, there was enhanced response (Nakae et al., 2001).

Also mice deficient in IL-1a have a brisk inflammatory response to turpentine-induced inflammation, whereas IL-1b-deficient mice have nearly no response.

Studies in IL-1RI-deficient mice As stated above, mice deficient in IL-1RI develop normally and exhibit no particular phenotype despite being housed in standard animal facilities (Labow et al., 1997). IL-1RI-deficient mice show no abnormal phenotype in health and exhibit normal homeostasis, similar to that observed in IL-1b- or IL-1a-deficient mice (Zheng et al., 1995; Horai et al., 1998), but distinctly different from mice deficient in IL-1Ra (Hirsch et al., 1996). They do, however, exhibit reduced responses to challenge with inflammatory agents. When given a turpentine abscess, for example, IL-1RIdeficient mice exhibited an attenuated inflammatory response compared with wild-type mice (Josephs et al., 2000). IL-1RI-deficient mice also had a reduced delayed-type hypersensitivity responses. Similar to wild-type mice treated with anti-IL-1 antibodies or IL-1Ra, IL-1RI-deficient mice were susceptible to infection with Listeria monocytogenes. Lymphocytes

TABLE 27.2 Effects in IL-1b-deficient mice Disease model

Effect

Reference

Endotoxin fever LPS-induced leptin Zymosan peritonitis

No Effect ↓ Circulating leptin ↓ Inflammation ↓ Mortality ↓ IL-6 and chemokines ↓ Inflammation ↓ Fever ↓ IL-6; ↓ SAA ↓ cortisone ↓ COX-2 ↑ Fever ↑ Cytokines ↓ Metastasis ↓ Neuronal death Resistant to disease development ↓ Neutrophil infiltration No effect on neutrophil infiltration ↓ Plasminogen activator inhibitor (i) ↓ Plasminogen activator i unchanged ↓ Delayed hypersensitivity ↓ Langerhans cell activation ↓ Levels and translocation

Fantuzzi et al. (1996) Faggioni et al. (1998)

Turpentine inflammation

IL-1a-induced fever Hepatic melanoma Brain ischemia Immune myasthenia gravis Fas-expressing tumors LPS-induced shock lung Turpentine coagulopathy LPS-induced coagulopathy Contact hypersensitivity Contact hypersensitivity Steady state p65 (NFjB)

THE CYTOKINES AND CHEMOKINES

Fantuzzi et al. (1997b) Zheng et al. (1995) Horai et al. (1998) Alheim et al. (1997) Vidal-Vanaclocha et al. (2000) Boutin et al. (2001) Huang et al. (2001) Miwa et al. (1998) Parsey et al. (1999) Seki et al. (1999) Seki et al. (1999) Shornick et al. (1996) Shornick et al. (2001) Reznikov et al. (2000)

IL - 1 RECEPTOR FAMILY

from IL-1RI-deficient mice with major cutaneous leishmanial infection produced more IL-4 and IL-10, but less IFNc, than did those from wild-type mice. Although mice deficient in IL-1RI do not exhibit significant disruption of reproduction aside from a somewhat reduced litter size (Abbondanzo et al., 1996), in some laboratories, however, the body weights of the IL-1RI-deficient mice were 30% less than wild-type, whereas the TNFRp55-deficient mice weighed 30% more than wild-type mice of equivalent age (Vargas et al., 1996). Although IL-1a is constitutively expressed in the skin, the barrier function of skin remains intact in mice deficient in IL-1RI (Man et al., 1999). Similarly, mice deficient in IL-1R-AcP appear normal, but have no responses to IL-1 in vivo (Cullinan et al., 1998). However, cells deficient in IL-1R-AcP have normal binding of IL-1a and IL-1Ra (binding to the IL-1RI being intact), but a 70% reduction in binding of IL-1b (Cullinan et al., 1998). In these cells, there is no biological response to IL-1a, despite binding of IL-1a The results suggest that IL-1R-AcP and not IL-1RI is required for IL-1b binding and biological response to IL-1. Mice injected with LPS have been studied. IL-1RIdeficient mice exhibit the same decrease in hepatic lipase, as do wild-type mice. However, injection of LPS directly into the eye of mice deficient in IL-1RI reveal a decrease in the number of infiltrating leukocytes, whereas there was no decrease in mice deficient in either TNF receptors (Rosenbaum et al., 1998). IL-1RIdeficient mice failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1bdeficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. Also similar to IL-1b-deficient mice (Shornick et al., 1996), IL-1RIdeficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. Mice deficient in IL-1RI did not develop trabecular bone loss following ovariectomy compared to wildtype controls (Lorenzo et al., 1998). Although mice deficient in both the TNF-RI and TNF-RII receptors develop experimental autoimmune encephalomyelitis (EAE) after immunization with central nervous system antigens, mice deficient in IL-1RI failed to develop inflammatory lesions in the central nervous system or evidence of clinical EAE. Mice deficient in

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IL-1RAcP, the essential component of the IL-1RI signaling complex, have also been generated. Although cells from IL-1RAcP-deficient mice bound IL-1, there was no activation of genes dependent on NF-kB or activator protein-1 (AP-1) (Cullinan et al., 1998). Interestingly, the binding affinity of IL-1b for cells deficient in IL-1RAcP was reduced by 70%, whereas the binding affinity of IL-1a was only moderately reduced. In general, mice deficient in the IL-1RI exhibit reduced disease severity as do wild-type mice injected with pharmacologic doses of IL-1Ra.

IL-1 RECEPTOR FAMILY Genes coding for IL-1 receptors The IL-1 receptor family now encodes nine distinct genes of which some remain orphan receptors. As shown in Table 27.3, these receptors have been assigned a nomenclature in the order of their discovery. The IL-18 binding protein (IL-18BP) is not listed due to its lack of being fixed to the cell via a transmembrane domain. However, the IL-18BP likely represents the former cell-bound decoy receptor for IL-18 similar to the decoy receptor for IL-1 (the IL-1 receptor type II, see below). In fact, there is limited but significant amino acid homology between the IL-18BP and the type II IL-1 receptor, particularly in the third domain (Novick et al., 1999). IL-1R1, IL-1R2 and IL-1R3 are the bona fide receptors for IL-1. IL-1R4

TABLE 27.3 Nomenclature of IL-1R family Name

New designation

Ligand

IL-1RI IL-1RII IL-1R Ac-P ST2/Fit-1 IL-18Ra/ IL-1Rrp1 IL-1Rrp2 IL-1R18b/ IL-1RAcPL IL-1RAPL IL-R9

IL-1R1 IL-1R2 IL-1R3 IL-1R4 IL-1R5

IL-1a, IL-1b, IL-1Ra IL-1b, IL-1a, IL-1Ra IL-1a, IL-1b unknown IL-18

IL-1R6 IL-1R7

?IL-1e, IL-1d IL-18

IL-1R8 IL-1R9

unknown unknown

THE CYTOKINES AND CHEMOKINES

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(also known as ST2 and Fit) remains an orphan receptor, although proteins have been reported that bind to this receptor (Gayle et al., 1996). Despite a lack of a specific ligand for this receptor, a number of studies have examined the distribution and gene regulation of this receptor in mast cells (Moritz et al., 1998). IL-1R5 was formerly an orphan receptor termed IL-1R related protein-1 (Parnet et al., 1996), but was subsequently discovered to be the ligand-binding chain of the IL-18 receptor (Torigoe et al., 1997), now termed IL-18Ra chain. The IL-1R related protein-2 (IL-1R6) has been proposed to be the receptor for a novel member of the IL-1 family, IL-1Fe (Debets et al., 2001). The activity of this ligand for the IL-1R6 was demonstrated in a luciferase NFjB assay; another member of the IL-1 family, IL-1Fd, appears to be its natural receptor antagonist for IL-1Fe binding to IL-1R6 (Debets et al., 2001). The IL-1R7, formerly the non-ligand binding chain of the IL-18 receptor, termed IL-1R AcPL (Born et al., 1998), is now named IL-18Rb chain. Similar to the IL-1R-AcP, the IL-18Rb is essential for IL-18 signal transduction (Born et al., 1998; Kim et al., 2001b). Two members of the IL-1 receptor family are particularly unique in that they are found on the  chromosome. These are IL-1R8 and IL-1R9, both being homologous to the IL-1 accessory protein receptor chains (IL-1R-AcP and IL-1R-AcPL). IL-1R9 (Sana et al., 2000) is highly homologous to IL-1R8 (Carrie et al., 1999). Both forms have no known ligands and receptors are found in the fetal brain. In fact, nonoverlapping deletions and a nonsense mutation in the IL-1R8 gene were found in patients with cognitive impairment (Carrie et al., 1999) where expression in the adult hippocampal area may play a role in memory or learning. The cytoplasmic domains of IL-1R8 and IL-1R9 are longer than the other accessory chains. The IL-1R9 may function as a negative receptor. This was shown in cells overexpressing this receptor, as well as the IL-1RI and IL-1R-AcP in which IL-1b signaling was blocked with a specific antibody to the IL-1R-AcP. In the presence of the antibody, IL-1binduced luciferase was suppressed, suggesting that a possible complex of the type I receptor with IL-1b plus IL-1R9 results in a negative signal (Sana et al., 2000).

IL-1 receptor type I The first studies on the specific receptor for IL-1a and IL-1b were based on the identification of an 80 kDa glycoprotein on T cells and fibroblasts, which, in retrospect, is now termed the IL-1 receptor type I (IL-1RI) (Bird et al., 1987; Scapigliati et al., 1989; Savage et al., 1989; Qwarnstrom et al., 1988). The molecular cloning of IL-1RI was first made in the mouse in 1988 (Sims et al., 1988) and then subsequently in the human (Chizzonite et al., 1989). The extracellular segment of the IL-1RI and nearly all members of the IL-1 receptor family have three Ig-like domains. The cytoplasmic segment of the IL-1RI is unique in that it contains the Toll-homology domain. This domain contains amino acids closely related to those of a gene found in Drosophila (Gay and Keith, 1991); the Drosophila gene is essential for the embryonic development of the fruit fly. This Toll-homology domain is also found in the cytoplasmic domains of each member of the Toll-like receptor (TLR), which transduce the signals of endotoxins and other microbial products in mammalian cells (Beutler, 2001). In mammalian cells, the Toll-homology domain of the IL-1R is necessary for signal transduction (Heguy et al., 1992). For several years following the molecular cloning of the IL-1RI, IL-1 signal transduction was thought to occur when IL-1 bound to the single chain IL-1RI. However, in 1995 Greenfeder and co-workers discovered that IL-1 signal transduction was initiated by the formation of a heterodimer with a second, different receptor chain; this second chain is now termed the IL-1 receptor accessory protein (IL-1RAcP) (Greenfeder et al., 1995). When IL-1 binds to the cell membrane IL-1RI, the IL-1:IL-1RI complex recruits the IL-1R IL-1R-AcP (Greenfeder et al., 1995). There is considerable amino acid homology between the IL-1RI and IL-1R-AcP in the extracellular, as well as cytoplasmic domains, including the Toll homology domain. Importantly, IL-1R-AcP does not bind IL-1 itself, but rather ‘wraps around’ the complex of IL-1:IL-1RI (Casadio et al., 2001). As shown by X-ray crystallization studies, IL-1RI exhibits a conformational change when binding IL-1b and apparently this shape change allows the IL-1R-AcP to form the heterodimer. The formation of the heterodimer of the IL-1RI with the IL-1R-AcP results in the physical approximation of

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the Toll homology domains of each chain in the cytoplasmic segments and initiates signal transduction (Figure 27.2). A similar event of approximately Toll homology domains takes place when IL-18 binds to its receptor, the IL-18Ra chain (Torigoe et al., 1997) and recruits the IL-18Rb chain (Born et al., 1998). The extracellular and cytoplasmic domains of the IL-1RI share homologies to IL-18Ra chain (Torigoe et al., 1997), which was previously an orphan receptor in the IL-1R family (Parnet et al., 1996). Like IL-1, IL-18 binding to the IL-18Ra chain recruits a second, non-ligand binding chain (IL-18Rb) (Born et al., 1998). Thus, IL-1 and IL-18 signal transduction are initiated by similar, if not identical, physical approximation of the Tollhomology domains, which initiates signal transduction for both cytokines. In both cases, the second chain, although not capable of binding the respective ligand, is essential for activity (Born et al., 1998; Kim et al., 2001b). Glycosylation of IL-1RI appears to be necessary for optimal activity. In fact, blocking the glycosylation sites reduces the binding of IL-1. In general, IL-1responsiveness is a more accurate assessment of receptor expression than ligand binding (Rosoff et al., 1988). The failure to show specific and saturable IL-1 binding to cells is often due to the low numbers of surface IL-1RI on primary cells. In cell lines, the number of IL-1RI can reach 5000 per cell, but primary cells usually express less that 200 receptors per cell. In some primary cells there are less than 50 per cell (Shirakawa et al., 1987) and IL-1 signal transduction has been observed in cells expressing less than 10 type I receptors per cell (Stylianou et al., 1992).

Inactive IL-18 Precursor Active, mature IL-18

N L E S D Y F G K L …… N-terminus

ICE Inactive IL-1β Precursor Active, mature IL-1β

Y V H D A P V………. N-terminus

ICE

FIGURE 27.2 IL-1 signal transduction (see text).

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Both chains of the IL-1, as well as IL-18 receptors are needed for signal transduction. For IL-1, this has been shown using specific neutralizing antibodies to IL-1RI or IL-1R-AcP (Yoon and Dinarello, 1998) and for IL-18, transfection of the IL-18Rb chain affords responsiveness (Born et al., 1998; Kim et al., 2001b). The cytoplasmic segment of IL-1RI or IL-1R-AcP have no apparent intrinsic tyrosine kinase activity, but when IL-1 binds to only a few receptors, the remaining unoccupied receptors appear to undergo phosphorylation (Gallis et al., 1989). However, the Toll-homology domain is essential for biological activity of IL-1 (Guida et al., 1992; Heguy et al., 1992). The Toll-like receptors (TLR) have extracellular domains that recognize microbial products, such as endotoxins and peptidolglycans, but the intracellular domains share significant sequences with the intracellular domains of IL-1RI and IL-1R-AcP. Therefore, it is not surprising that cellular responses to endotoxins, as well as to IL-1, are similar. For example, the portfolio of genes induced by either endotoxins or IL-1 are nearly the same. Differences exist in the binding affinity, association and dissociation rates of the mature forms of each member of the IL-1 family to cell-bound IL-1RI and soluble (extracellular domains) IL-1RI receptors. In some cells, there is a discrepancy between the dissociation constant of either form of IL-1 (usually 200–300 pM) and concentrations of IL-1 that elicit a biological response (10–100 fM) (Orencole and Dinarello, 1989). In cells expressing large amounts of IL-1R-AcP, the high affinity binding of the IL-1R/ IL-1R-AcP complex may explain which two classes of binding have been observed. Human IL-1a binds to cell surface and soluble type I receptors with approximately the same affinity (100–300 pM), as does IL-1Ra. If one examines the binding of IL-1Ra using BiaCore analysis, the affinity is even higher than that of IL-1a. IL-1Ra avidly binds to the surface type I receptor (50–100 pM). Although IL-1Ra binds less to the soluble form of this receptor it is, nevertheless, a high affinity binding. Of the three members of the IL-1 family (IL-1a, IL-1b and IL-1Ra), IL-1b has the lowest affinity for the cell bound form of IL-1RI (500 pM–1 nM). IL-1b binding to the soluble form (extracellular domains) of the IL-1RI is lower compared with the cell bound receptor. The greatest binding affinity of the three IL-1

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ligands for the IL-1RI is the IL-1Ra. In fact, the off-rate is slow and binding of IL-1Ra to the cell bound IL1RI is nearly irreversible. Compared with IL-1Ra, IL1a binds to IL-1RI with affinities ranging from 100 to 300 pM. By comparison, IL-1b binds more avidly to the non-signal transducing type II receptor (100 pM).

IL-1R type II The IL-1 receptor type II (IL-1RII) was described by several investigators (Symons et al., 1991, 1993) and the ability of IL-1b to preferentially bind to B cells likely represents binding to the type II receptor (Scapigliati et al., 1989; Ghiara et al., 1991). The amino acid sequence of the human IL-1RII was reported in 1991 (McMahon et al., 1991). The concept that this receptor functioned as a negative or ‘decoy’ receptor was demonstrated by Colotta and Mantovani in 1993 (Colotta et al., 1993, 1994). The extracellular segment of the IL-1RII has three typical Ig-like domains; there is a transmembrane segment and a short cytoplasmic domain (McMahon et al., 1991). The short cytoplasmic domain is unable to initiate signal transduction since there is no Toll-homology domain. Therefore, when IL-1 binds to the cell membrane, IL-1RII does not signal. Vaccinia and cowpox virus genes encode for a protein with a high amino acid homology to the type II receptor and this protein binds IL-1b (Alcami and Smith, 1992; Spriggs et al., 1992). These same viruses also code for IL-18 binding protein-like molecules (Novick et al., 1999). The viral form of the IL-1RII likely serves to reduce the inflammatory and immune response of the host to the virus. A soluble (extracellular) form of this receptor is released from the cell surface by the action of a protease, binds IL-1b and neutralizes the biological effects of IL-1b (Dower et al., 1994). Although the short cytoplasmic domain in the rat is longer than in the human (Bristulf et al., 1994), this receptor does not signal. In the human and mouse, the cytoplasmic domain of IL-1RII consists of 29 amino acids. In the rat, there are an additional six charged amino acids (Bristulf et al., 1994). IL-1b binds with a greater affinity to the type II receptor than does IL-1a and IL-1Ra binding to this receptor is the lowest of the three ligands (Arend et al., 1994; Sims et al., 1994; Dower et al., 1994). Although IL-1a binds to cell surface and soluble type I receptors with approximately the same affinity (200–300 pM),

IL-1a binding to surface and soluble type II receptors is nearly 100-fold less (30 and 10 nM, respectively). By comparison, IL-1b binds avidly to the non-signal transducing type II receptor (100 pM) and IL-1b binding to the soluble form of the this receptor is also high at 500 pM. Moreover, IL-1b binding to the soluble IL-1RII is nearly irreversible due to a long dissociation rate (2 h) (Symons et al., 1994; Dower et al., 1994; Arend et al., 1994). The precursor form of IL-1b also preferentially binds to the soluble form of IL-1RII (Symons et al., 1991, 1993). The function of the type II receptor as a ‘decoy’ receptor is based on the binding of IL-1b to the cell surface form of this receptor, thus preventing the ability of the ligand to form a complex with the type I receptor and the accessory protein (Colotta et al., 1993, 1994). Another and perhaps more efficient function of the decoy receptor is to form a trimeric complex of the IL-1b ligand with the type II receptor and the accessory protein (Lang et al., 1998; Neumann et al., 2000). This mechanism serves to deprive the functional receptor type I of the accessory chain.

Gene and surface regulation of IL-1RI The entire gene is distributed over 29 kb. The genomic organization of the human type I receptor reveals three distinct transcription initiation sites contained in three separate segments of the first exon. Each part of this first exon is thought to possess a separate promoter, which functions independently in different cells (Ye et al., 1993; Sims et al., 1995). Despite evidence that type I receptor gene expression can be upregulated in vitro (Koch et al., 1992; Ye et al., 1996), the most proximal (5) promoter region lacks a TATA or CAAT box (Ye et al., 1993). In fact, this promoter region for the human IL-1RI shares striking similarity to those of housekeeping genes rather than highly regulated genes. The transcription initiation start site contains nearly the same motif as that for the TdT gene (Ye et al., 1993). There is a guanosine-cytosine rich segment (75%) following the transcription initiation site of exon I which accounts for considerable secondary RNA structure. Low numbers of surface IL-1RI may, in fact, be due to multiple secondary RNA structures, which reduce optimal translation of the mRNA (Ye et al., 1996). Surface expression of IL-1RI clearly impacts upon

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the biological response to IL-1. Similar to IL-1b, cells can express high steady state levels of mRNA for IL-1RI, but low levels of the protein. This may be due to the amount of secondary structure in each of the polyadenylated RNA species. Studies on IL-1R surface expression have mostly used binding of labeled ligands rather than assessment of surface receptor density using specific antibodies. Nevertheless, phorbol esters, PGE2, dexamethasone, epidermal growth factor, IL-2 and IL-4 increase surface expression of IL-1RI. In cells which synthesize PGE2, IL-1 upregulates its own receptor via PGE2. However, when PGE2 synthesis is inhibited, IL-1 down-regulates IL-1RI in the same cells. Part of the immunosuppressive properties of TGFb may be due to downregulation of the IL-1RI on T cells. In the case of TH2 lymphocytes, IL-1 down-regulates IL-1RI surface expression and this is associated with a decrease in mRNA half-life. Therefore, despite the housekeeping nature of its promoters, IL-1RI is regulated in the context of inflammation and immune responses.

IL-18 binding protein There are limited amino acid homologies between the IL-18BP (Novick et al., 1999) and the type II IL-1R and both function as decoy receptors for their respective ligands. However, a transmembrane domain of the IL-18BP has apparently been deleted and this decoy receptor functions solely as a secreted protein. Another soluble receptor that has apparently lost its transmembrane domain is osteoprotegerin which binds and neutralizes RANK ligand. The IL-18BP has a single Ig-domain and limited homology to the IL-18Ra chain (Kim et al., 2000). Molecular modeling of IL-18 binding to IL-18BP has identified specific amino acids, which, when mutated decrease the ability of IL-18BP to bind and neutralize IL-18 (Kim et al., 2001a). The affinity of IL-18 for IL-18BP is high (Kd of 400 pM) and plasma levels of 3–4 ng/ml in healthy subjects (Novick et al., 2001) suggests that IL-18BP functions as a natural buffer against IL-18 and the TH1 response.

SIGNAL TRANSDUCTION Associated or intrinsic kinases Hopp reported a detailed sequence and structural comparison of the cytosolic segment of IL-1RI with the ras-family of GTPases (Hopp, 1995). In this analysis, the known amino acids residues for GTP binding and hydrolysis by the GTPase family were found to align with residues in the cytoplasmic domain of the IL-1RI. In addition, Rac, a member of the Rho family of GTPases, were also present in the binding and hydrolytic domains of the IL-1RI cytosolic domains. These observations are consistent with the observations that GTP analogues undergo a rapid hydrolysis when membrane preparations of IL-1RI are incubated with IL-1. Amino acid sequences in the cytosolic domain of the IL-1R-AcP also align with the same binding and hydrolytic regions of the GTPases. A protein similar to G-protein activating protein has been identified which associates with the cytosolic domain of the IL-1RI (Mitchum and Sims, 1995). This finding is consistent with the hypothesis that an early event in IL-1R signaling involves dimerization of the two cytosolic domains, activation of putative GTP binding sites on the cytosolic domains, binding of a G-protein, hydrolysis of GTP and activation of a phospholipase. In then follows that hydrolysis of phospholipids generates diacylglycerol or phosphatidic acids.

Cytoplasmic signaling cascades Signal transduction of IL-1 depends on the formation of a heterodimer between IL-1RI and IL-1R-AcP (Greenfeder et al., 1995). This interaction recruits MyD88, a cytoplasmic adapter molecule. This is followed by recruitment of the IL-1R activating kinase (IRAK) (Croston et al., 1995; Cao et al., 1996, Huang et al., 1997; Cao, 1998). Antibodies to IL-1RI block IL-1 activity. Although IL-1R-AcP does not bind IL-1, antibodies to IL-1R-AcP also prevent IL-1 activity (Yoon and Dinarello, 1998). Both the extracellular domain of the IL-1R-AcP and its cytoplasmic segment share homology with the IL-1RI. There is a perfectly conserved protein kinase C acceptor site in both cytoplasmic domains, although agents activating protein kinase C do not mimic IL-1 signal transduction. Limited sequence homology of the gp130 cytoplasmic

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domain with those of IL-1RI and IL-1R-AcP suggest that complex formation of the IL-1R/IL-1/IL-1R-AcP transduces a signal similar to that observed with ligands, which cause the dimerization of gp130. In fact, deletion of the gp130 shared sequences from the IL-1RI cytoplasmic domain results in a reduced response to IL-1. IL-1 shares some prominent biological properties with gp130 ligands; for example, fever, hematopoietic stem cell activation and the stimulation of the hypothalamic-pituitary-adrenal axis are common to IL-1 and IL-6. Other biological activities of IL-1 and IL-6 are distinctly antagonistic. High levels of IL-1R-AcP are expressed in mouse and human brain tissue. The discovery and function of the IL-1R-AcP has placed IL-1 receptor biology and signaling mechanisms into the same arena as other cytokines and growth factors. The IL-1R-AcP also explains previous studies describing low and high binding affinities of IL-1 to various cells. As shown in Figure 28.2, like other models of two chain receptors, IL-1 binds first to the IL-1RI with a low affinity. Although there is no direct evidence, a structural change may take place in IL-1 allowing for docking of IL-1R-AcP to the IL-1RI/IL-1 complex. Once IL-1RI/ IL-1 binds to IL-1R-AcP, a high affinity binding is observed. Antibodies to the type I receptor and to the IL-1R-AcP block IL-1 binding and activity. Therefore, IL-1 may bind to the type I receptor with a low affinity causing a structural change in the ligand followed by recognition by the IL-1R-AcP. Within a few minutes following binding to cells, IL-1 induces several biochemical events. It remains unclear which is the most ‘upstream’ triggering event or whether several occur at the same time. No sequential order or cascade has been identified, but several signaling events appear to be taking place during the first 2–5 min. Some of the biochemical changes associated with signal transduction are likely cell specific. Within 2 min, hydrolysis GTP, phosphotidylcholine, phosphotidylserine or phosphotidylethanolamine (Rosoff, 1989) and release of ceramide by neutral (Schutze et al., 1994), not acidic, sphingomyelinase (Andrieu et al., 1994) have been reported. In general, multiple protein phosphorylations and activation of phosphatases can be observed within 5 min (Bomalaski et al., 1992) and some are thought to be initiated by the release of lipid mediators. The release of ceramide has attracted attention

as a possible early signaling event (Kolesnick and Golde, 1994). Phosphorylation of PLA2 activating protein also occurs in the first few minutes (Gronich et al., 1994), which would lead to a rapid release of arachidonic acid. Multiple and similar signaling events have also been reported for TNF. Of special consideration to IL-1 signal transduction is the unusual discrepancy between the low number of receptors (10 in some cells) and the low concentrations of IL-1 which can induce a biological response. This latter observation, however, may be clarified in studies on high affinity binding with the IL-1R-AcP complex. A rather extensive ‘amplification’ step(s) takes place following the initial post-receptor binding event. The most likely mechanism for signal amplification is multiple and sequential phosphorylations (or dephosphorylations) of kinases which result in nuclear translocation of transcription factors and activation of proteins participating in translation of mRNA. IL-1RI is phosphorylated following IL-1 binding. It is unknown whether the IL-1R-AcP is phosphorylated during receptor complex formation. In primary cells, the number of IL-1RI type I is very low (100 per cell) and a biological response occurs when only as few as 2–3% of IL-1RI receptors are occupied (Gallis et al., 1989; Ye et al., 1992). In IL-1responsive cells, one assumes that there is constitutive expression of the IL-1R-AcP. With few exceptions, there is general agreement that IL-1 does not stimulate hydrolysis of phosphatidylinositol nor an increase in intracellular calcium. Without a clear increase in intracellular calcium, early post-receptor binding events nevertheless include hydrolysis of a GTP with no associated increase in adenyl cyclase, activation of adenyl cyclase (Mizel, 1990; Munoz et al., 1990), hydrolysis of phospholipids (Rosoff et al., 1988; Kester et al., 1989), release of ceramide (Mathias et al., 1993) and release of arachidonic acid from phopholipids via cytosolic phospholipase A2 (PLA2) following its activation by PLA2 activating protein (Gronich et al., 1994). Some IL-1 signaling events are prominent in different cells. Post-receptor signaling mechanisms may therefore provide cellular specificity. For example, in some cells, IL-1 is a growth factor and signaling is associated with serine/threonine phosphorylation of the MAP kinase p42/44 in mesangial cells (Huwiler and Pfeilschifter, 1994). The MAP p38 kinase, another member of the

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MAP kinase family, is phosphorylated in fibroblasts (Freshney et al., 1994), as is the p54 MAP kinase in hepatocytes (Kracht et al., 1994).

Characteristics of the cytoplasmic domain of the IL-1RI The cytoplasmic domain of the IL-1RI does not contain a consensus sequence for intrinsic tyrosine phosphorylation, but deletion mutants of the receptor reveal specific functions of some domains. There are four nuclear localization sequences which share homology with the glucocorticoid receptor. Three amino acids (Arg-431, Lys-515 and Arg-518), also found in the Toll protein, are essential for IL-1induced IL-2 production (Heguy et al., 1992). However, deletion of a segment containing these amino acids did not affect IL-1-induced IL-8 (Kuno et al., 1993). There are also two cytoplasmic domains in the IL-1RI which share homology with the IL-6-signaling gp130 receptor. When these regions are deleted, there is a loss of IL-1-induced IL-8 production (Kuno et al., 1993). The C-terminal 30 amino acids of the IL-1RI can be deleted without affecting biological activity (Croston et al., 1995). Two independent studies have focused on the area between amino acids 513 and 529. Amino acids 508–521 contain sites required for the activation of NFB. In one study, deletion of this segment abolished IL-1-induced IL-8 expression and in another study, specific mutations of amino acids 513 and 520 to alanine prevented IL-1-driven E-selectin promoter activity. This area is also present in the Toll protein domain associated with NFB translocation and previously shown to be part of the IL-1 signaling mechanism. This area (513–520) is also responsible for activating a kinase, which associates with the receptor. This kinase, termed ‘IL-1RI-associated kinase’ phosphorylates a 100 kDa substrate. Others have reported a serine/threonine kinase which co-precipitates with the IL-1RI (Martin et al., 1994). Amino acid sequence comparisons of the cytosolic domain of the IL-1RI have revealed similarities with a protein kinase C (PKC) acceptor site. Because PKC activators usually do not mimic IL-1-induced responses, the significance of this observation is unclear.

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Recruitment of MyD88 and IL-1 receptor activating kinases An event that may be linked to the binding of G-proteins to the IL-1 receptor complex is the recruitment of the cytosolic protein MyD88. This small protein has many of the characteristics of cytoplasmic domains of receptors, but MyD88 lacks any known extracellular or transmembrane structure. Mice deficient in MyD88 do not respond to IL-1 or IL-18. It is unclear exactly how this protein functions, since it does not have any known kinase activity. However, it may assist in the binding of the IRAKs to the complex and hence has been said to function as an adapter molecule. There are presently four IRAKs (Suzuki et al., 2002). In mice with a deletion in IRAK-4, there is reduced endotoxin, as well as IL-1 signaling (Suzuki et al., 2002). The binding of IRAKs to the IL-1R complex appears to be a critical step in the activation of NFB (Croston et al., 1995). The IL-1R-AcP is essential for the recruitment and activation of IRAK (Wesche et al., 1997; Huang et al., 1997). In fact, deletion of specific amino acids in the IL-1R-AcP cytoplasmic domain results in loss of IRAK association (Wesche et al., 1997). In addition, the intracellular adapter molecule, termed MyD88, appears to dock to the complex allowing IRAK to become phosphorylated (Croston et al., 1995; Cao, 1998). IRAK then dissociates from the IL-1R complex and associates with TNF receptor associated factor-6 (TRAF-6) (Cao et al., 1996). TRAF-6 then phosphorylates NIK (Malinin et al., 1997) and NIK phosphorylates the inhibitory B kinases (IKK-1 and IKK-2) (DiDonato et al., 1997). Once phosphorylated, IB is rapidly degraded by a ubiquitin pathway liberating NFB which translocates to the nucleus for gene transcription. Some studies suggest that NIK is not necessary for IL-1 signaling. However, in mice deficient in TRAF-6, there is no IL-1 signaling in thymocytes and the phenotype exhibits severe osteopetrosis and defective formation of osteoclasts (Lomaga et al., 1999). IRAKs also associate with the IL-18R complex (Robinson et al., 1997; Kojima et al., 1988). This was demonstrated using IL-12-stimulated T cells followed by immunoprecipitation with anti-IL-18R or antiIRAK (Kojima et al., 1998). Furthermore, IL-18triggered cells also recruited TRAF-6 (Kojima et al., 1998). Like IL-1 signaling, MyD88 has a role in IL-18

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signaling. MyD88-deficient mice do not produce acute phase proteins and have diminished cytokine responses. Recently, TH1-developing cells from MyD88-deficient mice were unresponsive to IL-18induced activation of NFB and c-Jun N-terminal kinase (JNK) (Adachi et al., 1998). Thus, MyD88 is an essential component in the signaling cascade that follows IL-1 receptor, as well as IL-18 receptor binding. It appears that the cascade of sequential recruitment of MyD88, IRAK and TRAF-6 followed by the activation of NIK and degradation of IBK and release of NFB are nearly identical for IL-1 as well as for IL-18. Indeed, in cells transfected with IL-18R (IL-1Rrp) and then stimulated with IL-18, translocation of NFjB is observed using electromobility shift assay (Torigoe et al., 1997). In U1 macrophages, which already expresses the gene for IL-1Rrp, there is translocation of NFjB and stimulation of the human immunodeficiency virus-type 1 (HIV-1) production (Shapiro et al., 1998b).

Activation of MAP kinases following IL-1 receptor binding Multiple phosphorylations take place during the first 15 min following IL-1 receptor binding. Most consistently, IL-1 activates protein kinases which phosphorylate serine and threonine residues, targets of the MAP kinase family. An early study reported an IL-1-induced serine/threonine phosphorylation of a 65 kDa protein clearly unrelated to those phosphorylated via PKC (Matsushima et al., 1987). As reviewed by O’Neill (O’Neill, 1995), prior to IL-1 activation of serine/threonine kinases, IL-1 receptor binding results in the phosphorylation of tyrosine residues (Freshney et al., 1994; Kracht et al., 1994). Tyrosine phosphorylation induced by IL-1 is likely due to activation of MAP kinase kinase which then phosphorylates tyrosine and threonine on MAP kinases. Following activation of MAP kinases, there are phosphorylations on serine and threonine residues of the epidermal growth factor receptor, heat-shock protein p27, myelin basic protein and serine 56 and 156 of b-casein, each of which has been observed in IL-1stimulated cells (Bird et al., 1991). TNF also activates these kinases. There are at least three families of MAP kinases. The p42/44 MAP kinase family is associated with signal transduction by growth factors including ras-raf-1 signal pathways. In rat mesangial cells, IL-1

activates the p42/44 MAP kinase within 10 min and also increases de novo synthesis of p42 (Huwiler and Pfeilschifter, 1994).

p38 MAP kinase activation The stress-activated protein kinase (SAPK), which is molecularly identified as Jun N-terminal kinase (JNK), is phosphorylated in cells stimulated with IL-1 (Stylianou and Saklatvala, 1998). In addition to p42/44, two members of the MAP kinase family (p38 and p54) have been identified as part of an IL-1 phosphorylation pathway and are responsible for phosphorylating hsp 27 (Kracht et al., 1994; Freshney et al., 1994). In rabbit primary liver cells, IL-1 selectively activates JNK without apparent activation of p38 or p42 p38 MAP kinases (Finch et al., 1997). These MAP kinases are highly conserved proteins homologous to the HOG-1 stress gene in yeasts. In fact, when HOG-1 is deleted, yeasts fail to grow in hyperosmotic conditions. However, the mammalian gene coding for the IL-1-inducible p38 MAP kinase (Kracht et al., 1994) can reconstitute the ability of the yeast to grow in hyperosmotic conditions (Galcheva-Gargova et al., 1994). In cells stimulated with hyperosmolar NaCl, LPS, IL-1 or TNF, indistinguishable phosphorylation of the p38 MAP kinase takes place (Han et al., 1994). In human monocytes exposed to hyperosmolar NaCl (375-425 milliosmoles/l), IL-8, IL-1b, IL-1a and TNFa gene expression and synthesis takes place, which is indistinguishable from that induced by LPS or IL-1 (Shapiro and Dinarello, 1995, 1997). Thus, the MAP p38 kinase pathways involved in IL-1, TNF and LPS signal transductions share certain elements that are related to the primitive stress-induced pathway. The dependency of Rho members of the GTPase family (see above) for IL-1-induced activation of p38 MAP kinases has been demonstrated (Zhang et al., 1995). This latter observation links the intrinsic GTPase domains of IL-1RI and IL-1R-AcP with activation of the p38 MAP kinase.

Inhibition of p38 MAP kinase The target for pyridinyl imidazole compounds has been identified as a homologue of the HOG-1 family (Lee et al., 1994). Its sequence is identical to that of the p38 MAP kinase activating protein-2 (Han et al.,

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1995). Inhibition of the p38 MAP kinase is highly specific for reducing LPS- and IL-1-induced cytokines (Lee et al., 1994). IL-1 induced expression of HIV-1 is suppressed by specific inhibition using pyridinyl imidazole compounds (Shapiro et al., 1998a). As expected, this class of imidazoles also prevents the downstream phosphorylation of hsp 27 (Cuenda et al., 1995). Compounds of this class appear to be highly specific for inhibition of the p38 MAP kinase in that there was no inhibition of 12 other kinases. Using one of these compounds, both hyperosmotic NaCl- and IL-1a-induced IL-8 synthesis was inhibited (Shapiro and Dinarello, 1995). It has been proposed that MAP kinase activating protein-2 is one of the substrates for the p38 MAP kinases and that MAP kinase activating protein-2 is the kinase which phosphorylates hsp-27 (Cuenda et al., 1995).

EFFECTS OF BLOCKING IL-1 A case of a cortisol-secreting adrenal adenoma causing Cushing’s syndrome in a 62-year-old woman has been described. The patient exhibited the classic clinical and laboratory findings of Cushing’s syndrome which abated once the tumor was removed. Examination of the tissue revealed high expression of IL-1RI (Willenberg et al., 1998). Moreover, unlike normal adrenal cells, the tumor did not respond to corticotropin-induced cortisol production, but rather responded to IL-1b stimulation of cortisol production. In contrast to the patient’s tumor, other adrenal tumors responded to corticotropin-induced cortisol production, but not IL-1b. There was abundant expression of the IL-1RI in the patient’s tumor, but not in other tumors. It was concluded that the unique expression of IL-1RI in this tumor and induction of cortisol by IL-1b resulted in the pathological disease. The extracellular domain of the type I receptor has been used in several models of inflammatory and autoimmune disease. Administration of murine IL-1sRI to mice has increased the survival of heterotopic heart allografts and reduced the hyperplasic lymph node response to allogeneic cells (Fanslow et al., 1990). In a rat model of antigen-induced arthritis, local instillation of the murine soluble IL-1RI reduced joint swelling and tissue destruction (Dower et al., 1994). When a dose of soluble receptor (1 mg)

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was instilled into the contralateral, unaffected joint, a reduction in the degree of tissue damage was observed in the affected joint. These data suggest that the amount of soluble IL-1RI given in the normal, contralateral joint was acting systemically. In a model of experimental autoimmune encephalitis, the soluble IL-1RI reduced the severity of this disease (Jacobs et al., 1991). Administration of soluble IL-1RI to animals has also been reported to reduce the physiologic response to LPS, acute lung injury, and delayed-type hypersensitivity (reviewed in Dower et al., 1994). However, there are also data suggesting that exogenous administration of soluble IL-1RI may be harmful. In mice, an i.v. injection of soluble IL-RI alone induced a rapid release of circulating IL-1a, but not of TNFa or IL-1b (Netea et al., 1999). The soluble receptor did not interfere with the IL-1 assay. Treatment of mice with soluble IL-1RI improved the survival during a lethal infection with Candida albicans. In the accelerated model of autoimmune diabetes induced by cyclophosphamide in the non-obese diabetic (NOD) mouse, repeated injections with soluble IL-1R protected NOD mice from insulin-dependent diabetes mellitus in a dose-dependent fashion; the incidence of the diabetes was 53.3% in the mice treated with 0.2 mg kg1 and only 6.7% in mice treated with 2 mg kg1. However, none of the doses of the soluble IL-1RI reduced the extent of insulitis in NOD mice. Splenic lymphoid cells from NOD mice treated with 2 mg/kg soluble IL-1R for 5 consecutive days showed a normal distribution of mononuclear cell subsets and maintained their capacity to secrete IFNc and IL-2 (Nicoletti et al., 1994).

Effect of soluble IL-1RI in humans Recombinant human soluble IL-1RI has been administered intravenously to healthy humans in a phase I trial without side-effects or changes in physiologic, hematologic or endocrinologic parameters. Thus, similar to infusions of IL-1Ra, soluble IL-1RI appears safe and reinforces the conclusion that IL-1 does not have a role in homeostasis in humans. Volunteers have been injected with LPS and pretreated with soluble IL-1RI. The basis for these studies is that in animal models, blocking IL-1 with IL-1Ra has reduced the severity of the response (reviewed in Dinarello, 1996). Pretreatment of subjects with 10 mg

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kg1 of IL-1Ra prior to intravenous endotoxin resulted in a statistically significant, but modest decrease (40%) in circulating neutrophils (Granowitz et al., 1993). Volunteers were also pretreated with soluble IL-1R type I or placebo and then challenged with endotoxin. There were no effects on fever or systemic symptoms noted. Although there was a decrease in the level of circulating IL-1b compared with placebotreated volunteers, there was also a decrease in the level of circulating IL-1Ra (P  0.001) due to complexing of the soluble receptor to endogenous IL-1Ra (Preas et al., 1996). This was dose-dependent and resulted in a 43-fold decrease in endotoxin-induced IL-1Ra. High doses of soluble IL-1R type I were also associated with higher levels of circulating TNFa and IL-8, as well as cell-associated IL-1b (Preas et al., 1996). These results support the concept that soluble IL-1R type I binds endogenous IL-1Ra and reduces the biological effectiveness of this natural IL-1 receptor antagonist in inhibiting IL-1. As discussed below, patients with rheumatoid arthritis treated with soluble IL-1R type I do not exhibit improved clinical outcome and the mechanism is likely to the binding of endogenous IL-1Ra with a reduction in its biological role. Soluble IL-1R type was administered subcutaneously to 23 patients with active rheumatoid arthritis in a randomized, double-blind, two-center study. Patients received subcutaneous doses of the receptor at 25, 250, 500 or 1000 lg m2 day1 or placebo for 28 consecutive days. Although four of eight patients receiving 1000 lg m2 day1 showed improvement in at least one measure of disease activity, only one of these four patients exhibited clinical improvement (Drevlow et al., 1996). Similar to the placebo-treated patients, lower doses of the receptor did not result in any improvement by acceptable criteria. Despite this lack of clinical or objective improvement in disease activity, cell surface monocyte IL-1 expression in all patients receiving the soluble IL-1R type I was significantly reduced. Other parameters of altered immune function in common in patients with rheumatoid arthritis also showed reduction. One possible explanation for the lack of clinical response despite efficacy in suppressing immune responses could be the inhibition of endogenous IL-1Ra. This was observed in volunteers receiving soluble IL-1R type I before challenge by endotoxin (Preas et al., 1996).

A phase I trial of soluble IL-1RI was conducted in patients with relapsed and refractory acute myeloid leukemia. Soluble IL-1RI was well tolerated. Serum levels of IL-1b, IL-6 and TNFa did not change. Circulating levels of soluble IL-1RI were elevated 360- and 25-fold after i.v. and s.c. administration, respectively. There were no complete, partial or minor responses to treatment (Bernstein et al., 1999). The goal of any anti-IL-1 strategy is to prevent IL-1 binding to surface receptors. Using soluble receptors to block IL-1 activity in disease is similar to using neutralizing antibodies against IL-1 and distinct from using receptor blockade with IL-1Ra. Since the molar concentrations of circulating IL-1 in disease are relatively low, pharmacologic administration of soluble IL-1RI to reach a 100-fold molar excess of the soluble receptor over that of IL-1 is feasible. The human trial of soluble IL-1RI in delayed hypersensitivity reactions supports the notion that low doses (100 lg patient1) can have anti-inflammatory effects. The fusion of two chains of extracellular domains of the type IL-1RI to the Fc portion of immunoglobulin enhances the binding IL-1 over that of monomeric soluble IL-RI (Pitti et al., 1994) and may have a greater plasma halflife compared with that of the monomeric form. However, as shown in the study of soluble IL-1RI in rheumatoid arthritis, binding of the endogenous IL-1Ra worsened disease. In contrast to neutralizing IL-1 itself, the goal of receptor blockade requires the condition of blocking all unoccupied IL-1 surface receptors since triggering only a few evokes a response. Receptor blockade is a formidable task and the large amounts of IL-1Ra required to reduce disease activity contributes to this conclusion. The potential disadvantage for using soluble IL-1 receptor type I therapy is the possibility that these receptors will either prolong the clearance of IL-1 or bind the natural IL-1Ra. The soluble form of IL-1RI and IL-1RII circulate in healthy humans at molar concentrations which are 10–50-fold greater than those of IL-1b measured in septic patients and 100-fold greater than the concentration of IL-1b following intravenous administration (Crown et al., 1991). Why do humans have a systemic response to an infusion of IL-1a (Smith et al., 1993) or IL-1b? One concludes that binding of IL-1 to the soluble forms of IL-1R types I and II exhibits a slow ‘on’ rate compared with the cell IL-1RI.

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In addition, naturally occurring neutralizing antibodies to IL-1a are present in many subjects and likely reduce the activity of IL-1a. Despite the portfolio of soluble receptors and naturally occurring antibodies, IL-1 produced during disease does, in fact, trigger the type I receptor since in animals and humans, blocking receptors or neutralizing IL-1 ameliorates disease. These findings underscore the high functional level of only a few IL-1 type I receptors. They also imply that the post-receptor triggering events are greatly amplified. It seems reasonable to conclude that treating disease based on blocking IL-1R needs to take into account the efficiency of so few type I receptors initiating a biological event.

ACKNOWLEDGMENTS This work was supported by NIH grant AI-15614.

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28 Interleukin-1 receptor antagonist [IL-1F3] William P. Arend1 and Christopher H. Evans2 1

University of Colorado Health Sciences Center, Denver, CO, USA 2

Harvard Medical School, Boston, MA, USA

It is better to be hated for what you are than to be loved for something you are not. André Gide

INTRODUCTION The pleiotropic and ubiquitous inflammatory activities of interleukin-1 (IL-1) suggested to many investigators that natural inhibitory mechanisms or molecules must exist. IL-1 inhibitors theoretically could work at the levels of synthesis, secretion, receptor, or post-receptor intracellular pathways in cells. Early studies had described IL-1 inhibitory bioactivities in human urine and in a variety of cell supernatants (reviewed in Arend, 1993). However, many of these activities turned out to be artifacts of bioassay systems or remained uncharacterized. At a symposium in Ann Arbor, MI, in June 1985, two different groups first reported bioactivities now known to be due to interleukin-1 receptor antagonist (IL-1Ra). Dayer and colleagues described that the dialyzed and concentrated urine of a patient with acute monocytic leukemia and fever inhibited the effects of IL-1 on induction of PGE2 and collagenase production by cultured human dermal fibroblasts. Arend and colThe Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

leagues reported that the supernatants of human monocytes cultured on adherent immune complexes exhibited an activity inhibitory towards IL-1 stimulation of proliferation of murine thymocytes. During the next 5 years both research groups further characterized these IL-1 inhibitory bioactivities. The semi-purified inhibitor from the urine of patients with monocytic leukemia also inhibited IL-1-induced PGE2 and collagenase production in human synovial cells, as well as proliferation of thymocytes. In addition, both serum and urine from febrile patients with systemic juvenile chronic arthritis contained an IL-1 inhibitor that was absent during afebrile periods. Further purification of the urine material indicated a size of 18–25 kDa with inhibitory activities towards both IL-1a and IL-1b induction of PGE2 production by fibroblasts. Most importantly, Dayer and colleagues first demonstrated that the urine-derived inhibitor blocked the binding of IL-1 to receptors on EL4-6.1 murine thymoma cells. The material in the supernatants of human monocytes stimulated by adherent

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immune complexes also was ~22 kDa in size and inhibited IL-1, but not IL-2, effects on both thymocytes and chondrocytes. Furthermore, similar to the IL-1 inhibitor in urine, the semi-purified monocytederived material also functioned as a specific IL-1 receptor antagonist. This period culminated with the description in 1990 of the purification, sequencing, cDNA cloning, and expression of the IL-1Ra molecule (reviewed in Arend, 1993). IL-1Ra is the first described naturally occurring specific receptor antagonist of any cytokine or hormonelike molecule. Thus, it occupies a unique position in biology. The pattern of production of IL-1Ra, immediately following that of IL-1 and as an acute phase protein by the liver, indicates that IL-1Ra may serve an important role in regulation of the potentially injurious effects of IL-1. Furthermore, the spontaneous development of vasculitis (Nicklin et al., 2000) or arthritis (Horai et al., 2000) in different inbred strains of mice rendered genetically deficient in IL-1Ra production suggests that maintenance of a balance between local levels of IL-1Ra and IL-1 is important in prevention of disease. Knowledge about the biochemistry and biology of the IL-1Ra family of molecules rapidly expanded during the 1990s (Arend, 1990, 1991, 1993; Arend et al., 1990, 1998; Dinarello, 1991, 1993; Dinarello and Thompson, 1991; Lennard et al., 1992; Bresnihan and Cunnane, 1998). At least four isoforms of IL-1Ra are now known: secreted IL-1Ra, or sIL-1Ra and types 1, 2 and 3 intracellular IL-1Ra, icIL-1Ra1, icIL-1Ra2 and icIL-1Ra3. Furthermore, during the 1990s the mechanism of action of IL-1Ra was established, the anti-inflammatory role of endogenous IL-1Ra was determined, and the therapeutic use of recombinant IL-1Ra in human diseases was explored. Sections in this chapter will review characteristics of the IL-1Ra gene and protein isoforms, biological role in normal physiology and in disease, potential therapeutic uses of exogenous administration, and areas of current inquiry and research.

STRUCTURE OF THE IL-1Ra GENE AND REGULATION OF TRANSCRIPTION The structure of the 16.5 kb IL-1Ra gene (IL1RN) is depicted in Figure 28.1 (Lennard et al., 1992; Arend,

1993). Four exons encode sIL-1Ra, with 6.4 kb comprising the region for sIL-1Ra DNA including a short 3 UTR. An additional exon encoding icIL-1Ra1 is located 9.6 kb upstream, with the intervening region constituting a large first intron for this structural variant. Human IL1RN contains three Alu repeats, two located in the intron 3 from the first exon for icIL-1Ra1 and the third located in intron 3 for sIL-1Ra. A potentially important allelic polymorphism is present in the sIL-1Ra second intron caused by variable numbers of an 86-bp tandem repeat (Tarlow et al., 1997). Allele 2 of this polymorphism is associated with a variety of human diseases, largely of epithelial cell origin, as discussed below. This intronic allele appears to influence production of different isoforms of IL-1Ra in a cell-specific manner. It is not known whether the allele affects transcription, translation, or both. IL1RN was mapped to the long arm of human chromosome 2 at band 2q14.2 (reviewed in Arend, 1993). The IL-1α and IL-1b loci are also located in this region of chromosome 2 and the genes for IL-1Ra, IL-1a and IL-1b all contain similar exon–intron structures. An analysis of sequence comparisons and mutation rates for these three genes suggest an origin by gene duplication from a primordial precursor. A physical map of the region on chromosome 2 described the genes for the three IL-1 family members to be present on a 430-kb restriction fragment flanked by two CpG islands (reviewed in Arend et al., 1998). The three genes were mapped relative to one terminal CpG island with the following intervals: IL1A between 0 and 35 kb, IL1B between 70 and 110 kb, and IL1RN between 330 and 430 kb. It is of interest that the genes for human IL-1 receptors, both types I and II, are also located on the long arm of chromosome 2, although not close to the genes for the three IL-1 ligands. The separate 5 regulatory regions for both sIL-1Ra and icIL-1Ra1 have been mapped, and some of the cis-acting DNA regions involved in regulation of transcription have been characterized (reviewed in Arend et al., 1998). To study the sIL-1Ra promoter, 1680 bp of 5 flanking DNA was isolated, sequenced and cloned into a luciferase expression vector for use in gene transfer experiments. The sIL-1Ra promoter possessed a TATAA box at 26, with consensus sequences for possible NF-jB-, NFIL-1bA-, AP-1- and CRE-

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Downstream ATG No leader peptide Intracellular

No leader peptide Intracellular

FIGURE 28.1 IL-1Ra gene structure, three mRNAs and four proteins. The extended IL-1Ra gene contains four originally described exons encoding sIL-1Ra. Two additional upstream exons were subsequently identified, producing mRNAs for icIL-1Ra1 and icIL-1Ra2 by alternative transcriptional splice mechanisms. The four IL-1Ra protein isoforms described to date result from translation of these three mRNAs, with icIL-1Ra3 formed by alternative translational initiation primarily from the sIL-1Ra mRNA. The three ic (intracellular) isoforms of IL-1Ra contain no leader peptides, are formed in the cytoplasm and generally remain within cells. (Reproduced by permission from Gabay, C. (2000). IL-1 inhibitors. Novel agents in the treatment of rheumatoid arthritis Exp. Opin. Invest. Drugs 9, 113–127). binding sites located further upstream. Cloning of this sIL-1Ra promoter construct into a variety of cell lines indicated a pattern of activity identical to that of the endogenous IL-1Ra gene, i.e. primarily in monocytes and macrophages. A series of 5-truncated mutants with a common 3 end at 27 were generated to further characterize active regions in the sIL-1Ra promoter. Removal of sequences between 294 and 148 led to a 90% decrease in both basal and LPSinduced promoter activity. Further deletion to 85 led to an almost complete loss of promoter activity. These early results indicated that sites in the proximal region of the sIL-1ra promoter potently regulated both basal and induced activity. Further studies indicated the presence of one inhibitory and three positive-acting LPS response elements within the proximal 294 bp of the sIL-1Ra promoter. An element between 294 and 250 masked the LPS response, representing an inhibitory region. Three positive LPSresponse elements were located between 250 and 200, 200 and 148, and 148 and 31, with co-

operativity demonstrated between these sites for maximal promoter activity. The most proximal LPSresponse element contained a NF-jB binding site between 93 and 84. Subsequent studies indicated the presence of a PU.1 binding site between 90 and 80 of the sIL-1Ra promoter, overlapping the NF-jB site (Smith et al., 1998). Moreover, this region demonstrated interactions with GA-binding protein, as well as with NF-jB and PU.1. Mutation studies indicated that cooperativity between all three transcription factors acting on this proximal region was involved in sIL-1Ra promoter activity. A second PU.1 binding site was centered at 230 with mutation of both PU.1 sites leading to an almost complete loss of promoter activity. Thus, the two PU.1 sites in the proximal sIL-1Ra promoter represent the major response elements for LPS-induced sIL-1Ra gene expression. To study transcriptional regulation of the icIL-1Ra1 gene, 4.5 kb of the 5 flanking region was isolated, sequenced and cloned into a luciferase expression vector. The promoter for icIL-1Ra1 lacked a traditional

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TATAA or CAAT motif so must use an alternative mechanism of transcriptional initiation. This promoter construct demonstrated a pattern of activity identical to that of the endogenous icIL-1Ra1 gene, with constitutive expression in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937 or the lymphocyte cell lines Raji and Jurkat. However, inducible expression was demonstrated in response to LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. Studies with deletional mutants indicated that constitutive expression of the icIL-1Ra1 promoter in epithelial cells was under the control of three positively acting regions located between bases 4525 to 1438, 288 to 156, and 156 to 49. In contrast, basal expression of the icIL-1Ra1 promoter in RAW 264.7 cells was regulated by an inhibitory element between 4525 to 1438 and a strong positive element between 156 and 49. Lastly, LPS induction of the icIL-1Ra1 promoter in RAW 264.7 cells was regulated by strong positive DNA regions between bases 1438 to 909 and 156 to 49. Thus, the proximal region of the icIL-1Ra1 promoter, between bases 156 to 49, contained positive cis-acting elements necessary for expression in both epithelial and macrophage cell lines. However, the ability of this proximal region in the icIL-1Ra1 promoter to regulate transcription was strongly influenced in both positive and negative manners by other upstream elements in a cell type-specific pattern. Recent studies have examined transcriptional regulation of the icIL-1Ra1 gene in IL-1a-stimulated primary mouse keratinocytes (La and Fischer, 2001). Regulatory elements for both C/EBP and NF-jB between –598 and –288 in the icIL-1Ra1 promoter were found to be involved in mediating the response to IL-1a. Post-transcriptional regulation of IL-1Ra production may be influenced by the 3-untranslated (3-UTR) region of the IL-1Ra mRNA (Yamshchikov et al., 2002). The human 3-UTR led to a 5.7-fold and 3.9-fold, respectively, decrease in expression of a luciferase reporter gene in the murine macrophage cell line RAW264.7 or in the human macrophage cell line U937. This reporter gene construct was under the control of the CMV promoter with the IL-1Ra 3-UTR placed downstream of the luciferase gene. The IL-1Ra mRNA levels were not changed significantly, suggesting that the 3-UTR influenced translation.

IL-1Ra PROTEIN ISOFORMS The purification to homogeneity of IL-1Ra from the supernatants of monocytes cultured on adherent IgG indicated a protein of 17 kDa and 152 amino acids (Arend, 1993). This isoform of IL-1Ra is secreted as variably glycosylated species of 22–25 kDa and is now known as secretory IL-1Ra (sIL-1Ra). Another laboratory subsequently purified the same molecule from the supernatants of the human myelomonocytic leukemia cell line U937 after differentiation with phorbol myristate acetate (PMA) and stimulation with granulocyte–macrophage colony-stimulating factor (GM-CSF). Both laboratories showed that this molecule functioned as a specific inhibitor of receptor binding of IL-1 and exhibited an absence of agonist properties. sIL-1Ra is produced in large amounts by monocytes, macrophages and neutrophils, but can be synthesized by virtually any cell that produces IL-1 with the possible exception of epithelial and endothelial cells. Sequencing the primary structure of this IL-1 inhibitor led to cloning using degenerate oligonucleotide probes. The IL-1Ra cDNA was cloned from a kgt10 library prepared from monocytes cultured on adherent IgG or from U937 cells (Arend, 1993; Carter et al., 1990). The cDNA contained an open reading frame encoding a protein of 177 amino acids, a short 5 UTR of 14 nucleotides, and a long 3 UTR of 1133 nucleotides. The N-terminal 25 residues possessed an internal hydrophobic stretch representing a signal peptide. Recombinant IL-1Ra was obtained after cloning and expression in E. coli. The purified recombinant nonglycosylated protein showed identical structure as the purified native molecule. IL-1Ra exhibited 26% amino acid sequence homology to IL-1b and 19% homology to IL-1a, similar to the degree of homology between the two forms of IL-1. Subsequent structural studies on IL-1Ra from other species indicated that it is a highly conserved molecule, again similar to IL-1. A single Asn-linked glycosylation site is located at 333–339, and the native sIL-1Ra is variably glycosylated accounting for the size range of this secreted molecule. However, glycosylation is not thought to influence receptor binding of this molecule as both native purified sIL-1Ra and the recombinant molecule appeared to have identical biological properties in early studies.

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Intracellular isoforms of IL-1Ra have been described which possess variations in the N-terminal amino acid sequence (Table 28.1) (reviewed in Arend et al., 1998). The first intracellular isoform of IL-1Ra, now termed type 1 or icIL-1Ra1, was identified from a cDNA isolated from a human blood cell library. The cDNA was identical to that for sIL-1Ra except at the 5 end where 85 bp were replaced by a different sequence of 130 bp derived from an upstream exon. This segment was spliced into an internal splice acceptor site within the exon encoding the leader sequence (bp 87 and 88) of the sIL-1Ra cDNA. The resultant protein lacked the N-terminal 21 amino acids of the 25-residue signal sequence of sIL-1Ra, which was replaced by three different amino acids. icIL-1Ra1 is a non-glycosylated intracellular protein of 159 amino acids and 18 kDa which exhibits binding to IL-1 receptors with the same characteristics as sIL-1Ra (Malyak et al., 1998a). This intracellular isoform of IL-1Ra is produced primarily by keratinocytes and other epithelial cells, but is a delayed transcriptional product of monocytes and macrophages (Malyak et al., 1998b). Additional species of intracellular IL-1Ra have subsequently been described. The distance between the 5 UTR of sIL-1Ra and the alternative exon for

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icIL-1Ra1 is 9.6 kb. A cDNA identical to that for icIL-1Ra1 except for an additional in-frame 63-bp sequence located three codons downstream of the translation start site of icIL-1Ra1 was cloned from human polymorphonuclear cells. This additional sequence is inserted between the first and second exons of icIL-1Ra1 and is coded by an extra exon located 2 kb downstream from the first exon for icIL-1Ra1. This second isoform of intracellular of IL-1Ra was termed type 2 or icIL-1Ra2 with the predicted protein of 25 kDa and 180 amino acids possessing an extra sequence of 21 amino acids in the N-terminal region. However, icIL-1Ra2 has never been shown to exist as a naturally occurring protein in vivo and may not be translated to any degree in living cells. A third intracellular isoform of IL-1Ra, now termed type 3 or icIL-1Ra3, was originally described as a 143 amino acid and 16 kDa protein in a variety of cells (Malyak et al., 1998a). This protein is derived by alternative translational initiation from the mRNAs for both sIL-1Ra and icIL-1Ra1, but particularly from the former. Thus, icIL-1Ra3 lacks a signal peptide and is found only in the cytoplasm of cells. In direct binding studies using surface immobilized receptors, this low molecular weight isoform of IL-1Ra bound to IL-1 receptor type I (IL-1RI) four- to five-fold less avidly

TABLE 28.1 IL-1Ra isoforms Secretory IL-1Ra (sIL-1Ra) Gene contains four exons Synthesized as a protein of 177 amino acids with a signal peptide of 25 residues Processed to a 17 kDa molecule containing 152 amino acids Secreted as variably glycosylated species of 22–25 kDa 26% sequence homology to IL-1b and 19% homology to IL-1a Produced by monocytes, macrophages, neutrophils, and other cells Intracellular IL-1Ra (icIL-1Ra) Type 1 icIL-1Ra (icIL-1Ra1) An additional upstream exon is spliced into an acceptor site within the exon encoding the leader sequence for sIL-1Ra cDNA Sythesized in the cytoplasm as an 18 kDa molecule containing 159 amino acids Binds to IL-1 receptors equally as well as sIL-1Ra A major product of keratinocytes and other epithelial cells, endothelial cells, and fibroblasts Type 2 icIL-1Ra (icIL-1Ra2) cDNA found in human cells encoded by an additional upstream first exon Predicted protein of 25 kDa and 180 amino acids may not be translated in vivo Type 3 icIL-1Ra (icIL-1Ra3) Produced by alternative translation initiation or by an alternative transcriptional splice Synthesized in the cytoplasm as a non-glycosylated 16 kDa molecule of 143 amino acids Binds poorly to type I IL-1R, but binds well to type II IL-1R A major protein in hepatocytes and neutrophils with smaller amounts found in macrophages THE CYTOKINES AND CHEMOKINES

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than did sIL-1Ra or icIL-1Ra1. However, all three isoforms of IL-1Ra bound equally well to immobilized IL-1RII. IL-1RI is the functional form of the IL-1 receptor as IL-1RII exhibits no induction of intracellular responses after ligand binding. icIL-1Ra3 is found in monocytes, macrophages and epithelial cells, but is also present in large amounts in hepatocytes and neutrophils (Malyak et al., 1998b; Gabay et al., 1999). A recently described variant IL-1Ra cDNA, cloned from human articular cartilage and subsequently identified in keratinocytes, theoretically could give rise to an intracellular IL-1Ra species of 143 amino acids and 16 kDa by alternative transcriptional splicing and translation from an internal methionine (Weissbach et al., 1998). Thus, icIL-1Ra3 may arise by both alternative transcriptional and translational mechanisms. Additional structural variants of the IL-1 family have been described that have most similarity to IL-1Ra, although none of these proteins to date have been shown to have IL-1 receptor antagonist biologic activity. The results of initial studies on the effects of IL-1Ra in IL-1-stimulation of target cells indicated that a great excess of IL-1Ra was necessary to inhibit IL-1. Up to 100-fold or greater amounts of IL-1Ra over IL-1 were required to give 50% inhibition of IL-1augmented proliferation of thymocytes or IL-1induced production of PGE2 and collagenase by cultured human synovial cells (Arend, 1990; Arend et al., 1990). This requirement is because cells are exquisitely sensitive to very small amounts of IL-1, exhibiting full biological responses with occupancy of only a few IL-1 receptors per cell. Since cells have an excess of IL-1 receptors, and IL-1Ra binds with near the same affinity as IL-1, 100-fold or greater amounts of IL-1Ra need to be present to effectively inhibit the binding of only a few molecules of IL-1.

PRODUCTION AND TISSUE LOCALIZATION The characteristics of production of the major IL-1Ra isoforms, as well as cell and tissue localization, have been determined. sIL-1Ra is primarily a product of monocytes and tissue macrophages, but also can be secreted by neutrophils, hepatocytes, dendritic cells and other cells. icIL-1Ra1 is a major constitutive pro-

tein in keratinocytes and epithelial cells lining the entire gastrointestinal tract, but also can be produced by fibroblasts and endothelial cells. icIL-1Ra2 appears not to be produced in detectable levels by any cell in vivo. icIL-1Ra3 is created by alternative translational initiation primarily from the sIL-1Ra mRNA, and in much lower amounts from the icIL-1Ra1 mRNA. Thus, icIL-1Ra3 can theoretically be found in any cell secreting sIL-1Ra, but is present in particularly large levels in neutrophils and hepatocytes. The stimuli for sIL-1Ra production by monocytes and macrophages have been summarized in previous reviews (Dinarello, 1991; Arend, 1993; Arend et al., 1998). The major inducing factors are adherent IgG, GM-CSF and IL-1 itself. Early studies showed that IL-1 and IL-1Ra were both produced by the same cell. In fact, IL-4 stimulation of human monocytes led to reciprocal regulation of IL-1 and IL-1Ra production, inhibited IL-1 while enhancing IL-1Ra. High levels of icIL-1Ra1 are present constitutively in keratinocytes with enhanced production during cell differentiation. Both TNF-a and IL-1a also increase icIL-1Ra1 production in keratinocytes (Kutsch et al., 1993; La and Fischer, 2001). icIL-1Ra1 is produced constitutively in small amounts by fibroblasts, but is up-regulated by PMA, LPS, TNF-a and IL-1 (Krzesicki et al., 1993; Martel-Pelletier et al., 1993; La and Fischer, 2001). Studies on the tissue localization of IL-1Ra isoforms in the mouse indicated that sIL-1Ra mRNA was not present in any tissue in control mice, but was upregulated primarily in the lung, spleen and liver after LPS injection (Gabay et al., 1997a). In contrast, icIL-1Ra1 mRNA was present constitutively in the skin and epithelial cells lining the gastrointestinal tract. A unique characteristic of sIL-1Ra is production by the liver as an acute phase protein. Both HepG2 hepatoma cells and freshly isolated human hepatocytes produced sIL-1Ra mRNA and protein in response to stimulation with IL-1b and IL-6. Both NF-jB and C/EBP binding sites on the sIL-1Ra promoter mediated transcription of the sIL-1Ra gene. Furthermore, IL-4 enhanced the stimulatory effects of IL-1b on sIL-1Ra production by HepG2 cells and hepatocytes (Gabay et al., 1999). This IL-4 effect on transcription was mediated through a STAT6 binding element within the sIL-1Ra promoter. In vivo studies showed that after either LPS injection or induction of local inflammation with turpentine injection, hepatic

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production of sIL-1Ra was up-regulated and paralleled the rise in serum levels (Gabay et al., 2001a, 2001b). The total amount of sIL-1Ra present in the liver after LPS injection was six- and ten-fold higher than in the lung and spleen, respectively. By in situ hybridization, icIL-1Ra1 mRNA, but not sIL-1Ra mRNA, was present in hepatocytes, but not Kupfer cells, after LPS injection. These studies proved conclusively that sIL-1Ra was produced by the liver as an acute phase protein, explaining the high levels of this protein found in the circulation of patients with inflammatory, infectious, or neoplastic diseases (see below).

CRYSTAL STRUCTURE, RECEPTOR BINDING AND MECHANISM OF ACTION The results of detailed studies on the crystal structure of the three IL-1 ligands (IL-1a, IL-1b, and IL-1Ra) and the two IL-1 receptors has clarified how the ligands bind to the receptors and the mechanism, whereby IL-1Ra fails to stimulate cells (reviewed in Arend et al., 1998). Examination of the crystal structure of sIL-1Ra at 2.0-Å resolution revealed the presence of the same b-trefoil structure as IL-1a and IL-1b as well as a similar hydrophobic core. However, structural differences were observed between sIL-1Ra and the two IL-1 agonists, particularly at the open end of the b-barrel. Structural studies by NMR spectroscopy yielded similar results, suggesting that differences in side chains may contribute to the difference in biological properties between IL-1Ra and IL-1. Additional studies on the structure of sIL-1Ra by NMR spectroscopy indicated conservation of residues between sIL-1Ra and IL-1b thought to be important in receptor binding, but lack of conservation of residues critical for receptor activation. Analysis of crystals of sIL-1Ra bound to soluble type I IL-1 receptors revealed a 1:1 receptor–ligand complex, suggesting that sIL-1Ra bound a single receptor and did not induce receptor aggregation. The binding of sIL-1Ra to type I IL-1 receptors on murine EL4 thymoma cells indicated an affinity equal to that of IL-1a and IL-1b (Kd  150 pM). Surfacebound IL-1Ra failed to activate the protein kinase activity responsible for down-modulation of the

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EGF receptor on the murine 3T3 fibroblast cell line and did not undergo receptor-mediated internalization. Extensive site-directed mutagenesis experiments described candidate regions in IL-1b and IL-1Ra that might be involved in receptor binding. Conversion of IL-1Ra into a partial agonist was seen following mutation of Lys-145(K) into Asp(D), suggesting the possible importance of the region surrounding Asp-145 in IL-1b for triggering biological responses after receptor binding. Additional studies indicated that the insertion of the b-bulge of IL-1b at the corresponding region of IL-1Ra K145D resulted in a three to four-fold increase in agonist activity, with a further enhancement in activity following the coexpression of a second substitution of His-54 to Pro. Most importantly, these investigators also described that the bioactivity of the triple mutant IL-1Ra K145D/H45P/b-bulge was dependent upon an interaction with the IL-1 accessory protein (IL-1R AcP). Cloning and characterization of the IL-1R AcP demonstrated that this molecule was present in the plasma membrane of cells responsive to IL-1 and formed a complex with the single chain type I IL-1R and either IL-1a or IL-1b, but not IL-1Ra (Greenfeder et al., 1995). IL-1R AcP was shown to be indispensable in IL-1 induction of signal transduction pathways (Wesche et al., 1996; Korherr et al., 1997; Cullinan et al., 1998). An early event in IL-1 signaling is the recruitment of the Ser/Thr kinase IRAK to the intracellular domain of the type I IL-1 receptor; IRAK was recruited to the IL-1 receptor complex through its association with IL-1R AcP (Huang et al., 1997). Over-expression of an IL-1R AcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-jB activation. Thus, the mechanism whereby IL-1Ra inhibits cell responses to IL-1 is by competitively binding to type I IL-1 receptors and preventing the interaction of the IL-R AcP molecule with the single chain receptor. The crystal structure at 2.7Å resolution of the soluble extracellular portion of type I IL-1 receptor complexed to sIL-1Ra indicated that the receptor possessed three Ig-like domains. Residues of all three domains contacted the sIL-1Ra molecule, including five critical residues previously identified by sitedirected mutagenesis as important in receptor binding. However, a region in IL-1b thought to be

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important in biological function, the receptor trigger site, was not in direct association with the receptor in the IL-1Ra complex. The possibility exists that a small structural change in the IL-1Ra molecule conformationally separates the receptor trigger site from association with the type I IL-1 receptor in a manner that blocks interaction of the IL-1R AcP with the complex. Type II IL-1 receptors possess a short cytoplasmic domain and fail to trigger cells after ligand binding (Colotta et al., 1994; Sims et al., 1994). Thus, type II IL-1 receptors on the cell surface act as further inhibitors of IL-1 effects by competing for binding of the IL-1 agonists. The extracellular portions of both types of IL-1 receptors are enzymatically cleaved from the plasma membrane of activated cells and are present in the microenvironment of cells and in body fluids, particularly soluble type II IL-1 receptors. The results of binding studies demonstrated that IL-1b bound more avidly to soluble IL-1RII than IL-1a or IL-1Ra, primarily because of a slow dissociation rate (Arend et al., 1994; Burger et al., 1995). In contrast, IL-1Ra bound more avidly to soluble IL-1RI than either IL-1 agonist, again because of a very slow dissociation rate. Further experiments indicated that some IL-1Ra and IL-1 may bind soluble IL-1R in body fluids in vivo, obscuring measurement of these cytokines by ELISA. A further implication is that naturally occurring soluble IL-1RI may bind to endogenous or exogenously administered IL-1Ra, reducing its IL-1-inhibitory effects. Lastly, IL-1R AcP interacts with both IL-1RI and IL-1RII in the plasma membrane of cells (Lang et al., 1998; Malinowsky et al., 1998). Upon IL-1 binding, IL-1RII may recruit IL-1R AcP into a nonfunctional complex, removing the IL-1R AcP from possible interaction with IL-1RI. Thus, IL-1RII may function as a natural IL-1 inhibitor through three different mechanisms that all involve reduction in binding of IL-1a or IL-1b to membrane-bound IL-1RI: competing on the cell surface for ligand binding, competing for ligand binding as a soluble receptor in the cell microenvironment, and competing on the cell surface for interaction with IL-1R AcP.

EXPRESSION IN NORMAL ANIMALS AND HUMANS Tissue distribution Examination of tissues recovered from normal mice suggests the constitutive expression of icIL-1Ra1 mRNA and protein in the skin (Gabay et al., 1997a). This is consistent with data from analysis of human keratinocytes and epithelial cells, which also spontaneously synthesize icIL-1Ra1. Analysis of normal human skin by immunohistochemistry has also identified IL-1Ra in the sebaceous glands, eccrine sweat glands, dermal dendritic cells and upper dermal blood vessels. The intestine is an additional site of constitutive icIL-1Ra1 production in mice and rabbits (Cominelli et al., 1994). The secreted isoform of IL-1Ra, in contrast, does not appear to be synthesized spontaneously in mice. However, IL-1Ra has been detected in normal, human synovial fluid (Cameron et al., 1994), tears (Solomon et al., 2001), blood and cerebrospinal fluid (Tarkowski et al., 2001), using ELISA methods that do not distinguish between the different isoforms of IL-1Ra. Moreover, IL-1Ra has been identified by immunohistochemistry in certain neurons in the neocortex and hippocampus of the normal brain (Yasuhara et al., 1997). Low levels of icIL-1Ra1, but not sIL-1Ra, are synthesized ex vivo by orbital fibroblasts recovered from the eyes of normal donors (Muhlberg et al., 1997). In addition, the epithelial cells of the normal human cornea synthesize both sIL-1Ra and icIL-1Ra1, while the stromal cells produce only the intracellular variant (Kennedy et al., 1995). Human mast cells recovered from normal bronchoalveolar lavage fluid constitutively synthesize IL-1Ra (Hagaman et al., 2001). As described below, IL-1Ra is produced by the normal ovary in a cyclical manner around the time of ovulation, and during gestation and child birth. Human milk also contains high concentrations of IL-1Ra that may contribute to the improved development and immune function of breast-fed infants.

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IL-1Ra in blood IL-1Ra cannot be detected in the blood of normal mice (Gabay et al., 1997a), despite the fact that sIL-1Ra mRNA is constitutively present in whole blood RNA as a result of its expression in neutrophils. Plasma levels of IL-1Ra increase dramatically in mice within 4 h of LPS injection. The major sites of sIL-1Ra expression following the administration of LPS to mice are the lung, spleen and liver. The major blood cell contributing to circulating sIL-1Ra in response to LPS appears to be the neutrophil (Gabay et al., 1997a). Equivalent results have been obtained using rats. Unlike the case with mice, sIL-1Ra is detectable in human serum recovered from normal individuals, usually at concentrations of 1 ng/ml or less. Administration of LPS to healthy volunteers leads to a rapid increase in circulating IL-1Ra levels in approximately the same time-frame as seen with mice (Granowitz et al., 1991). TNF-a, IL-1 and IL-6 have also been injected intravenously into normal volunteers, with a rapid and marked increase in plasma IL-1Ra concentrations; a slower elevation occurs following injection of IFN-c. The rapid induction of IL-1Ra by LPS, IL-1 and IL-6 is explained by the demonstration (Gabay et al., 1997b) that IL-1Ra is an acute phase protein. In cell culture, both normal human hepatocytes and the hepatoma cell line HepG2 produce sIL-1Ra in response to IL-1 and IL-6. As with other acute phase proteins, synthesis of sIL-1Ra is increased by dexamethasone. Infection and the onset of inflammatory conditions lead to rapid induction of sIL-1Ra synthesis by the liver, resulting in serum concentrations in excess of 50 ng/ml in certain patients with sepsis (Rogy et al., 1994). Concentrations of sIL-1Ra are also strongly elevated in the peripheral blood of patients with conditions such as juvenile rheumatoid arthritis, polymyositis–dermatomyositis, systemic lupus erythematosis, rheumatoid arthritis (RA), active multiple sclerosis, peripheral artery disease, heart failure and Gaucher disease, as well as following trauma, burns, hemodialysis, stroke, vigorous exercise and acute mental stress. The effects of trauma, stress and exercise may be secondary to the influence of adrenaline, the infusion of which elevates plasma IL-1Ra concentrations. Plasma levels of IL-1Ra have been reported to increase markedly during pregnancy.

The role in biology of sIL-1Ra appears to be to regulate the effects of IL-1 in the cell microenvironment. IL-1Ra is synthesized and released from cells immediately following IL-1, and the ratio of IL-1Ra found locally to IL-1 influences the relative stinulatory potential of the IL-1 present. This ratio may be imbalanced either because of sustained stimulation of production of IL-1 or inadequate local production of sIL-1Ra. An imbalance in the IL-1Ra/IL-1 ratio may lead both to abnormalities in normal cyclical organ function, or to actual disease where the imbalance is prolonged. The role in biology of the intracellular isoforms of IL-1ra is less clear. These molecules may play unique roles inside cells that do not involve receptor binding. Precursor IL-1a possesses a nuclear localization sequence in the propiece, which mediates movement to and entry into the nucleus of cells (Wessendorf et al., 1993). Pre-IL-1a acts as a negative regulator of the growth of endothelial cells (McMahon et al., 1997) and as a transforming nuclear oncoprotein (Stevenson et al., 1997). However, it is not known if icIL-1Ra isoforms influence these intracellular functions of pre-IL-1a. The presence of icIL-1Ra1 in ovarian carcinoma cells was associated with a decrease in mRNA stability for the chemokines IL-8 and GRO (Watson et al., 1995). No more recent reports have investigated this observation further, and its relevance must remain unclear. In addition, scleroderma fibroblasts overexpress both pre-IL-1a and icIL-1Ra1, with the pre-IL-1a actually inducing expression of icIL-1Ra1 (Higgins et al., 1999). The relationship of this finding to the abnormal phenotype of scleroderma fibroblasts is not known. Under certain conditions icIL-1Ra1 may be secreted from keratinocytes and other epithelial cells and function as a competitive inhibitor of IL-1 receptor binding. Larger, more differentiated keratinocytes secrete icIL-1Ra1 in culture, suggesting that these cells in the upper layers of the skin may also carry out this function (Corradi et al., 1995). Human airway epithelial cells also release icIL-1Ra1 in response to stimulation with IL-4, IL-13 or IFN-c, all cytokines that also induce production of sIL-1Ra in monocytes and macrophages (Levine et al., 1997). Lastly,

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icIL-1Ra1 is released from respiratory epithelial cells in vivo and in vitro after infection with rhinovirus, and may help resolve the symptoms of rhinovirus infections of the upper respiratory tract (Yoon et al., 1999). In all of these studies, the release of icIL-1Ra1 by the epithelial cells was not due to cell death, as cytoplasmic enzymes were not found in the cell supernatants. Although these results indicate that one function of icIL-1Ra1 may be to block extracellular IL-1, other as yet poorly characterized intracellular effects of icIL-1Ra may exist.

Reproductive system The concept that ovulation resembles a cyclical, inflammatory process driven by IL-1 has become popular in recent years. Among the evidence for this hypothesis is the ability of IL-1 to induce ovulation and to synergize with luteinising hormone (LH) in this regard (Brannstrom et al., 1993), the ability of IL-1Ra to inhibit ovulation (Simon et al., 1994b), and the expression of IL-1 in the ovary around the time of ovulation (Hurwitz et al., 1991). This being so, IL-1Ra may serve as an endogenous modulator of ovulation. IL-1Ra is not expressed in the immature rat ovary, but is rapidly induced after treatment with chorionic gonadotrophin. Transcripts encoding sIL-1Ra and icIL-1Ra are localized to the mural, antral and cumulus granulosa cells, as well as the oocytes. It is likely that IL-1Ra synthesis is stimulated by prior induction of IL-1 in the ovary. In agreement with the studies of rat ovaries, analysis of cervical mucus in humans revealed that IL-1Ra was constitutively expressed during the entire menstrual cycle, but concentrations were higher in the ovulatory than in the follicular phase (Kanai et al., 1997). Immunohistochemistry identified epithelial cells of the endometrium as a source of IL-1Ra (Fukuda et al., 1995). Macrophages within the endometrial stroma also stained positively for IL-1Ra. RT-PCR analysis confirmed that stromal and glandular cells of the human endometrium expressed icIL-1Ra (Simon et al., 1995). IL-1 is also intimately involved with embryonic implantation. At the two-cell stage, murine embryos do not express IL-1Ra transcripts. However, starting at the eight-cell stage, embyos start to express IL-1Ra at

increasing frequencies, such that 74% of blastocytes give a positive RT-PCR signal for IL-1Ra (Kruessel et al., 1997). This timing is likely to be of great significance, as IL-1Ra inhibits embryonic implantation (Simon et al., 1994a), possibly by inhibiting the expression of adhesion molecules on the endometrial epithelium. IL-1Ra is further likely to play a key role in gestation and delivery. Maternal serum levels of IL-1Ra increase with increasing gestational age and during labor activity (Ammala et al., 1997). IL-1Ra is synthesized by the placenta at the time of birth; cells within the amnion, chorion and decidua are sources of IL-1Ra. Amniotic fluid also contains high concentrations of IL-1Ra, but much of this is thought to originate from the fetal urine. According to one study, urinary IL-1Ra concentrations are higher in female newborns than male newborns, even though newborn serum concentrations of IL-1Ra are not dependent upon gender (Bry et al., 1994).

Host defense and inflammation IL-1 is a central orchestrator of the body’s response to infection, being involved in both innate and acquired immunity at various levels (see Chapter 27). As well as activating macrophages, IL-1 serves as a costimulatory molecule for lymphocyte proliferation, is involved in antigen presentation, and increases T celldependent antibody production. By modulating the activities of IL-1, the IL-1Ra molecule plays a key role in regulating these processes. Its importance in the normal homeostasis of immune and inflammatory reactions is indicated by the phenotypes of mice with disrupted IL-1Ra genes. Such animals spontaneously develop inflammatory joint diseases (Horai et al., 2000), arterial inflammation (Nicklin et al., 2000), and are more sensitive to the lethal effects of endotoxin.

Central nervous system There is growing evidence that IL-1 is involved in normal sleep regulation, and that IL-1Ra is a physiological regulator of this process. Administration of IL-1Ra into the lateral cerebral ventricle of normal rabbits transiently reduced sleep, induced non-rapid eye movements, and blocked fever (Opp et al., 1992).

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IL - 1 R a IN DISEASE

IL-1Ra IN DISEASE Infection IL-1 is a key component of host defense responses (see Chapter 27). There are numerous examples confirming the marked increase in circulating IL-1Ra levels that occurs during viral and bacterial infections. Intravenous administration of endotoxin or TNF into normal human volunteers provokes a rapid rise in serum IL-1Ra concentrations, peaking 3–6 h after injection (Granowitz et al., 1991). As noted above, IL-1Ra is an acute phase protein synthesized rapidly in the liver in response to infection and endotoxin (Gabay et al., 1997b). Concentrations of IL-1Ra may also be elevated by local synthesis within various organs during infection. Much of the IL-1Ra produced under these conditions is likely to be by infiltrating leukocytes, particularly macrophages, or by the cells that are normally resident within the tissue. Plasma levels of IL-1Ra can be particularly high during sepsis. The protective role of IL-1Ra has been well established in experimental animals where disruption of the IL-1Ra gene, or administration of neutralizing antibodies to IL-1Ra, worsens disease and increases mortality. Conversely, administration of recombinant IL-1Ra has the opposite effect (Wakabayashi et al., 1991) and has led to clinical trials, described below, of its use in human sepsis.

Arthritis The role of IL-1 in both the inflammatory and erosive components of RA is well established (reviewed in van den Berg, 2001). IL-1 synthesized within the synovium by both resident synoviocytes and infiltrating leukocytes contributes to the recruitment of inflammatory cells, the formation of pannus, and the invasion of the adjacent bone and cartilage. Moreover, IL-1 produced by the articular chondrocytes appears to be the major stimulus for endogenous breakdown of the cartilagenous matrix (‘chondrocytic chondrolysis’). It also inhibits synthesis of the catilagenous matrix, and thus serves as a particularly powerful agent of cartilage damage. IL-1Ra is present in the synovial fluid of normal joints, and its concentration rises considerably in rheumatoid arthritis and other inflammatory joint conditions. Macrophages and neutrophils are proba-

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bly the major sources of the IL-1Ra found in the synovial fluid; the amounts produced by chondrocytes are quite low. Although synovial fibroblasts synthesize considerable quantities of IL-1Ra, it is predominantly the intracellular isoform(s) that are produced (Firestein et al., 1994). The fact that RA persists in many patients despite the intraarticular presence of considerable quantities of IL-1Ra probably reflects the high molar ratio of IL-1Ra:IL-1 that is needed to suppress the biological activities of the latter. This problem is highlighted by a study of Firestein et al., who measured the ex vivo synthesis of IL-1 and IL-1Ra by explants of articular tissues recovered from the joints of patients with RA (Firestein et al., 1994). IL-1Ra:IL-1 ratios of 1.2–3.6 were noted, values far below those needed to provide strong protection from the intraarticular activities of IL-1. An improvement in the ratio of IL-1Ra:IL-1 synthesized by peripheral blood monocytes from patients whose rheumatoid arthritis responded to treatment by methotrexate and gold drugs, has been noted (Seitz et al., 1995). The importance of IL-1Ra has also been demonstrated in a study of acute knee arthritis in patients with Lyme disease, where those who recovered more rapidly had higher synovial fluid ratios of IL-1Ra:IL-1 (Miller et al., 1993). IL-1 may be particularly important in osteoarthritis (OA), especially in view of its unique potency as a mediator of cartilage breakdown, the major pathology in this condition. There are no convincing studies in which IL-1Ra:IL-1 ratios have been measured in OA, but administration of recombinant IL-1Ra (Caron et al., 1996), or delivery of a cDNA encoding IL-1Ra (Pelletier et al., 1997; Frisbie et al., 2002), reduces disease activity in animal models. In rotator cuff diseases of the shoulder, there is expression of icIL-1Ra1 by the synovial lining cells and of sIL-1Ra by cells of the sublining; expression of IL-1Ra correlates with shoulder pain (Gotoh et al., 2001). IL-1Ra is also elevated in patients with temporomandibular joint disease. Blood levels of IL-1Ra are elevated in RA (Chikanza et al., 1995), suggesting an involvement in modulating the extraarticular and systemic components of the disease. High serum levels of IL-1Ra are also associated with systemic lupus erythematosus (Suzuki et al., 1995), where IL-1Ra concentrations correlate with disease severity. Response to immunoglobulin therapy in patients with the multisystem autoimmune

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disease cicatricial pemphigoid is also associated with increased levels of circulating IL-1Ra (Kumari et al., 2001). Elevated concentrations of circulating IL-1Ra have been reported for patients with juvenile rheumatoid arthritis (Prieur et al., 1987) and polymyositis (Gabay et al., 1994). Interestingly, synovial fluid concentrations of IL-1Ra have been reported to fall following rupture of the anterior cruciate ligament, and to remain chronically reduced in symptomatic patients (Cameron et al., 1994). Whether this is related to the secondary development of OA in such patients is a matter for speculation.

Nervous system There is evidence to suggest that IL-1 is involved in hypoxia-reperfusion injury and the sequelae of blunt trauma to the brain (Sanderson et al., 1999). IL-1Ra is expressed constitutively in neocortex and hippocampus, and is induced by IL-1 and ischemia (Loddick et al., 1997) in additional areas of the brain, including the cerebellum. Administration of IL-1Ra cDNA reduces the severity of the response to blunt trauma in the rat brain (DeKosky et al., 1996). There is growing appreciation of the role of inflammatory processes in neurodegenerative conditions. Of interest in this regard is the recent finding that the cerebrospinal fluids of patients with Alzheimer’s disease contain lower concentrations of IL-1Ra than those of unaffected control individuals (Tarkowski et al., 2001). IL-1b was not detectable in fluids recovered from patients or unaffected controls. Nevertheless, in a separate study of the immunohistochemical localization of IL-1Ra in brain tissue, Alzheimer’s disease was associated with increased numbers of positively staining neurons, as well as increased levels of staining (Yasuhara et al., 1997). IL-1Ra was additionally expressed in globular deposits in senile plaques and some extracellular neurofibrillary tangles. Increased expression of IL-1Ra was also noted in Pick’s disease, another neurodegenerative condition (Yasuhara et al., 1997). IL-1Ra has been implicated in Guillain-Barré syndrome, an autoimmune disease affecting the peripheral nervous system. During the development of a rodent model of this condition, the Schwann cells surrounding the sciatic nerves start to express both IL-1a and IL-1b during the pre-clinical stage of the

disease (Skundric et al., 2001). IL-1Ra is not detectable at this time, but its expression occurs as the disease becomes clinically manifest. Of possible significance is the observation that IL-1Ra immunoreactivity co-localizes with myelin-associated glycoprotein, a marker for paranodal regions essential for proper impulse transmission (Skundric et al., 2001). Multiple sclerosis, an autoimmune condition affecting the central nervous system, is treated by the injection of IFN-b .Treatment with weekly, intramuscular injections of 30 mg IFN-b increased serum levels of IL-1Ra, suggesting a role for IL-1Ra in the clinical improvement noted in these patients (Nicoletti et al., 1996). Experiments in various animal models suggest that IL-1 is an important contributor to neuropathic pain. This leads to the suggestion that IL-1Ra may serve an analgesic function under the appropriate conditions. Using a rodent L5 spinal nerve transection model, it was shown that, although administration of IL-1Ra alone failed to reduce pain, it improved the ability of soluble TNF receptors to do so. This was associated with reduced expression of IL-6, but not IL-1, in the spinal cord (Sweitzer et al., 2001). Patients with fibromyalgia, a condition associated with chronic pain, have higher levels of circulating IL-1Ra. Expression of IL-1Ra correlates with the degree of shoulder pain experienced by individuals with rotator cuff disease (Gotoh et al., 2001). IL-1Ra may also be involved in other aspects of neurological function. For instance, associations with autism and attention deficit hyperactivity have been suggested, indicating that the IL-1/IL-1Ra axis may play subtle roles in pyschobiological development and function.

Skin Expression of IL-1Ra appears to be disturbed in psoriasis. The expression of IL-1Ra increases in the lesions of psoriatic skin (Debets et al., 1997). Of interest is the finding that although levels of IL-1Ra protein appeared to increase, as judged by immunohistochemistry, levels of IL-1Ra message, as judged by in situ hybridization, did not (Debets et al., 1997). Serum levels of IL-1Ra are higher in patients with psoriatic arthritis. The expression of IL-1Ra during keratinocyte cell division and differentiation has been studied with normal and psoriatic epidermis (Hammerberg et al.,

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1998). IL-1Ra expression increases as keratinocytes differentiate from basal stem cells into transient amplifying cells. Production of IL-1Ra by the latter cells normally increases throughout the cell cycle, but in cells derived from psoriatic skin, IL-1Ra production falls slightly (Hammerberg et al., 1998). Dermal fibroblasts recovered from patients with systemic sclerosis express higher levels of icIL-1Ra1 and intracellular pre-IL-1a than controls (Higgins et al., 1999). These events may be coupled, as transduction of normal skin fibroblasts with a cDNA encoding pre-IL-1a increases expression of icIL-1Ra1 (Higgins et al., 1999). Serum levels of IL-1Ra increase during flares of contact allergy, and other conditions involving dermatitis, such as exposure to sunlight, and atopic dermatitis (Pastore et al., 1998). The expression of IL-1Ra increases in murine hair follicular cells as they cycle from early anagen to telogen (Tokura et al., 1997). At the latter stage, contact photosensitivity is much lower, possibly due to the elevated levels of IL-1Ra that are present. In agreement with this, administration of IL-1Ra inhibits contact hypersensitivity in mice (Kondo et al., 1995).

Lung diseases In asthma, levels of IL-1Ra increase both in the serum (Yoshida et al., 1996) and locally in the bronchial epithelium (Sousa et al., 1996). Moreover, serum concentrations of IL-1Ra are higher in patients during asthma attacks than under stable conditions (Yoshida et al., 1996). In vitro studies suggest that mast cells within the lung increase synthesis of IL-1Ra when stimulated with IgE (Hagaman et al., 2001), suggesting one way in which asthmatic attacks may result in increased IL-1Ra levels. Corticosteroids induce the synthesis of icIL-1Ra by human bronchial epithelium (Levine et al., 1996), and this may contribute to their efficacy in treating asthma. Alterations in the pulmonary production of IL-1Ra have also been associated with a number of inflammatory and fibrotic diseases of the lung, including tuberculosis, smoking, malignancy, interstitial lung disease, pneumonia, acute respiratory distress syndrome, cystic fibrosis, high altitude edema, chest trauma, panbronchiolitis, and sarcoidosis. In sarcoidosis, elevated IL-1Ra production is localized to

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the sarcoid granulomas. In patients with idiopathic pulmonary fibrosis, IL-1Ra production has been localized by immunohistochemistry and in situ hybridization to hyperplastic type II pneumocytes, macrophages and local stromal cells (Smith et al., 1995). Human bronchogenic carcinoma cells also produce elevated quantities of IL-1Ra (Smith et al., 1993). Intraperitoneal administration of IL-1Ra protects mice from the pulmonary fibrotic response to bleomycin or silica (Piguet et al., 1993). Moreover, low serum concentrations of IL-1Ra and IL-10 are associated with a poor prognosis in patients with acute respiratory distress syndrome (Parsons et al., 1997).

Cardiovascular diseases There is currently much interest in the relationship between inflammation and atherosclerosis. Inflammatory events within atherosclerotic plaques are considered to be major determinants of disease progression and clinical outcome. Baseline plasma levels of IL-1Ra are increased in patients with atherosclerosis (Fiotti et al., 1999). According to Fiotti et al. (1999), these levels correlate with the occurrence and stage of the disease so strongly as to be predictive. Expression of icIL-1Ra1 is elevated in the endothelial lining of human, atherosclerotic cardiomyopathic arteries. When patients were subjected to a treadmill test, IL-1Ra levels increased in both control subjects and those with atherosclerosis (Fiotti et al., 1999). Administration of IL-1Ra is able to inhibit the formation of fatty streaks on the intimal surfaces of blood vessels in apolipoprotein E-deficient mice (Elhage et al., 1998). Moreover, mice lacking a functional IL-1Ra gene spontaneously develop arterial inflammation (Nicklin et al., 2000). Circulating levels of IL-1Ra also increase after severe, acute myocardial infarction and rise to higher levels than those found in patients with uncomplicated acute myocardial infarctions (Shibata et al., 1997). Moreover, plasma concentrations of IL-1Ra correlate closely with the severity of hemodynamic changes, and are significantly higher in non-survivors than survivors (Shibata et al., 1997). Serum IL-1Ra levels are also elevated in patients with severe chronic congestive heart failure and correlate with their symptom-limited oxygen consumption. The possible correlation between high plasma levels of IL-1Ra and

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a poor prognosis for heart conditions of this nature is supported by a study of patients hospitalized for angina. Those patients with an uneventful course had lower levels of IL-1Ra at entry, and these levels further declined during the hospital stay. Those patients with in-hospital complications had higher levels of IL-1Ra at entry, and these levels increased during the hospital stay (Biasucci et al., 1999).

Kidney diseases Renal clearance represents the major excretion pathway of IL-1Ra, and elevated plasma levels occur in patients with renal failure. Serum levels of IL-1Ra increase in patients with pyelonephritis, chronic renal failure, end-stage renal disease, and other conditions that severely impair kidney function. Conversely, urinary levels are lower than controls as a result of impaired IL-1Ra renal clearance. IL-1Ra is synthesized by the glomerular cells of the kidney during experimental glomerulonephritis in rats (Karkar et al., 1995), indicative of a local contribution to the increase in circulating IL-1Ra that occurs under these conditions. Low circulating levels of IL-1Ra, in contrast, are correlated with kidney involvement in patients with lupus (Sturfelt et al., 1997). Plasma concentrations of IL-1Ra increase during hemodialysis, and it has been suggested that the level of IL-1Ra synthesis by peripheral blood mononuclear cells of patients receiving dialysis may have value as a predictor of morbidity (Balakrishnan et al., 2000).

Reproductive system As noted earlier in this chapter, IL-1 and IL-1Ra play important roles in ovulation, implantation, gestation and childbirth. Disruption of their interplay is associated with various disorders of reproduction. Plasma levels of IL-1Ra are elevated in women with preeclampsia (Greer et al., 1994). These levels do not correlate with disease severity, but have 58% positive predictivity, 100% negative predictivity, 50% specificity and 100% sensitivity for pre-eclampsia during gestational weeks 20–25 (Kimya et al., 1997). IL-1Ra expression in the placenta is increased during chorioamnionitis, preterm labor, and intrauterine infection (Romero et al., 1994). However, IL-1Ra levels in cord blood are not affected by labor pain or fetal

distress (Hata et al., 1996). Reduced concentrations of IL-1Ra in the plasma of pregnant women have been associated with maternal depression and higher rates of maternal complications after childbirth (Schmeelk et al., 1999).

Osteoporosis IL-1, along with TNF and IL-6, is a powerful inducer of osteoclastic activity. There is good evidence that these mediators are responsible for the loss of bone that occurs following human menopause and ovariectomy in both humans and experimental animals (Pacifici, 1996). The importance of IL-1 to the process has been demonstrated by administration of IL-1Ra, as a recombinant protein or by gene transfer, to ovariectomized rats and mice (Kimble et al., 1994b; Kitazawa et al., 1994; Baltzer et al., 2001). Bone loss in these animals is strongly inhibited. Mice lacking IL-1b or type I IL-1 receptors are completely protected from the loss of bone that accompanies ovariectomy (Lorenzo et al., 1998). Clinical studies are largely, but not unanimously, in support of the relevance of these animal studies to osteoporosis in humans. IL-1Ra plasma levels tend to be lower in osteoporotic women compared with age-matched non-osteoporotic controls, although the differences are quite small and not apparent in all studies. In a recent analysis, cytokine mRNA levels were measured in bone biopsies recovered from early post-menopausal women and related to measurements of bone mineral density. Slower bone loss was associated with higher IL-1Ra:IL-1b mRNA levels (Abrahamsen et al., 2000b). Of interest is the suggestion that the actions of estrogen on bone may be associated with an influence on IL-1:IL-1Ra ratios. Thus, the cells in whole blood recovered from women receiving hormone replacement therapy produce IL-1 and IL-1Ra at a lower ratio than controls (Abrahamsen et al., 2000a). Addition of 17b-estradiol to blood recovered from post-menopausal women decreases the ratio of IL-1:IL-1Ra that is spontaneously produced in vitro (Rogers and Eastell, 2001). In the bone biopsy study discussed in the previous paragraph, hormone replacement therapy in post-menopausal women normalized IL-1b mRNA levels without affecting the abundance of IL-1Ra message (Abrahamsen et al., 2000b).

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an attempt by the body to limit the disease (Sandberg et al., 1994).

Gastrointestinal tract and inflammatory bowel disease Plasma levels of IL-1Ra are higher than normal in patients with active ulcerative colitis, Crohn’s disease and other inflammatory bowel diseases. Levels are lower in patients with inactive disease, but remain above normal (Kuboyama, 1998). In a study of biopsied colonic material, IL-1Ra secretion was elevated in moderately to severely involved tissue samples, compared with non-inflamed tissue (Dionne et al., 1998). IL-1Ra:IL-1b ratios were lower in involved inflammatory bowel disease tissue (Dionne et al., 1998). Similar imbalances have been found in Crohn’s disease, ulcerative colitis, diverticulitis and infectious colitis. Immunohistochemical studies have identified cells within the lamina propria, especially macrophages, as the major source of IL-1Ra in inflammatory bowel disease tissue (Nishiyama et al., 1994). Administration of antibodies to IL-1Ra exacerbates and prolongs disease in a rabbit model of colitis (Ferretti et al., 1994). Mice lacking a functional IL-10 gene develop spontaneous inflammatory changes in the gastrointestinal tract, and this may be related to IL-1Ra (Kuhn et al., 1993). It is interesting to note that IL-10 down-regulates IL-1 and up-regulates IL-1Ra in mononuclear phagocytes and lamina propria present in biopsies of colon taken from patients with inflammatory bowel disease (Schreiber et al., 1995). A similar response was noted following administration of an IL-10 enema (Schreiber et al., 1995). According to one study, serum levels of IL-1Ra are reduced in patients with colorectal cancer (Iwagaki et al., 1997). However, the results from a similar type of investigation suggested increased IL-1Ra in patients with this disease (Ito and Miki, 1999). The incidence of sIL-1Ra mRNA increases in tumorous tissue recovered from patients with gastric cancer, and correlates with lymph node and liver metastasis (Iizuka et al., 1999).

Insulin-dependent diabetes mellitus Circulating concentrations of IL-1Ra are elevated in patients with longstanding insulin-dependent diabetes mellitus (Netea et al., 1997). Data from experiments in mice suggest that this response may indicate

Cancer There is considerable evidence that expression of IL-1Ra is dysregulated in various cancers (Kurzrock, 2001). In esophageal squamous cell carcinoma, for instance, expression of IL-1Ra is reduced two-fold (Hu et al., 2001). This also occurs in chronic myelogenous leukemia, where high leukocyte levels of IL-1b and low levels of IL-1Ra are seen in advanced disease and correlate with reduced survival (Kurzrock, 2001). In view of this, it is interesting that transfection of a murine skin carcinoma cell line with icIL-1Ra1 reduced its growth rate in vitro and slowed the development of tumors in vivo (La et al., 2001). Treatment of children with hematological malignancies using the chemotherapeutic agent cytarabine increases IL-1Ra blood levels (Ek et al., 2001). It is possible that IL-1 serves as a growth factor for certain types of tumors, with IL-1Ra acting to restrain tumor growth. Nevertheless, high levels of IL-1Ra are found in the sera of patients with Hodgkin’s lymphoma (Shin et al., 1995), and IL-1Ra is produced by bronchogenic carcinoma cells (Smith et al., 1993). It is also expressed by 20% of human hepatocellular carcinomas, and in pituitary tumor cells (Sauer et al., 1994). In these cases, IL-1Ra may help the tumor evade the immune system. Serum levels of IL-1Ra are altered in patients with soft tissue sarcomas (Ruka et al., 2001).

Transplantation The allograft response that leads to the rejection of transplanted organs is partly mediated by IL-1. Hepatic expression of IL-1b and IL-1Ra is increased during rejection following liver transplantation in humans. Moreover, low production of IL-1Ra is associated with steroid resistance of acute rejection (Conti et al., 2000). Acute graft-versus-host disease (GVHD) is a serious complication of marrow transplantation. Circulating levels of IL-1Ra are depressed 3–5 days after transplantation, but become elevated with the development of GVHD (Schwaighofer et al., 1997). The magnitude of the increase correlates with disease severity. Because IL-1 is thought to be an important

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component of the GVH response, this response has been interpreted as an attempt by the body to limit GVHD. In mice, administration of recombinant IL-1Ra reduces the immunosuppression and mortality of GVHD without impairing the engraftment of hematopoietic stem cells (McCarthy et al., 1991). These results have led to a clinical trial of IL-1Ra in patients with GVHD (see below).

IL-1Ra ALLELIC POLYMORPHISMS AND DISEASE A polymorphism in intron 2 of the IL-1Ra gene, caused by two to six copies of an 86 bp tandem repeat, is associated with a variety of human diseases (Tarlow et al., 1993) (Table 28.2). Two single nucleotide polymorphisms exist within exon 2 of IL1RN, but polymorphisms at these sites are always linked with the allelic polymorphism in intron 2. Allele A2 containing two repeats is found in 21.4% of the normal Caucasian population and is present in increased frequencies in diseases largely of epithelial cell or tissue origin TABLE 28.2 IL-1Ra allelic polymorphisms and disease Human diseases associated with IL-1Ra gene allele A2 (IL1RN*2) Ulcerative colitis in certain population groups Severity of alopecia areata Lichen sclerosis Early-onset psoriasis Multiple sclerosis in certain population groups Systemic lupus erythematosus, particularly skin lesions Sjögren’s disease Juvenile chronic arthritis Hyochlorhydria and gastric cancer Diabetic nephropathy Susceptibility to sepsis Henoch-Schönlein purpura IgA nephropathy Early-onset periodontitis Bronchial asthma Fibrosing alveolitis Silicosis Severity of acute graft-versus-host disease in bone marrow transplant patients Idiopathic recurrent miscarriage

(Arend and Guthridge, 2000; Witkin et al., 2002). However, all of the associated diseases are likely polygenic and IL1RN allele A2 (IL1RN*2) represents only one of many genes that may predispose to a disease. Furthermore, IL1RN*2 may be more linked with the severity of the disease, and the association may not actually be with the IL-1Ra gene itself, but with another gene in close linkage disequilibrium. An association of IL1RN*2 with an increased prevalence of ulcerative colitis, but not always with regional enteritis or Crohn’s disease, was described in some, but not all population groups (Mansfield et al., 1994; Andus et al., 1997; Roussomoustakaki et al., 1997; Bouma et al., 1999; Tountas et al., 1999; Craggs et al., 2001; Ishizuka et al., 2001). The association of IL1RN*2 with ulcerative colitis has been found in Americanbased Hispanic and Jewish populations, but the disease may be more heterogeneous in Northern European ethnic groups. However, in many of these reported studies, the number of subjects may have been too small to uncover a relatively weak association with IL1RN*2. A meta-analysis of eight European studies showed a significant association between carriage of allele 2 and UC with an odds ratio of 1.23 (Carter et al., 2001). IBD patients with IL-1Ra allele 2 demonstrated a decreased mucosal concentration of IL-1Ra protein (Andus et al., 1997; Carter et al., 1998). IL1RN*2 is also associated with some skin diseases, including the severity of alopecia areata (Tarlow et al., 1994), lichen sclerosis (Clay et al., 1994), and early onset psoriasis (Tarlow et al., 1997). IL1RN 2 was associated with multiple sclerosis in Dutch, Spanish and Italian patients (Crusius et al., 1995; de la Concha et al., 1997; Sciacca et al., 1999), particularly with a more aggressive disease (Schrijver et al., 1999). However, no association of IL1RN*2 with MS was demonstrated in French, American, Japanese or Finnish patients (Semana et al., 1997; Kantarci et al., 2000; Luomala et al., 2001; Niino et al., 2001). An association of IL1RN*2 was also described with HenochSchönlein nephritis (Liu et al., 1997), idiopathic recurrent miscarriage (Unfried et al., 2001), and a reduced risk for duodenal ulcer disease (Garcia-Gonzalez et al., 2001). No association with IL1RN*2 was found with endometriosis (Hsieh et al., 2001), sarcoidosis or idiopathic pulmonary fibrosis (Hutyrova et al., 2002), or with changes in serum levels of IL-1b or IL-1Ra after yellow fever vaccination (Hacker et al., 2001).

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The possible association of IL1RN*2 with rheumatic diseases has been explored in different population groups. In Caucasian patients in the UK, an increase in both frequency and carrier rate of IL1RN*2 was associated with systemic lupus erythematosus (SLE) (Blakemore et al., 1994). This association was strengthened with the presence of extensive disease and with discoid skin lesions. In a similar fashion, IL1RN*2 was associated with SLE in Japanese patients, particularly with photosensitive skin lesions (Suzuki et al., 1997). In a Swedish study, the presence of IL1RN*2 increased the susceptibility to SLE, and was associated with arthritis, although not with renal disease or overall severity (Tjernstrom et al., 1999). In all of these studies on IL-1Ra alleles and SLE, there was no relationship between the levels of IL-1Ra in serum and the presence or activity of disease. An association of definite Sjögren’s syndrome with IL-1Ra allele A2 was described in a French study; in these patients the levels of IL-1Ra in serum were elevated, but the levels in salivary fluid were low (Perrier et al., 1998). Three studies failed to describe an association between any IL-1Ra allele and rheumatoid arthritis (Perrier et al., 1998; Cantagrel et al., 1999; Cvetkovic et al., 2002). However, IL-1b allele 2 (3954) was associated with more severe RA and was accompanied by decreased serum levels of IL-1Ra (Buchs et al., 2000). IL1RN*2 was associated with juvenile chronic arthritis in Czech and Turkish patients, particular with patients exhibiting an extended oligoarticular course (Vencovsky et al., 2001). British patients with ankylosing spondylitis exhibited a significant increase in the carriage of IL1RN*2 compared with local controls (odds ratio 2.3) (McGarry et al., 2001). Lastly, IL1RN*1, but nor IL1RN*2, was found to be associated with juvenile idiopathic inflammatory myopathies in Caucasians, with an odds ratio of 2.5 (Rider et al., 2000). Additional studies on the association between IL-1Ra, IL-1 and disease have emphasized the importance of the ratio between these opposing cytokines. Linkage disequilibrium was demonstrated between IL1RN*2 and an IL-1b-31T diallelic polymorphism that enhances IL-1b production (Santtila et al., 1998; El-Omar et al., 2000). This genetic combination was associated with hypochlorhydria and gastric cancer (El-Omar et al., 2000). Furthermore, either IL1RN*2

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alone or in association with an IL-1b allele has been associatedwithdiabeticnephropathy(Blakemoreetal., 1996), susceptibility to sepsis (Fang et al., 1999), IgA nephropathy (Shu et al., 2000; Syrjanen et al., 2002), early-onset periodontitis (Parkhill et al., 2000), bronchial asthma (Mao et al., 2000), fibrosing alveolitis (Whyte et al., 2000), silicosis (Yucesoy et al., 2001), and severity of acute graft-versus-host disease in bone marrow transplant patients (Cullup et al., 2001). The mechanism of IL1RN*2 association with this variety of diseases, primarily of epithelial cell origin, remains unclear, but probably relates to a change in the ratio of IL-1Ra to IL-1b. In early studies, increased secretion of sIL-1Ra, but not levels of cellassociated icIL-1Ra isoforms, were observed in cytokine-stimulated monocytes from IL1RN*2 normal donors (Danis et al., 1995). However, the results of more recent studies have not substantiated these findings as total IL-1Ra production, both secreted and cell-associated, was decreased in resting or stimulated monocytes from either normal donors or patients with ulcerative colitis each carrying the IL1RN*2 allele (Tountas et al., 1999). Studies in collagen-induced arthritis in mice, an animal model resembling rheumatoid arthritis, indicated that the disease activity subsided commensurate with a large increase in production of icIL-1Ra1 in the joint tissue and a marked decrease in IL-1 production (Gabay et al., 2001b). This finding supported the concept that disease suppression was possibly secondary to an increase in the local ratio of icIL-1Ra1 to IL-1. The possibility exists that an association between IL1RN*2 and disease is not related to changes in production of sIL-1Ra by monocytes and other cells, but to decreased production of icIL-1Ra1 by epithelial and endothelial cells. This hypothesis is supported by the observations from many laboratories that icIL-1Ra1 levels are low in the intestinal lining of IL1RN*2 patients with ulcerative colitis. In addition, a genetic deletion in IL-1Ra production predisposes to the spontaneous development of arteritis (Nicklin et al., 2000) or arthritis (Horai et al., 2000), in different inbred strains of mice. These organs are rich in endothelial cells or fibroblasts where icIL-1Ra1 is the major isoform produced. The importance of decreased icIL-1Ra1 production in predisposition to disease is further supported by studies describing an association between IL1RN*2 and single-vessel

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coronary artery disease (Francis et al., 1999). Only icIL-1Ra1 mRNA, not sIL-1Ra mRNA, was demonstrated in different types of endothelial cells both in vivo and in vitro (Dewberry et al., 2000). Most importantly, cultured human umbilical vein endothelial cells from individuals carrying the IL1RN*2 allele produced less icIL-1Ra1 mRNA and protein in comparison with cells from donors possessing the other IL1RN alleles (Dewberry et al., 2000). However, further work is necessary to prove the hypothesis that the association of IL1RN*2 with the reported variety of human diseases is secondary to decreased production of icIL-1Ra1 and to a reversal in the local ratio of total IL-1Ra to IL-1.

injected intravenously or into a body cavity such as the joint (Granowitz et al., 1992; Barrera et al., 2000). Subcutaneous injection provides a more sustained release, because the extracellular matrix of the dermis captures the IL-1Ra and releases it progressively over the course of several hours (Campion et al., 1996); this is the route of administration of IL-1Ra in the treatment of RA. These issues are of particular concern to the therapeutic adminstration of IL-1Ra, because it needs to be present in a very large molar excess over IL-1 to suppress the latter’s biological properties (Arend et al., 1998). As discussed below, transfer of the IL-1Ra gene offers considerable advantages as a delivery strategy for achieving high, sustained concentrations of IL-1Ra within the body (Evans and Robbins, 1994).

POTENTIAL THERAPEUTIC USES OF IL-1Ra TREATMENT OF ANIMAL MODELS OF DISEASE WITH IL-1Ra

Conceptual issues IL-1Ra has obvious therapeutic potential in conditions where dysregulated IL-1 activity is a key pathophysiological mechanism. A variety of inflammatory and degenerative diseases falls into this category, as do the sequelae of acute trauma. As discussed below, impressive data from a wide range of different animal models of disease support this conclusion. Nevertheless, application to human disease has been questioned on two main fronts. The first concern draws attention to the complexity of cytokine interactions in human diseases, where large numbers of interacting cytokines, with overlapping, pleiotrophic properties, appear to offer substantial redundancy and resistance to simple therapeutic manipulation. These circumstances led to the view that there was little therapeutic benefit to be gained by blocking the activities of a single cytokine, as other cytokines within the system would compensate. The recent, remarkable success of TNF-a antagonists in treating RA and Crohn’s disease (Feldmann et al., 2001; Emery and Buch, 2002) has provided a powerful argument against this opinion, and has reinvigorated the development of cytokine antagonists as drugs. The second concern is delivery. Like other proteins, IL-1Ra does not lend itself readily to convenient, longterm administration. It cannot be taken orally, and has a biological half-life of less than an hour when

Table 28.3 lists those animal models of disease that respond to recombinant IL-1Ra. A wide range of inflammatory, allergic and degenerative conditions, as well as responses to trauma, ischemia/reperfusion and injury, are represented. In most cases the molTABLE 28.3 Animal models of diseases and disorders successfully treated with IL-1Ra Sepsisa Rheumatoid arthritisa Osteoarthritis Lupus (specific symptoms in certain models) Osteoporosis Traumatic, ischemic and excitotic brain injury Allergic reactions: Contact dermatitis Asthma Intestinal anaphylaxis Lung fibrosis Atherosclerosis Glomerulonephritis Colitis and other inflammatory bowel conditions Pancreatitis Diabetes Graft-versus-host diseasea Allograft responses Pain a

Has been tested in human clinical trials.

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ecule has been administered by frequent injection, or by the insertion of a pumping device that ensures constant delivery.

Sepsis IL-1 is a major secondary mediator of the effects of endotoxins released during sepsis. Considerable research confirms the protective effect of IL-1Ra in experimental sepsis in a range of laboratory animals, including rats, rabbits, piglets and baboons. In septic rats, a variety of physiological disturbances are normalized by the intravenous infusion of IL-1Ra, including bradycardia, hypothermia, hypotension and arteriole vasoconstriction, leading to reduced mortality (Alexander et al., 1992). Various cellular and biochemical sequelae of sepsis are also responsive to IL-1Ra, including increased muscle protein breakdown, decreased muscle synthesis, altered IGF-1 concentrations in blood, liver, kidney and skeletal muscle, altered IGF-1 binding protein-1 in blood and liver, structural changes in the aortic endothelium, and elevated circulating TNF (Norman et al., 1995; Lang et al., 1996). However, the effects of IL-1Ra are not uniform in all cases. For instance, in rats treated with E. coli endotoxin, IL-1Ra attenuated synthesis of TNF-a, but not of nitric oxide (Norman et al., 1995). In a separate study, IL-1Ra was found to prevent the increase of IGF-1 binding protein-1 in blood and liver, but not muscle (Lang et al., 1996). Likewise, in rats with an abdominal abscess, IL-1Ra did not restore hepatic protein synthesis, despite doing so in the kidney and small intestine (Cooney et al., 1996). Moreover, in the latter it did so only in the seromuscular layer. In newborn rats infected with Klebsiella pneumonia, IL-1Ra reduced or enhanced mortality, depending on the dose and duration of IL-1Ra administration (Mancilla et al., 1993). In rabbits injected with Staphylococcus epidermidis, IL-1Ra inhibited the fall in mean arterial pressure and systemic vascular resistance, and the increase in circulating TNF and IL-1. However, leukopenia and thrombocytopenia were unaffected. Circulating levels of TNF and IL-1 were not responsive to IL-1Ra in rabbits infused with E. coli, but blood pressure was preserved and lethality reduced (Wakabayashi et al., 1991). In baboons treated with a LD100 of live E. coli, infusion of IL-1Ra attenuated the decrease in mean

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arterial blood pressure and cardiac output, and improved survival. IL-1Ra also reduced circulating levels of IL-1 and IL-6, but not TNF (Fischer et al., 1992). A related study demonstrated that IL-1Ra also reduced levels of thrombin, plasminogen activator inhibitor, and neutrophil elastase in septic baboons (Jansen et al., 1995). As discussed below, IL-1Ra has been evaluated in the treatment of sepsis in humans.

Arthritis and autoimmune diseases Animal models of RA are the most comprehensively investigated in the present context, and have led to the use of IL-1Ra to treat human RA. The results from these studies reveal interesting variations in the responses of different pathophysiological processes occurring within a single disease entity. IL-1 is thought to play an important role in both inflammatory and erosive events in RA. In almost all animal models of RA yet tested (e.g. Kuiper, 1998; Bendele, 1999, 2000; Joosten, 1996), administration of IL-1Ra strongly protects the articular cartilage from destruction, and is more powerful than TNFa antagonists in this regard. However, the effects of IL-1Ra on inflammation and bone loss depend upon the disease model. For example, a weak antiinflammatory effect has been noted in antigen-induced arthritis in mice and rabbits, and adjuvant-induced arthritis in rats, despite almost complete protection of the articular cartilage (van de Loo et al., 1995; Otani et al., 1996). However, IL-1Ra has marked antiinflammatory properties in collagen-induced arthritis and streptococcal wall-induced arthritis in rats and mice (Joosten et al., 1996; Makarov et al., 1996; Bendele et al., 1999). Of interest is the observation that IL-1Ra had powerful anti-fibrotic properties in the knee joints of rabbits with antigen-induced arthritis (Lewthwaite et al., 1995). This agrees with the strong anti-fibrotic effects noted for IL-1Ra in experimental lung fibrosis (Piguet et al., 1993) and indicates a pathology against which IL-1Ra might be particularly effective. Because of the short biological half-life of injected IL-1Ra and the substantial amounts needed to inhibit strongly the biological actions of IL-1, the most dramatic anti-arthritic effects are obtained with continuous administration; implantable pumps are frequently used for this purpose (e.g. van de Loo, 1995;

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Bendele, 1999). Data from experiments using rats and mice suggest that sustained IL-1Ra serum concentrations in excess of 1 lg/ml are necessary for a maximal anti-arthritic effect (van de Loo et al., 1995; Bendele et al., 1999). It is interesting that in a model such as collagen-induced arthritis, where high doses of IL-1Ra block both inflammation and erosion, a suboptimal dose of IL-1Ra retains its anti-erosive properties, while failing to control inflammation (Bendele et al., 1999). Such data reinforce the conclusion that IL-1 is a very important mediator of cartilage destruction in arthritic joints, but contributes less critically to articular inflammation. These pre-clinical findings are important, because a single, subcutaneous injection of IL-1Ra in humans does not sustain a serum concentration of IL-1Ra above 1 lg/ml for a full 24 h (Bendele et al., 1999). Data from rat studies further suggest that sub-optimal doses of TNF-a soluble receptors and IL-1Ra act synergistically in controlling collagen-induced and adjuvant arthritis (Bendele et al., 2000). Repeated, intraarticular injection of IL-1Ra also inhibits the development of OA in the canine, anterior cruciate transection model (Pelletier et al., 1997). This effect is consistent with a body of data from the study of experimental animals and human biopsy material suggesting that IL-1 is an important mediator of cartilage loss in OA (Pelletier et al., 2001). IL-1Ra has also beneficial effects in murine models of systemic lupus erythematosus. In NZB/W F1 mice, that spontaneously develop lupus-like symptoms, multiple, intraperitoneal injections of IL-1Ra at 100 lg per mouse prevented the development of nephritis, reducing kidney damage and proteinuria (Sun et al., 1997). Serum levels of IL-1 were also reduced. However, IL-1Ra had no therapeutic effect in established disease in MRL/ mice (Kiberd and Stadnyk, 1995). In experimental autoimmune encephalomyelitis, a model of multiple sclerosis, IL-1Ra delayed disease onset, reduced the severity of paralysis and weight loss, and shortened the duration of the disease (Martin and Near, 1995). The mechanism may involve an influence on the activation and proliferation of encephalitogenic cells (Badovinac et al., 1998). Sustained administration of IL-1Ra using an osmotic pump protected against hyperglycemia and insulitis in a mouse model of diabetes (Sandberg, 1994). In a different approach related to the treatment of dia-

betes, IL-1Ra prolongs mouse islet allograft survival (Sandberg et al., 1993).

Nervous system IL-1Ra is a powerful neuroprotective agent in animal models of brain injury, whether damage is traumatic, ischemic or excitotic (Relton and Rothwell, 1992). In seizures, neuronal cell death results from persistent stimulation of N-methyl-D-aspartate receptors, a process that is enhanced by IL-1. Intrahippocampal application of IL-1Ra in mice strongly inhibits behavioral and EEG seizures induced by bicuculline (Vezzani et al., 2000). Intravenous injection of IL-1Ra also increases the survival time of rats exposed to heat stroke (Lin et al., 1995). Furthermore, continuous intravenous infusion of IL-1Ra after the onset of heat stroke dramatically enhances resistance to further development of heat stroke (Chiu et al., 1996). Death from heatstroke is associated with neuronal cell death secondary to cerebral ischemia. Several independent studies have confirmed that IL-1Ra protects against neuronal cell death during cerebral ischemia (e.g. Garcia, 1995). Intracranial administration of IL-1Ra also attenuates neuronal death after trauma, and maintains cognitive function (Sanderson et al., 1999). In certain animal models, IL-1Ra inhibits the hyperalgesia that accompanies inflammation. Thus in rats, IL-1Ra inhibits hyperalgesic responses to LPS, carrageenin, bradykinin, TNF and IL-1, but not those to IL-8, PGE2, and dopamine (Cunha et al., 2000). In mice, IL-1Ra inhibits the nociceptive response to intraperitoneal injection of acetic acid. Intrathecal delivery of IL-1Ra in combination with soluble TNF receptor inhibits pain in a rat model of neuropathic pain (Sweitzer et al., 2001). Administration of IL-1Ra also influences other aspects of neuronal function. For example, injection of 25 ng of IL-1Ra into the hypothalamus of rats increases food intake in tumor bearing rats (Laviano et al., 2000). This is of relevance to cancer-related anorexia.

Skin Local injection of IL-1Ra into sensitized mice inhibits contact hypersensitivity, as measured by ear swelling. At the highest intradermal dose of 100 lg per ear, swelling was reduced by 43% and both edema and the

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degree of inflammatory cellular infiltration were reduced. The sensitization and elicitation phases of contact hypersensitivity were also inhibited, but there was no effect on the non-specific inflammation elicited by phenol (Kondo et al., 1995).

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in this regard. TNF-a soluble receptors, in contrast, inhibited fatty streak formation in female, but not male, mice (Elhage et al., 1998). Nevertheless, IL-1Ra was unable to prevent the development of aortic abdominal aneurysms in rat model, unlike TNF-a soluble receptors (Hingorani et al., 1998)

Lung A 50 lg bolus of aerosolized IL-1Ra protects sensitized guinea pigs from bronchial hyperreactivity and pulmonary eosinophilia following antigen challenge (Watson et al., 1993). This observation suggests the possible utility of IL-1Ra in the treatment of asthma. Intraperitoneal administration of IL-1Ra is also effective in a guinea pig pulmonary hyperreactivity model (Selig and Tocker, 1992). IL-1Ra also reduces allergic reactions in the gastrointestinal tract and in the eye (Theodorou et al., 1993). IL-1 is known to promote the synthesis of type I collagen by fibroblasts. Consistent with this property, IL-1Ra protects mice from the pulmonary fibrosis that accompanies intratracheal administration of bleomycin or silica (Piguet et al., 1993). Not only does the continuous intraperitoneal infusion of IL-1Ra inhibit the development of fibrosis, but surprisingly, it also reduces the pulmonary hydroxyproline content in established fibrosis. IL-1Ra treatment has also been evaluated in three different rat models of chronic pulmonary hypertension induced by adminstration of monocrotaline, inflammation, or hypoxia equivalent to that experienced at an altitude of 16 000 feet. Hypertension provoked by monocrotaline or inflammation, but not hypoxia, is reduced by IL-1Ra (Voelkel et al., 1994).

Cardiovascular disease IL-1 has been implicated in the development of atherosclerosis. Mice deficient in the apolipoprotein E gene develop atherosclerotic lesions spontaneously. IL-1Ra was administered to these mice at a dose of 25 mg kg1 day1 using an osmotic pump implanted in a dermal subcutaneous pocket (Elhage et al., 1998). This produced serum levels of over 2 lg IL-1Ra/ml in females and over 1 lg/ml in males. These concentrations of circulating IL-1Ra dramatically reduced the formation of fatty streaks in the aortic sinus. IL-1Ra was as effective as the positive control, estradiol-17b

Kidney diseases Several animal models of antibody-mediated glomerulonephritis are responsive to treatment with IL-1Ra. In a model of crescentic glomerulonephritis induced by the adminstration of anti-glomerular basement mambrane antibodies, rats with established disease responded to constant infusion of IL-1Ra. Glomerular cell proliferation, crescent formation, glomerular sclerosis, tubular atrophy and interstitial fibrosis were all suppressed, with an impressive recovery of normal renal function (e.g. Lan et al., 1995). Protection was also seen in a spontaneously occurring IgA nephropathy in mice (Chen et al., 1997), but only modest effects occurred in rat anti-Thy-1 nephritis (Tesch et al., 1997). Nephritis is a prominent feature in murine models of lupus. In NZB/W F1 mice, intermittent intraperitoneal injection of IL-1Ra reduces renal damage and proteinuria, as well as normalizing certain aspects of immune function. Serum levels of TNF and IL-6, however, are not affected (Sun et al., 1997). Established nephritis in the MRL/ mouse, however, does not respond to continuous, intraperitoneal infusion of IL-1Ra (Kiberd and Stadnyk, 1995).

Osteoporosis There is increasing evidence that the loss of bone associated with menopause is driven by IL-1 and other cytokines. Intravenous infusion of IL-1Ra strongly reduces bone loss in ovariectomized rats and mice (Kimble et al., 1994b; Kitazawa et al., 1994). Of interest is the observation that the protective effect of IL-1Ra persisted for several weeks after discontinuation of the treatment (Kimble et al., 1994a). We have observed a similar effect following IL-1Ra gene therapy in ovariectomized animals (Baltzer et al., 2001) (see below). The reason for this is not known. IL-1Ra may also help to regulate strain-induced remodeling of bone. In one study using gerbils, IL-1Ra

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inhibited resorption, while having no significant effects on rates of bone apposition. Inhibition of resorption appeared to be related to a decrease in osteoclast cell surface area in response to IL-1Ra (Chole et al., 1995). Bone destruction by a multiple myeloma cell line in mice, in contrast, was not affected by IL-1Ra (Ferguson et al., 2002).

Gastrointestinal tract Inflammatory conditions in various compartments of the gastrointestinal tract are responsive to treatment with IL-1Ra. In a dose-related manner, the severity of experimentally induced colitis in both rats and rabbits is markedly reduced by administration of IL-1Ra (Thomas et al., 1991; Cominelli et al., 1992; Ferretti et al., 1994). This molecule also attenuates experimental pancreatitis in mice and rats, and reduces mortality (Norman, 1995). IL-1Ra also prevents the colonic motor and secretory changes induced by intestinal anaphylaxis in guinea pigs, suggesting a possible therapeutic role in food hypersensitivity (Thomas et al., 1991). There is also evidence that IL-1Ra may be of use in the treatment of hepatic inflammation secondary to amyloidosis (Grehan et al., 1997). IL-1Ra reduces liver injury and mortality following hepatic ischemia/reperfusion injury in the rat.

Diabetes Continuous infusion of IL-1Ra prevents the development of hyperglycemia and insulitis in mice treated with streptozotocin (Sandberg et al., 1994). Once the IL-1Ra is withdrawn, the disease returns. IL-1Ra has also been shown to prolong the survival and function of pancreatic islets after transplantation in mice (Sandberg et al., 1993).

Transplantation The influence of exogenously supplied IL-1Ra in the context of transplantation has been most comprehensively studied in GVHD. Early experiments demonstrated the ability of IL-1Ra to reduce the immunosuppression and mortality of GVHD in mice, without impairing engraftment of hematopoietic stem cells (McCarthy et al., 1991). Later research,

however, found that although IL-1Ra reduced the severity of GVHD in mice where the recipient and donors had only minor histocompatibility mismatch, there was no effect on disease in the presence of a major histocompatibility mismatch (Vallera et al., 1995). As discussed below, clinical trials of IL-1Ra in patients with GVHD have been carried out. IL-1Ra has also been evaluated experimentally as a way of improving the outcome of solid organ transplants. In a murine corneal transplant model, topical application of IL-1Ra improves the survival of corneal allotransplants (Dana et al., 1997). This is associated with reduced corneal inflammation, and attenuated infiltration of the graft by Langerhans cells. In subsequent studies, IL-1Ra did not alter the accelerated rejection that occurs in presensitized animals (Dekaris et al., 1999), and did not induce tolerance (Yamada et al., 2000). Nevertheless, in a murine model it sustains ocular immune privilege through a suppressive effect on Langerhans cell function. Locally applied IL-1Ra also inhibits the post-operative inflammation that accompanies intraocular lens implantation (Nishi et al., 1994). IL-1Ra prolongs the survival of cardiac allografts in rats, and is particularly effective when used in conjunction with cyclosporin. Survival of the allografts is associated with strongly reduced leukocytic infiltration (Shiraishi et al., 1995). IL-1Ra also prolongs the survival of pancreatic islet allografts in mice and prevents recurrence of diabetes in these animals (Sandberg et al., 1993, 1997).

HUMAN TRIALS WITH IL-1Ra Sepsis The first clinical studies were conducted in patients with sepsis. As described above, studies in mice, rats, rabbits and baboons with septic shock confirmed that administration of recombinant IL-1Ra dramatically reduced mortality. Because of the acute nature of the disease, it was possible to administer IL-1Ra to patients by continuous intravenous infusion. During the phase I component of this study, IL-1Ra was infused into both patients and healthy volunteers to establish safety and dosing. This conclusively established the remarkably acute safety of IL-1Ra. Over

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the course of a 3-day infusion, certain groups of healthy volunteers received tens of grams of recombinant IL-1Ra without any change in the various physiological parameters and blood chemistries that were monitored (Granowitz et al., 1992). Whether IL-1Ra will prove to be equally safe upon chronic administration will become clear from the results of its use in RA. In a subsequent, phase II, multicenter trial, recombinant IL-1Ra or placebo was infused intravenously for 3 days into 99 patients with sepsis syndrome or septic shock (Fisher et al., 1994b). Patients in the treatment arm were administered an intravenous loading dose of 100 mg IL-1Ra, and then one of three different doses: 17, 67 or 133 mg h1. Patients were evaluated for 28-day mortality. There was 44% mortality in the placebo group. Mortality was reduced to 32% among patients receiving the lowest dose of IL-1Ra, 25% among the middle dose group, and 16% in patients receiving the highest dose. These differences were statistically significant (Fisher et al., 1994b). In a phase clinical III trial, a total of 893 individuals with sepsis syndrome took part in a randomized, double-blind, placebo-controlled, multicenter, multinational trial (Fisher et al., 1994a). As in the phase II trial, patients were loaded with 100 mg IL-1Ra or placebo. They then received 72-h infusion of 1 or 2 mg kg1 IL-1Ra or placebo. The 28-day incidence of mortality was again used as the end-point. This study failed to demonstrate a statistically significant increase in survival time for the IL-1Ra-treated patients (Fisher et al., 1994a). Secondary and retrospective analyses of efficacy suggested that IL-Ra did increase survival in those patients who entered the study with the most severe disease (predicted risk of mortality of 24% or greater) (Knaus et al., 1996). In a second phase III study involving 696 patients with severe sepsis or septic shock, patients received standard supportive care and antimicrobial therapy in addition to IL-1Ra or placebo (Opal et al., 1997). IL-1Ra was delivered as a 100 mg intravenous loading dose, followed by infusion of 2 mg kg1 h1 for 72 h. Although 28-day mortality was the primary outcome measure, the study was stopped after an interim analysis showed that it was unlikely that this outcome would be met (Opal et al., 1997). No further clinical development of IL-1Ra for treatment of sepsis has occurred.

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Graft-versus-host disease There have been two clinical trials to evaluate the safety and efficacy of IL-1Ra in graft-versus-host disease. In a small, phase I/II open label trial, 17 patients with steroid-resistant GVHD received a continuous infusion of 400–3200 mg day1 IL-1Ra over 7 days (Antin et al., 1994). Stage-specific improvement occurred in the skin, gut and liver. Overall, acute GVHD improved by at least one grade in 63% of patients, associated with a decrease in TNF-a mRNA levels in blood mononuclear cells. However, of the 17 patients treated, five died (Antin et al., 1994). IL-1Ra treatment was further evaluated in a doubleblind, placebo-controlled, randomized trial of allogeneic stem cell transplantation in 186 patients. Patients received either saline placebo or IL-1Ra 0.5 mg kg1 h1 by continuous intravenous infusion from 4 days prior to 10 days after the transplantation. The results indicated no effect of IL-1Ra on the incidence of GVHD or on mortality (Antin et al., 1999).

Rheumatoid arthritis In an initial dose-ranging study, Campion et al. enrolled 175 patients with RA into a randomized, double-blind trial of IL-1Ra administered by subcutaneous injection (Campion et al., 1996). During an initial 3-week phase, patients were treated with 20, 70 or 200 mg IL-1Ra by subcutaneous injection once, three times or seven times per week. This was followed by a 4-week maintenance phase during which patients received IL-1Ra or placebo on a daily basis. Background NSAIDs or corticosteroids were permitted. IL-1Ra was well tolerated, with adverse injection site reactions being the most common side-effect. These were severe enough to cause 5% of patients to withdraw from the study. Insofar as it was possible to tell, daily dosing appeared to provide greater efficacy than weekly dosing (Campion et al., 1996). Under the name Anakinra, IL-1Ra has been evaluated in 472 European patients with active and severe RA of between 6 months and 8 years duration, recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study (Bresnihan et al., 1998). Patients were randomized into one of four groups receiving placebo, or 30, 75 or 150 mg IL-1Ra/ day self-administered by subcutaneous injection.

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DMARDs were prohibited during the study, but continuation of NSAIDs and corticosteroids was permitted. The primary outcome measure for this study was an ACR 20 response that was achieved by 27% of the placebo group and 43% of patients receiving the highest dose of IL-1Ra (P  0.014). Statistically significant improvement was recorded for several indices of disease, including number of swollen joints, number of tender joints, Health Assessment Questionnaire scores, erythrocyte sedimentation rate, and levels of C-reactive protein. Clinical responses occurred after 2 weeks of therapy. Radiologic damage assessed by Genant’s modification of Sharp’s grading system, revealed a 58% reduction in the rate of joint space narrowing, and a 38% reduction in the rate of erosions (Jiang et al., 2000). Synovial biopsy, performed in a small number of participants, showed that Anakinra reduced the numbers of intimal layer macrophages and subintimal macrophages and lymphocytes (Cunnane et al., 2001). This may reflect the down-regulation of various adhesion molecules. Those patients who experienced arrest of radiologic disease progression were those with the largest decline in macrophage infiltration. The initial study was followed by a further, 24-week extension phase in which 76 participants who had formerly received placebo were randomized among the three treatment groups (Bresnihan, 2001). Patients who had previously received Anakinra continued treatment at their previous levels. A total of 309 patients completed the extension phase of the study. Of the patients who had previously received placebo, 71% of the group now receiving IL-1Ra at 150 mg day1 achieved an ACR 20 response. Of the patients who were already receiving Anakinra, 49% maintained an ACR 20 response. Radiologic assessment of patients in the extension study confirmed that the reduction in the rates of joint space narrowing and erosion was sustained during the second 24-week period. In a separate recent study, 419 RA patients on maintenance doses of methotrexate for at least 6 months were randomized into six groups receiving 0, 0.04, 0.1, 0.4, 1.0 or 2.0 mg Anakinra kg1 day1 as a subcutaneous injection (Cohen et al., 2002). After 12 weeks, 19% of the placebo group had achieved an ACR 20 response. Forty-six percent of the group receiving 1 mg kg1 day1 Anakinra achieved an ACR 20 response, and 38% of those receiving 2 mg kg1 day1

did so. These improvements occurred after 2–4 weeks. Moreover, 24% of patients in the highest dose groups achieved an ACR 50 response, compared with 4% of the control group; 10% of the high-dose patients achieved an ACR 70 response. Monitoring of subjects in these trials indicated that Anakinra is safe. Injection site reactions were the most common side-effects, and occurred in 81% of individuals receiving the highest dose. Such reactions were usually mild and transient, but resulted in approximately 5–10% of participants withdrawing from the study. The FDA recently approved Anakinra, under the trade name Kineret, for the treatment of moderate to severe RA in adults who have failed one or more DMARDs. Kineret may be used alone, or in combination with DMARDs other than the TNF-a antagonists. Collectively, these data suggest that IL-1Ra holds promise in the treatment of patients with RA. However, the response is modest and much weaker than that seen in experimental animals subjected to continuous infusion with IL-1Ra. Of relevance is the likelihood that, despite daily self-injection of Anakinra at high concentrations, IL-1Ra levels did not remain high enough for long enough within the study subjects. Daily subcutaneous dosing at the highest level yet tested (150 mg), may fail to provide an anti-erosive dose of IL-1Ra for a substantial part of the day, and may barely achieve an anti-inflammatory dose at any time (Bendele et al., 1999). This could well explain the modest effects of Anakinra in clinical trials, and highlights the potential advantage of using gene transfer to achieve sustained, endogenous 24-h production of therapeutic quantities of IL-1Ra (Evans and Robbins, 1994).

GENE THERAPY WITH IL-1Ra General principles Athough IL-1Ra has therapeutic potential in numerous diseases, its usefulness as a drug is limited by several biological factors. These include its oral unavailability, its short biological half-life, and the need to maintain a large molar excess of IL-1Ra over IL-1 at its sites of action. A gene transfer approach to the administration of IL-1Ra arose in response to these challenges (Evans and Robbins, 1994). The basic

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strategy is to transfer a cDNA encoding IL-1Ra to the appropriate organ, so that it becomes a sustained, endogenous source of therapeutic quantities of IL-1Ra. Depending upon the vector system employed, and the physiology of the target cells, it is possible to achieve prolonged IL-1Ra gene expression, potentially in a regulated fashion. There is also the possibility that the native molecule synthesized from the transgene has superior properties to the recombinant protein produced in bacteria. The general principles of gene transfer have been described in Chapter 59 by Paul Robbins, Walter Storkus and Andrea Gambotto, and will not be repeated here. For present purposes, it is sufficient to note that various viral and non-viral vectors can be used to transfer genes by direct, in vivo or indirect, ex vivo administration (Ghivizzani et al., 2001). Recombinant adenoviruses are most commonly used in expriments requiring in vivo delivery, and recombinant retroviruses are most commonly used for ex vivo delivery. Non-viral vectors, such as plasmid DNA and DNA–liposome complexes, are typically used in an in vivo fashion. Generally speaking, viral transgene delivery is more efficient than non-viral delivery. This makes viral vectors particularly useful in experimental work with animals, but they raise greater safety issues when human application is envisaged. Present vector technology permits the efficient transfer and high in vivo expression of transgenes in many organs. However, it is not always possible to achieve long-term gene expression, which is a limitation for the treatment of chronic conditions. Technologies permitting close regulation of the level of transgene expression are under development.

IL-1Ra GENE TRANSFER IN ANIMAL MODELS OF DISEASE Arthritis IL-1Ra gene therapy experiments were first attempted in the context of RA (Evans et al., 1999). Two approaches have been evaluated. In so-called systemic delivery, the IL-1Ra that is synthesized as a result of gene transfer gains ready access to the systemic circulation. In so-called local delivery, the

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IL-1Ra gene is transferred to sites, typically the joints, where local accumulation of the transgene product occurs. For treating RA, the former strategy has the advantage of exposing multiple joints simultaneously to the transgene product and enabling the treatment of systemic and extraarticular manifestations of disease. It has the disadvantage of exposing non-target organs to high concentrations of the transgene product and thus facilitating unwanted side-effects. Moreover, non-genetic technologies can, or will within the foreseeable future, be able to deliver proteins systemically in an equivalent manner. With local delivery, in contrast, the highest concentrations of the transgene occur locally within the joint, with minimal exposure of extraarticular organs. This is something that no other delivery system can conveniently achieve, or is likely to be able to achieve soon. There is also the possibility that IL-1Ra synthesized locally at sites of pathology within the joint has a more powerful effect than IL-1Ra which diffuses into the joint from an extraarticular location. Systemic delivery of IL-1Ra has been achieved by retroviral transfer of the gene to the hematopoietic stem cells of mice (Boggs et al., 1995). This led to lifelong circulating levels of several hundred nanograms IL-1Ra per ml plasma. Although these mice were not subjected to detailed pathological scrutiny, they showed no obvious signs of distress, gained weight normally, had normal peripheral blood leukocyte profiles, lived a normal lifespan, and did not seem more prone to disease or infection. This observation agrees with the earlier safety data obtained for the recombinant protein. Most progress towards an IL-1Ra gene therapy for arthritis, however, has been made with local gene delivery to the synovium (Bandara et al., 1992). In vivo IL-1Ra gene transfer to the joints of experimental animals has been accomplished with various vectors, including adenovirus, herpes simplex virus, retrovirus, adeno-associated virus and lentivirus. In each case, levels of IL-1Ra production were sufficient to suppress experimental models of RA in these animals. It is of interest that, in several instances, the chondroprotective effects of IL-1Ra were more powerful than the anti-inflammatory effects (Ghivizzani et al., 1998), a result consistent with much of the data obtained using recombinant protein in animal models of RA.

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Despite impressive progress, the vectors used for the in vivo delivery of IL-1Ra to the joints of experimental animals are not yet suitable for application in human joints. However, ex vivo IL-1Ra gene delivery using a retrovirus has been implemented in a recent phase I clinical study (Evans et al., 1996, 2000). This protocol stemmed from a considerable body of work using the rabbit knee joint as the model system. A process was developed whereby a synovial biopsy was surgically recovered from the rabbit and used as a source of autologous synovial fibroblasts (Bandara et al., 1992). These were cultured in vitro, transduced with a retrovirus carrying the IL-1Ra cDNA, and returned to the knees of the individual donor animals by intraarticular injection. The injected cells engrafted in the recipient synovium and continued to produce elevated amounts of IL-1Ra, at a declining rate, for several weeks (Bandara et al., 1993). The amounts produced were sufficient to suppress antigen-induced arthritis in rabbits, with a particularly impressive protective effect on the articular cartilage (Hung et al., 1994). Similar protocols have been applied successfully to streptococcal cell wallinduced arthritis in rats and zymosan- and collageninduced arthritis in mice (Makarov et al., 1996; Bakker et al., 1997). As a demonstration of the advantages of gene therapy, Makarov et al. (1996) have calculated that local, ex vivo delivery of the IL-1Ra gene is 104-fold more potent than the recombinant protein in treating streptococcal cell wall-induced arthritis in rats. In agreement with this, we have noted a strong anti-arthritic effect of the IL-1Ra gene in rabbit antigen-induced arthritis (Otani et al., 1996; Ghivizzani et al., 1998), whereas Lewthwaite et al. found little effect of recombinant IL-1Ra (Lewthwaite et al., 1994), even though comparable intraarticular concentrations of human IL-1Ra accumulated in the knee joints. Extensive testing has confirmed that the ex vivo delivery of the IL-1Ra gene to the synovium is safe, and there is no gene transfer to the germ-line (Evans et al., 1996). Moreover, studies in SCID mice have confirmed that delivery of the IL-1Ra gene to human synoviocytes inhibits chondrocytic chondrolysis in human cartilage (Muller-Ladner et al., 1997). Local, intraarticular transfer of the IL-1Ra gene to the synovium may also be of value in treating osteoarthritis (OA). Using ex vivo, retroviral gene delivery, Pelletier et al. retarded the early loss of carti-

lage that follows transection of the anterior cruciate ligament in dogs (Pelletier et al., 1997). It was not possible to determine the long-term effects of the gene therapy because of loss of gene expression. In a subsequent study, the same group reported similar results in a rabbit model of OA in which naked DNA encoding IL-1Ra was injected into the joint space (Fernandes et al., 1999). This is a remarkable result given the low level and transience of transgene expression that typically follow the intraarticular administration of plasmid vectors, as well as the inflammatory response of synovium to large amounts of naked DNA. A recent experimental study in horses has provided a convincing demonstration of the potential of IL-1Ra gene therapy in treating OA. Frisbie et al. (2002) cloned equine IL-1Ra and used an adenoviral vector to deliver it to the joints of horses with experimental OA. This experimental model mimics a condition seem commonly in race horses, where an osteochondral fragment has become dislodged and remains within the joint to provoke synovitis and loss of articular cartilage. Delivery of the equine IL-1Ra cDNA 10 weeks after induction of disease provided powerful protection of the articular cartilage. As noted in several other settings, IL-1Ra had a more modest antiinflammatory effect. Most importantly, gene therapy provided clinical improvement, evidenced by a reduction in the horses’ lameness scores (Frisbie et al., 2002). In an alternative approach to therapy, the IL-1Ra gene has been transferred to articular chondrocytes (Baragi et al., 1995). Chondrocytes transduced with an adenovirus carrying the IL-1Ra cDNA were transferred on to cartilage fragments, and exposed to IL-1. Under these in vitro conditions, the underlying cartilage resisted the catabolic effects of IL-1. The IL-1Ra cDNA has also been transferred to chondrocytic cells from the end plates of the spine, as a strategy for influencing intervertebral disc disease and other conditions of the the spine (Wehling et al., 1997).

Central nervous system IL-1Ra gene transfer shows beneficial effects in animal models of brain injury resulting from trauma or ischemia and reperfusion. The inflammatory reaction to blunt trauma in the rat brain is dramatically

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reduced by the transfer of fibroblasts that have been retrovirally modified to express large amounts of IL-1Ra (DeKosky et al., 1996). In a rat model of stroke, the intraventricular, adenoviral delivery of IL-1Ra reduced cerebral infart volume by 64% (Betz et al., 1995). Equivalent results were reported with a mouse model of ischemia-reperfusion. In this case, delivery of the IL-1Ra gene reduced, in addition to the infarct size, the disruption of the blood–brain barrier and expression of ICAM-1 (Yang et al., 1999).

Insulin-dependent diabetes mellitus Transfer of the IL-1Ra gene to pancreatic islets is of interest both as a means of suppressing the autoimmune destruction of these cells during type I diabetes, and as a means of improving the survival of allografts. Human islets, transduced in vitro with adenovirus or lentivirus carrying the IL-1Ra gene, were protected from IL-1 mediated nitric oxide formation, impaired glucose-stimulated insulin production, and Fas-triggered apoptosis (Giannoukakis et al., 1999). However, liposomally mediated transfer of IL-1Ra cDNA to syngeneically transplanted murine islets failed to confer resistance to islet destruction in recipient NOD mice (Saldeen et al., 2000). It is possible that the level and duration of transgene expression were too low to achieve a protective effect.

Cardiovascular system The ability of the IL-1Ra gene to influence ischemiareperfusion injury during cardiac allograft has been evaluated in a rat model. In this system, the hearts were transfected with the gene by intracoronory infusion and allografted to the abdomen of a recipient rat. While in the abdomen, the transplanted heart was subjected to 30 min of ischemia followed by 24 h of reperfusion. In the presence of the IL-1Ra gene, infarct size, neutrophil infiltration, and cardiomyocyte apoptosis were strongly inhibited (Suzuki et al., 2001). In another application, mice were inoculated intraperitoneally with encephalomyocarditis virus. Immediately afterwards, the IL-1Ra gene was electroporated into the tibialis anterior muscles of mice leading to peak circulating levels of 10.5 ng IL-1Ra

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ml1. This procedure lowered expression of TNF-a and nitric oxide, and reduced mortality (Nakano et al., 2001).

Bone As noted above, administration of recombinant IL-1Ra protein is able to reduce bone loss in ovariectomized rodents. Baltzer et al. have demonstrated a similar effect using the IL-1Ra gene (Baltzer et al., 2001). These investigators treated ovariectomized mice with adenovirus carrying IL-1Ra cDNA by intramedullary injection. This procedure transduced lining osteoblasts, osteocytes and marrow cells, as well as adjacent muscle and draining lymph nodes. Transgene expression persisted for approximately 12 days, but bone loss was attenuated during the entire 5-week experiment. Why the effect should persist in this manner is unknown, but a similar response had been noted with the recombinant protein (Kimble et al., 1994a). Moreover, the protective effect of the IL-1Ra gene was not limited to bones receiving the intermedullary injection, but occurred in all bones that were evaluated. Another potential use of IL-1Ra gene therapy in bone is to inhibit the aseptic loosening of prosthetic joints. This process is thought to be triggered by mediators released from periprosthetic cells in response to wear debris. Transduction of macrophage cultures with IL-1Ra cDNA inhibits the cellular responses to wear particles in vitro (our unpublished data), and also suppresses in vivo responses to wear debris in a murine air pouch model (Sud et al., 2001).

Kidneys Two methods have been employed to transfer the IL-1Ra gene to the renal glomerulus. In one ex vivo approach, cultured rat mesangial cells were stably transfected and delivered to the glomeruli of rats via the renal circulation. Responses of the recipient glomeruli to IL-1 were blunted (Yokoo and Kitamura, 1996). In a second ex vivo approach, marrow derived CD11bCD18 cells were transduced with a recombinant adenovirus carrying the IL-1Ra gene (Yamagishi et al., 2001; Yokoo et al., 2001). The genetically modified cells were injected intravenously into mice with

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an experimental ureteral obstruction. Expression of ICAM-1, the counterligand for CD11b and CD18, increases in the renal interstitium during this disease, thus retaining the genetically modified cells and enhancing local synthesis of IL-1Ra. Under these conditions, macrophage infiltration and other aspects of inflammation were reduced (Yamagishi et al., 2001; Yokoo et al., 2001). In a related application, marrow cells were transduced with a recombinant retrovirus carrying IL-1Ra and infused into lethally irradiated recipients. After successful marrow transplant, an experimental glomerulonephritis was induced in the recipient mice. In those mice receiving the IL-1Ra gene, renal function and histology were maintained and the survival rate improved (Yamagishi et al., 2001; Yokoo et al., 2001).

Lungs Recombinant retrovirus has been used to transfer the IL-1Ra gene to the lungs of sheep in utero (Pitt et al., 1995). Recombinant adenovirus has been used for the same purpose in the lungs of adult mice and pigs (McCoy et al., 1995; Morrison and Murtaugh, 2001). In mice, the IL-1Ra produced as a result of gene transfer fails to inhibit the inflammatory response to the adenovirus (McCoy et al., 1995). In pigs, however, no inflammatory response occurred (McCoy et al., 1995).

HUMAN TRIALS OF GENE THERAPY WITH IL-1Ra

retrovirally transferred to one half of the cells, while the remaining cells served as unmodified controls. In a double-blind fashion, one week before MCP joint replacement surgery two of these joints received the genetically modified cells and the other two received unmodified cells by intraarticular injection. Articular tissue recovered at the time of MCP joint arthroplasty was then examined for evidence of successful gene transfer and gene expression. All nine subjects completed the study without problems, and no adverse events related to the procedure were noted. Expression of the transgene was detected by RT-PCR in all joints that received it. Elevated IL-1Ra protein synthesis was also noted in tissues recovered from most of the transduced joints. In situ hybridization and immunohistochemistry confirmed the presence of engrafted, transduced cells on the recipient synovia. Certain patients reported symptomatic relief during the study, but these anecdotal observations could be attributed to a placebo effect. A similar phase I study, with an intraarticular dwell time of the IL-1Ra gene of 1 month, is under way in Germany (Evans et al., 2000). The preliminary data from the German trial are similar to those of the American trial. A phase II study to determine efficacy is being planned. Future development of a convenient and effective IL-1Ra gene treatment for RA requires a vector that can be administered by direct, intraarticular injection and achieves prolonged transgene expression at therapeutic levels. Recent data suggest that novel lentiviral vectors hold promise in this regard (our unpublished data).

Arthritis IL-1Ra gene delivery to synovium ex vivo using autologous synovial fibroblasts in conjunction with a Moloney-based retroviral vector, was employed in a phase I clinical trial to determine safety and efficacy (Evans et al., 1996). Nine post-menopausal women with advanced RA were recruited to the study. Among the entry requirements was the need for surgical replacement of the 2nd–5th metacarpophalangeal (MCP) joints on one hand, and surgery on at least one other joint. The latter procedure provided the opportunity to harvest autologous synovium from which to grow synovial fibroblasts. Cultures of autologous cells were divided into two. The human IL-1Ra cDNA was

AREAS OF ACTIVE RESEARCH AND INQUIRY Role of IL-1Ra in normal physiology and in host defense The fact that IL-1Ra can be detected in normal individuals suggests a role for this molecule in normal physiology. As discussed above, there is evidence that the IL-1/IL-1Ra axis plays important roles in the normal CNS, in bone remodelling and in host defense, among others. Furthermore, the spontaneous development of arthritis or arteritis in IL-1Ra knockout

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mice, in each case on a specific genetic background, indicates that IL-1Ra in tissues plays a counterbalancing role to IL-1 in preventing disease. Possibly host responses to injury or infection, which normally would be controlled, lead to a clinical disease in the presence of a cytokine imbalance. Determining what are the other predisposing genes in these animal models of disease may clarify some of the genes and their interacting proteins in polygenic human diseases. Particularly intriguing are the roles of the intracellular isoforms of IL-1Ra, and how they may perform unique roles in host defense. It has been known for over a decade that, in addition to triggering signal transduction by binding to cell surface receptors, IL-1/receptor complexes can be internalized and translocated to the cell nucleus. Precursor IL-1a also affects cell growth through movement to the nucleus without ever being externalized. Since intracellular isoforms of IL-1Ra predominate in certain cells, it is tempting to ascribe a role for icIL-1Ra in regulating the intracellular activities of IL-1. However, exactly what this role might be remains unknown.

Delivery of IL-1Ra in therapy It is clear from the examples given above, that IL-1Ra has wide therapeutic potential in a large number of diseases. Delivery is a major impediment to developing IL-1Ra as a drug, especially in chronic conditions where therapeutic concentrations need to be sustained within the patient for an extended period. As noted, for the treatment of patients with RA, daily injections of very large amounts of recombinant protein are required. Various slow-release formulations and pumping devices are being investigated as improved delivery systems. These approaches to therapy with IL-1Ra may allow better pharmacokinetics with more sustained blood and tissue levels of the delivered agent. However, the kinetics of delivery from slow release vehicles tend to be biphasic with most of the dose liberated into the system relatively quickly, followed by a slow, non-uniform leaching of the residual protein. There is also the issue of the stability of the protein under physiological conditions, as it remains stored within the delivery device. Pumps solve the problems associated with non-uniform release, but not protein lability, and they are invasive

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and complex. Small organic molecules, which mimic the receptor blocking effects of IL-1Ra, have been sought unsuccessfully, probably because of the complex interactions between IL-1 or IL-1Ra and the IL-1 receptor. As discussed above, gene therapy is also being investigated as a means of solving problems related to delivery. Moreover, as a result of gene transfer and expression, it is the native, authentically processed molecule that is produced. The biological properties of the native and recombinant forms of IL-1Ra have not been compared in a detailed fashion, and important differences may exist. In addition to facilitating the administration of sIL-1Ra in various diseases, gene transfer approaches hold particular promise in the delivery of the icIL-1Ra1 isoform, as this molecule will need to accumulate within the target cells. Although the principle of IL-1Ra gene transfer is now well-established, there remain the problems of tissue or cell specificity of delivery and of long-term gene expression. Considerable research into these problems is under way, with particular attention being paid to vector design, cell turnover and the immunological constraints to transgene expression. Inducible transgene expression in specific tissues has become a reality in animal systems and may become applicable to gene therapy of arthritis. This would allow prolonged and regulated expression of a transgene in arthritic joints only at times of disease activity.

Combination therapy with IL-1Ra The biological activities of IL-1 are inhibited physiologically not only by IL-1Ra, but also by soluble forms of the IL-1 type I and type II receptors. Which of these holds the greatest therapeutic promise is a matter of importance in treating IL-1-driven diseases. Because the affinity of the soluble type I receptor for IL-1Ra exceeds that of its affinity for IL-1, it is a poor candidate, and experimental data confirm this. The soluble type II receptor, however, preferentially binds IL-1. Recent data from gene transfer experiments confirm that the type I soluble receptor is a far weaker inhibitor of the actions of IL-1 on chondrocytes than either the type II soluble receptor or sIL-1Ra. The latter are of approximately equal potency both in vitro and in a rabbit model of RA; whether they act synergistically is unknown.

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Most inflammatory and degenerative diseases are complex and involve multiple cytokines. There is consequently much interest in combining IL-1Ra with other biological agents to improve treatment. Data from many animal experiments suggest that the combination of IL-1Ra with a TNF-a antagonist is particularly powerful, but the potential side-effects of employing two such potent therapeutic agents will require thorough evaluation. Other combinations under consideration are IL-1Ra plus a type-2 cytokine (e.g. IL-4, IL-10) or IL-1Ra plus a traditional drug such as, in the case of RA, methotrexate. It is also possible that IL-1Ra may be combined in the future treatment of RA with other new therapies with different mechanisms of action, such as enzyme inhibitors or chemokine blockers. Combinations may allow each therapeutic agent to be used at lower and potentially less toxic concentrations.

Clinical trials in additional diseases

FIGURE 28.2 IL-1Ra gene transfer to human, rheumatoid, metacarpophalangeal joints. (1) Synovial tissue is removed from a non-MCP joint. Synovial cells are isolated and cultured. (2) Half the cells are transduced with a retrovirus carrying the human IL-1Ra cDNA; the other half of the cells remain as controls. (3) The majority of each population of cells is cryopreserved; aliquots are subjected to safety testing. (4) Safetytested cells are thawed, recultured and prepared for injection. Two MCP joints are injected with transduced cells and two are injected with controls, untransduced cells. (5) Seven days later, the injected joints are surgically replaced with prosthetic joints, as previously indicated for the management of the disease. (6) Tissues retrieved at surgery are analyzed for evidence of successful IL-1Ra gene transfer and intraarticular IL-1Ra transgene expression. (Reproduced, with permission, from Evans et al. (1998). Blocking cytokines with genes. J. Leukocyte Biol. 64, 55–61.)

As described above, recombinant IL-1Ra has been subjected to clinical trials in sepsis, GVHD and RA. Only the last of these has progressed to the point of being approved by the FDA as a drug. Yet it is clear that many more conditions potentially stand to benefit from treatment with IL-1Ra, including OA and a range of inflammatory and degenerative diseases. Given the time and cost of conducting large-scale clinical studies, the challenge is to select carefully from the list of candidate diseases. The recent success of TNF-a antagonists in psoriatic arthritis, ankylosing spondylitis and juvenile chronic arthritis, suggests that IL-1 inhibition also may be efficacious in these conditions.

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combination with methotrexate. Arthritis Rheum. 46, 614–624. Colotta, F., Dower, S.K., Sims, J.E. and Mantovani, A. (1994). The type II ‘decoy’ receptor: a novel regulatory pathway for interleukin 1. Immunol. Today 15, 562–566. Cominelli, F., Nast, C.C., Duchini, A. and Lee, M. (1992). Recombinant interleukin-1 receptor antagonist blocks the proinflammatory activity of endogenous interleukin1 in rabbit immune colitis. Gastroenterology 103, 65–71. Cominelli, F., Bortolami, M., Pizarro, T.T. et al. (1994). Rabbit interleukin-1 receptor antagonist. Cloning, expression, functional characterization, and regulation during intestinal inflammation. J. Biol. Chem. 269, 6962–6971. Conti, F., Breton, S., Batteux, F. et al. (2000). Defective interleukin-1 receptor antagonist production is associated with resistance of acute liver graft rejection to steroid therapy. Am. J. Pathol. 157, 1685–1692. Cooney, R.N., Owens, E., Slaymaker, D. and Vary, T.C. (1996). Prevention of skeletal muscle catabolism in sepsis does not impair visceral protein metabolism. Am. J. Physiol. 270, E621–E626. Corradi, A., Franzi, A.T. and Rubartelli, A. (1995). Synthesis and secretion of interleukin-1 alpha and interleukin-1 receptor antagonist during differentiation of cultured keratinocytes. Exp. Cell. Res. 217, 355–362. Craggs, A., West, S., Curtis, A. et al. (2001). Absence of a genetic association between IL-1RN and IL-1B gene polymorphisms in ulcerative colitis and Crohn disease in multiple populations from northeast England. Scand. J. Gastroenterol. 36, 1173–1178. Crusius, J.B., Pena, A.S., Van Oosten, B.W. et al. (1995). Interleukin-1 receptor antagonist gene polymorphism and multiple sclerosis. Lancet 346, 979. Cullinan, E.B., Kwee, L., Nunes, P. et al. (1998). IL-1 receptor accessory protein is an essential component of the IL-1 receptor. J. Immunol. 161, 5614–5620. Cullup, H., Dickinson, A.M., Jackson, G.H. et al. (2001). Donor interleukin 1 receptor antagonist genotype associated with acute graft-versus-host disease in human leucocyte antigen-matched sibling allogeneic transplants. Br. J. Haematol. 113, 807–813. Cunha, J.M., Cunha, F.Q., Poole, S. and Ferreira, S.H. (2000). Cytokine-mediated inflammatory hyperalgesia limited by interleukin-1 receptor antagonist. Br. J. Pharmacol. 130, 1418–1424. Cunnane, G., Madigan, A., Murphy, E. et al. (2001). The effects of treatment with interleukin-1 receptor antagonist on the inflamed synovial membrane in rheumatoid arthritis. Rheumatology (Oxford) 40, 62–69. Cvetkovic, J.T., Wallberg-Jonsson, S., Stegmayr, B. et al. (2002). Susceptibility for and clinical manifestations of rheumatoid arthritis are associated with polymorphisms of the TNF-alpha, IL-1beta, and IL- 1Ra genes. J. Rheumatol. 29, 212–219. Dana, M.R., Yamada, J. and Streilein, J.W. (1997). Topical interleukin 1 receptor antagonist promotes corneal transplant survival. Transplantation 63, 1501–1507. Danis, V.A., Millington, M., Hyland, V.J. and Grennan, D. (1995). Cytokine production by normal human monocytes: inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1Ra) gene polymorphism. Clin. Exp. Immunol. 99, 303–310. Debets, R., Hegmans, J.P., Croughs, P. et al. (1997). The IL-1 system in psoriatic skin: IL-1 antagonist sphere of influ-

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29 Interleukin-18 [IL-1F4] Haruki Okamura1, Michael T. Lotze 2, Hiroko Tsutsui1, Shin-ichiro Kashiwamura1, Haruyasu Ueda1, Tomohiro Yoshimoto1, and Kenji Nakanishi1 1

Hyogo College of Medicine, Nishinomiya, Japan

2

Molecular Medicine Institute 100 Technology Dr. Pittsburgh, PA, USA

Eccentricity has always abounded when and where strength of character has abounded; and the amount of eccentricity in society has always been proportioned to the amount of genius, mental vigour and moral courage which it contained. That so few men now dare to be eccentric marks the chief danger of our age. John Steward Mill

INTRODUCTION The identification of an IFN-inducing factor (IGIF) in 1995, subsequently termed Interleukin-18 (IL-18), or Interleukin-1 Family 4 (IL-1F4) (Sims et al., 2001) led ultimately to studies revealing that IL-18, like IL-1 and TNF, is a proinflammatory cytokine. It plays an important role in immune and inflammatory reactions, and is notably present in several autoimmune disorders. Recent investigations have brought its uniqueness into relief. For example, IL-18, in collaboration with IL-12, strongly induces type I cytokines such as IFN leading to the production of molecules destructive to tissues, including nitric oxide, reactive oxygen species, and TNF. In addition, IL-18 also induces production of type II cytokines, such as IL-4, IL-5, and IL-13, which may exacerbate allergic conditions. Indeed, IL-18 seems to exert its functions not by itself but rather in concert with other factors, promoting many of the biologic activities associated with these other cytokines. The information obtained so The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

far in in vitro experiments reflects only part of the underlying complexity now demonstrated in vivo. IL18 is abundant in non-immune tissues including the skin and and the gut and it likely provides an important role in the defense against mucosal and commensal organisms. There have been many excellent recent reviews of IL-18 biology (Dinarello et al., 1998; Okamura et al., 1998; Gillespie and Horwood, 1998; Dayer, 1999; Akira, 2000; Dinarello, 2000; Golab, 2000; Lebel-Binav et al., 2000; McInnes et al., 2000; Tsutsui et al., 2000; Nakanishi et al., 2001a,b). In addition to IFN- induction, IL-18 induces Fas ligand expression on T and NK cells, augments the cytolytic activity of T and NK cells, and induces production of several chemokines and cytokines. The major biological activities of IL-18 clarified to date are summarized in Table 29.1. These activities suggest that IL-18 plays important roles in the host defense against infections and malignant transformation, but is also involved in the pathogenesis of various inflammatory diseases. IL-18 receptor Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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TABLE 29.1 Major Biological Activities of IL-18 in vitro Induction of IFN- (T and NK cells, synergism with IL-12) Induction of Fas ligand expression (T and NK cells) Activation of NK cells Induction of other cytokines (GM-CSF, TNF, IL-4, -5, -8, -10 and -13) Induction of iNOS (macrophages, chondrocytes) Induction of cyclooxygenase; COX-2 (chondrocytes) in vivo Anti-microbial action (Cryptococcus, Salmonera, Yersinia, Leishmania) Anti-tumor action (sarcoma, carcinoma, melanoma) is a member of the IL-1/FGF/TLR beta trefoil cytokine family, and is composed of two peptides. Signaling pathways of IL-18 are similar to those of other proinflammatory cytokines including IL-1 and IL-1b (Dinarello et al., 1996; Dinarello, 1998). Upon binding its receptor, IL-18 activates several transcription factors including NFB and AP-1. Those reviews also indicated that there are many questions to be answered about the regulatory mechanism’s processing, biological action, and usage of signal pathways, as well as physiological and pathological roles. The role of IL-18 binding protein and regulatory mechanisms for IL-18 receptor expression on various cell types is also still not fully defined. IL-18 is associated with differentiation of various cells, mediating both immune and non-immune phenomena. IL-18 may play other roles that are still to be determined. Like IL-1, IL-18 connects the immune, endocrine, and nervous systems.

GENOMIC STRUCTURE, REGULATION, AND CHROMOSOMAL LOCALIZATION OF IL-18 The cDNA for murine IL-18 encodes a protein of 192 amino acids (Okamura et al., 1995). The murine IL-18 gene is composed of seven exons that distribute over a 26kb region (Tone et al., 1997). Exons 1 and 2 are non-coding. The human IL-18 gene is composed of six exons and five introns spanning about 19.5 kb. Encoding a 193 amino acid protein (Ushio et al., 1996; Kalina et al., 2000). Homology in the b sheet structure

between IL-18 and IL-1b is revealed by a fold recognition strategy, making IL-18 a distant member of the beta trefoil family of cytokines, including fibroblast growth factor (Welch et al., 1995; Dinney et al., 1998; Holland and Varmus, 1998; Xu et al., 1999; Robak et al., 2002) and the extended family of IL-1 family members. Like IL-1b [IL-1F2] and IL-1H4 [IL-1F7] (Kumar et al., 2002), IL-18 does not contain leader sequences and is secreted in the form of microvesicles.

Gene mapping The genes for IL-18 and its receptor map to different chromosomes. The human IL-18 gene is located on chromosome 11q22.2-22.3, closely linked to the locus of the dopamine receptor D2 (DRD2) and the stress responsive protein ATM (Bar-Shira et al., 2002; Chang et al., 2002; Yin et al., 2002; Zhou et al., 2002), while the IL-18R gene maps to chromosome 2q13-D21, on which the other human IL-1 family members and receptors (IL-1, IL-1b, IL-1R, IL-1 IL-1 IL-1R type I, IL-1R type II, T1/ST2, IL-1Rrp2, and IL-1R accessory protein (AcP)) are located (Parnet et al., 1996; Nolan et al., 1998; Debets et al., 2001). The relative gene order on the chromosomal region where the human IL-18 gene is located is ATM-IL-18-DRD2-THY1, which is the same as that of murine IL-18 (Rothe et al., 1997a). Preliminary experiments on non-obese diabetic (NOD) mice revealed that the murine IL-18 gene localizes within the Idd2 interval, which suggests that IL-18 may be a NOD-susceptible gene on chromosome 9 (Rothe et al., 1997a). The human chromosomal region 11q22-q23 is also a target for deletion in the development of various solid tumors (Baysal et al., 2001).

Regulatory regions The regulatory mechanism for IL-18 expression is not yet fully clarified. One of the promoters of murine IL-18 gene is in the upstream region of non-coding exon l, where the consensus sequence for ICSBP (IFN consensus sequence binding protein) is detected, and is up-regulated by LPS (Kim et al., 1999). The other is upstream of exon 2, where there is an element for PU.1 binding which is constitutively active. A potential NFB recognition sequence and two potential Oct1 recognition sequences are located upstream of exon 1

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suggesting interaction with the viral transcriptional activator protein VP16 and up-regulation of the expression of IL-18 by viruses and bacteria. Upstream of exon 1, the human IL-18 gene contains a threeconsensus sequence for STAT3 binding, to which activated STAT3, however, does not bind (Kalina et al., 2000). Using a luciferase reporter gene assay, co-transfection of STAT5 into macrophage cells, U937 and THP-1, was shown to increase the expression of IL-18. The STAT5 protein also binds to the IL-18 promoter region (Kalina et al., 2000). Thus, expression of the IL-18 gene may be up-regulated by the signal activating molecule STAT5. Both of the promoters in the murine IL-18 gene are TATA-less and GC poor, suggesting that IL-18 expression is not restricted to lymphocytes. In addition, emphasizing the uniqueness of IL-18, the IL-18 gene contains no RNA-destabilizing elements that are associated with many other cytokine genes (Tone et al., 1997).

Hormonal regulation It is probable that IL-18 expression is regulated by hormones, because the level of IL-18 mRNA is elevated by cold stress or by adrenocorticotropic hormone production acting on the adrenal gland where glucocorticoid is produced (Conti et al., 2000). Since the size and the 5-end of IL-18 mRNA in the adrenal cortex and immune cells are different, there may be tissue-specific usage of promoter regions (Sugama et al., 2000). It appears that IL-18 may also be important in stress responses and obesity (Kalina et al., 2000; Esposito et al., 2002).

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24kDa

18kDa

FIGURE 29.1 Processing of IL-18 by Caspase-1. that pro-IL-1b and pro-IL-18 are not the only substrates for this enzyme (Bras et al., 1997). Other caspases, caspase-3 and caspase-4, can cleave pro-IL-18, but produce smaller-sized fragments than those produced by caspase-1, which are biologically inactive (Akita et al., 1997; Gu et al., 1997). Caspase-1 is regulated at the level of gene expression. Processing of its precursor protein to an active form as well as the regulatory mechanism for its activation remains to be fully clarified. It may be processed by other caspases as well, because mice deficient for caspase-11 are resistant to endotoxic shock and incapable of producing IL-1b (Wang et al., 1998). LPS-induced, caspase-1dependent IL-18 secretion by macrophages requires TLR4, a member of the Toll-like receptor family, but does not require MyD88, a molecule required for the intracelluar signal transduction from TLR4 (Seki et al., 2001). This indicates that the activation of caspase-1 occurs independently of gene expression.

Processing of IL-18 Caspase-1 cleavage of pro-IL18

Alternative processing of pro-IL-18

Production of IL-18 is regulated at the stage of gene transcription and of post-translational processing. IL-18 protein is stored in an inactive precursor form in various cell types and extracellularly secreted rapidly upon stress. However, the mechanism for processing and secretion of IL-18 remains somewhat of a mystery. As shown in Figure 29.1, IL-18 is processed by the protease IL-1b-converting enzyme (ICE, caspase-1, Ghayur et al., 1997; Gu et al., 1997). Caspase-1 also cleaves several protein kinase C isoforms indicating

IL-18 may also be processed by stimuli other than caspase-1. FasL stimulates macrophages to release mature IL-1b accompanying an apoptotic response in a caspase-1-independent manner (Miwa et al., 1998). Likewise, mature IL-18 is secreted from Fas-expressing macrophages on stimulation with FasL, while secretion is inhibited by general caspase-inhibitors (Tsutsui et al., 1999). Macrophages from caspase-1-deficient mice similarly secrete IL-18, whereas the same macrophages do not secrete IL-18 after stimulation

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with LPS. Caspase-1 gene targeting also does not completely impair IL-1b release, and some of the metalloproteinases (MMP-2, -3, -4) cleave pro-IL-1b into a biologically active form (Fantuzzi et al., 1997; Schönbeck et al., 1998). Proteinase 3, a serine protease stored in the granules of neutrophils and macrophages, also participates in the processing of IL-1b and TNF (Coeshott et al., 1999). Recombinant proteinase 3 has the capacity to cleave pro-IL-18 (Fantuzzi and Dinarello, 1999), but at a different site from caspase-1. Viral infection causes macrophages to secrete biologically active IL-18 dependent on the activity of caspase-1 and caspase-3 (Pirhonen et al., 1999). Nitric oxide, a biologically active gas, essential for clearance of intracellular bacteria, including Listeria monocytogenes, directly inhibits caspase-1 to prevent IL-1b and IL-18 secretion (Kim et al., 1998).

Rapid release of IL-18 in microvesicles Recently, a mechanism for rapid release of the leaderless cytokine IL-1b was proposed (MacKenzie et al., 2001). P2X7 receptor, an ion channel, activated by the presence of extracellular ATP, is involved with rapid microvesicle shedding following stimulation. Bioactive IL-1b is released from monocytes by exogenously added ATP after priming by LPS (Humphreys and Dubyak, 1998; Grahames et al., 1999). Addition of ATP also remarkably enhances secretion of mature IL-18 from monocytes. ATP-mediated release of IL-18 also requires the functional P2X(7) receptor, but not caspase-1 (Perregaux et al., 2000; Mehta et al., 2001).

Receptor-mediated release of IL-18 Other receptors may also be involved. Bioactive IL-18 is secreted from epithelial cells by stimulation of the neutrophil proteinase 3 together with LPS (Sugawara et al., 2001). Pretreatment of these cells with IFN- is required and the induction is not dependent on caspase-1. It is probable that the induction signal is transduced by protease-activated receptors (PARs), likely involving and regulated by increases in intracellular calcium concentration. Calcium may also play a role in the processing of IL-18 in macrophages, because calcium channel blockers inhibit IL-18 secretion from dendritic cells (DCs) stimulated by antigen specific T cells (Gardella et al., 2000). The IL-18 in the

cytosol of DCs exists in organelles co-fractionating with endolysosomes. Following stimulation of DC with T lymphocytes or calcium ionophore, the particulated IL-18 precursor disappears from the cytosol and appears in the culture medium. However, the IL-18 released by exocytosis is still in its precursor form.

Cellular expression of IL-18 IL-18 mRNA is expressed in a variety of organs and tissues including pancreas, skeletal muscle, skin and kidney, where conversely, expression of IL-18R/ IL-1Rrp mRNA is very low (Ushio et al., 1996). Activated macrophages were first shown to express high levels of IL-18, but it is ubiquitously produced by a wide range of cell types, both immune and nonimmune (Nakanishi et al., 2001a). Furthermore, both IL-18 and IL-18R are expressed in lesions of tissues in various disease states. Cells that express IL-18 are listed in Table 29.2.

Expression in the skin and gut Epidermal keratinocytes as well as Langerhans cells are abundant sources of IL-18 in the skin (Stoll et al., 1997). Other epithelial cells including corneal epithelial cells and airway epithelial cells also express IL-18 (Cameron et al., 1999; Burbach et al., 2001). Cutaneous IL-18 expression remains a great mystery since which role it plays in the skin and how it is processed is largely unknown. IL-18 expression and processing is augmented by contact allergens (Stoll et al., 1997, 1998; Xu et al., 1998a; Naik et al., 1999; Konishi et al., 2002). Although keratinocytes usually lack active caspase-1, they may be activated by contact allergens to process IL-18 just as they process IL-1b (Zepter et al., 1997). On the other hand, cytokines including TABLE 29.2 Cells that express IL-18 Macrophages (peritoneal macrophages, Kupffer cells, alveolar macrophages) Keratinocytes (augmented with TNBS) Osteoblast (suppress osteoclast) Intestine epithelial cells Synovial cells (RA) Chondrocytes Adrenal cortex cells (respond to cold stress) Mammary gland

THE CYTOKINES AND CHEMOKINES

GENOMIC STRUCTURE , REGULATION , AND CHROMOSOMAL LOCALIZATION OF IL - 18

IL-1b, TNF and IFN fail to induce processing of pro-IL-18 in keratinocytes (Companjen et al., 2000; Mee et al., 2000). Thus, regulatory mechanisms for processing of pro-IL-18 to the active form as well as that for caspase-1 activation in keratinocytes are complex. The possibility that IL-18 plays a role in pathogenesis of skin diseases has been suggested. Skin-specific caspase-1 transgenic mice manifest chronic dermatitis accompanied by high levels of IL18 and IgE, but not IL-1 in the circulation; thus, IL-18 may also be involved in other skin diseases (Yamanaka et al., 2000; Tanaka et al., 2001). IL-18, together with IL-12, stimulates the expression of IFN in murine dendritic epidermal T cells, leading to production of potentially harmful molecules, including nitric oxide and reactive oxygen intermediates (Sugaya et al., 1999). IL-18 expression is increased in the lesions of psoriasis and may be involved in the pathogenesis associated with activated TH1 responses (Mee et al., 2000; Ohta et al., 2001). Expression of IL-18 is augmented in common skin tumors such as squamous cell carcinoma and melanoma, which may provide an important clue for the understanding of pathogenesis of skin tumors (Park et al., 2001). IL-18 has also been suggested to play a role in the repair of cutaneous wounds (Kampfer et al., 1999, 2000). Mature IL-18 and active caspase-1 are detected in the active lesions of patients with Crohn’s disease, suggesting the possible involvement of IL-18 in this disease (Monteleone et al., 1999; Pizarro et al., 1999). Although IL-18 is expressed in intestinal epithelial cells of the villi (Takeuchi et al., 1997), the major cells expressing IL-18 in the lesions are macrophages (Monteleone et al., 1999; Pizarro et al., 1999; Kanai et al., 2000, 2001). Combined administration of IL-12 and IL-18 to mice induces severe colitis as well as pulmonary edema and liver injury (Carson et al., 2000; Chikano et al., 2000; Nakamura et al., 2000).

Expression in endocrine organs Pituitary gland and adrenal cortex both express IL-18 (Conti et al., 1996). Levels of IL-18 mRNA are elevated by cold stress or by adrenocorticotropic hormone treatment in the zona reticularis and fasciculata of the adrenal gland where glucocorticoid is produced (Conti et al., 2000). Tissue-specific usage of unique

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promoter regions has been suggested, because the size and the 5’-end of IL-18 mRNA in adrenal cortex and immune cells are different (Sugama et al., 2000). Further characterization of the IL-18 expressing cells and analysis of the role of IL-18 in these endocrine organs are awaited.

Expression in bone and cartilage IL-18 is produced by osteoblastic cells and suppresses osteoclast formation (Udagawa et al., 1997; Horwood et al., 1998, 2001). IL-18 induces osteoprotegerin, which also suppresses osteoclast survival in response to RANK ligand (Makiishi-Shimobayashi et al., 2001). Expression of IL-18 in bone marrow hematopoietic cells is induced by M-CSF, but the significance of this is as yet unclear (Cappellen et al., 2002). In addition, IL-1b induces IL-18 mRNA in chondrocytes, and mature IL-18 is secreted following stimulation with IL-1b (Olee et al., 1999). Both active caspase-1 and IL-18 are detected in osteoarthritic cartilage (Saha et al., 1999). In the synovial tissues of joints involved with osteoarthritis, the IL-18 producing cells are localized below the synovium (Tanaka et al., 2001). Thus IL-18 may affect the balance of positive and negative bone metabolism, and may also regulate chondrocyte function and contribute to cartilage degradation.

Expression in the CNS and other sites Microglial cells also produce and respond to IL-18, although the role of IL-18 in these cells is not clear (Conti et al., 1999; Prinz and Hanisch, 1999; Suk et al., 2001; Jander et al., 2002). IL-18 expression in microglia is induced in a distinct manner from that of IL-1b in the brain of animals causing focal brain ischemia (Jander et al., 2002; Yatsiv et al., 2002) and is detected in myasthenia gravis, autoimmune encephalitis, as well as acute inflammatory demyelinating polyneuropathy. IL-18-producing cells in nonimmune tissues preferentially reside at those sites where cells turn over rapidly or in tissues that include secretory cells. However, there is little in common between these cells and immune cells producing IL-18. Moreover, the mechanism for processing of IL-18 in these cells is unclear.

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IL-18 RECEPTOR AND IL-18 RECEPTOR BINDING PROTEIN Chromosome, gene and subunits IL-18R is a member of the IL-1R/TLR (Toll-like receptor) family (Torigoe et al., 1997; Dale and Nicklin, 1999; Bowie and O’Neill, 2000; Daun and Fenton, 2000). Similar to IL-1R, the IL-18 receptor is composed of two subunits, IL-18 formerly designated as an IL-1Rrp (Parnet et al., 1996; Torigoe et al., 1997) and IL-18Rb which was initially called IL-1RacPL (Born et al., 1998). Genes for IL-18R map to chromosomes distinct from those encoding the IL-18 ligand. Human IL-18R maps to chromosome 2q13–21, where genes encoding IL-1, IL-1b, IL-1R IL-1R, and IL-1 receptors (type 1, a binding component, and type 2, a decoy receptor, IL-1RAcP), and T1/ST2 are located (Nolan et al., 1998; Aizawa et al., 1999; Dale and Nicklin, 1999). Human IL-18 maps to chromosome 11q22. The gene for IL-18 binding protein (IL-18 BP) is located on chromosome 11q13 (Novick et al., 1999; Aizawa et al., 1999).

Murine receptor localization and homology Murine IL-18R is homologous to human IL-18R (65%), murine IL-18Rb (31%), murine T1/ST2 (30%), and murine IL-1R type I (27%) in overall amino acid sequence. The cytoplasmic domains have slightly greater sequence homology (36–44%) than the extracellular portions (20–27%) (Parnet, P. et al., 1996). Human IL-18Rb shows 25% homology to human IL-1R type I, 27% to IL-1R type II, and 26% to IL-18R (Born et al., 1998). Murine IL-18 binding protein (BP) is 65.7% identical to human IL-18BP at the amino acid level. The Ig domain of IL-18BP is homologous to the third Ig domain of IL-1R type II. IL-18 BP is significantly homologous to putative proteins encoded by several poxviruses (Novick et al., 1999).

Receptor composition IL-18R consists of a ligand binding subunit (IL-18R/ IL-1Rrp) and a signaling component (IL-18Rb/IL1RAcPL) (Born et al., 2000; Debets et al., 2000). Splenocytes from IL-18R-deficient mice do not bind IL-18 ligand, hence NFB and c-Jun-terminal kinase

are not activated, and they do not produce IFN or up-regulate NK cell functions in response to IL-18 (Hoshino et al., 1999a). Thus, IL-18R is needed for the binding and signal transduction of IL-18. However, IL-18R alone does not bind to IL-18 directly, and co-expression of IL-18R/IL-1Rrp and IL-18Rb/ AcPL is required for IL-18 responsiveness (Born et al., 1998, 2000). It was further demonstrated by cotransfection experiments that the expression of both IL-18R and IL-18Rb results in high affinity binding to IL-18 and enables the full biological activity of IL-18, while neither component alone does (Debets et al., 2001). Studies using IL-18R-deficient mice and neutralizing anti-IL-18R antibodies have also revealed requirements for both the IL-18R and IL18Rb to reveal the biologic activity of IL-18 (Hoshino et al., 1999a; Kim et al., 2001). A novel IL-1-related molecule, IL-1H or IL-1F9 has 36% homology to the IL-1 receptor antagonist (IL-1R) and may be another ligand for IL-18R (Pan et al., 2001). Whether IL-1H/IL-1F9 acts on the IL-18R remain to be clarified. The novel IL-1H4/IL-1F7 interacts with the IL18R and may bind other factors including the IL-18BP to mediate biologic effects (Sims et al., 2001; Kumar et al., 2002). Human papilloma virus 16, an oncogenic virus causing human cervical cancer, has oncoproteins homologous to the IL-18 binding protein. These competitively inhibit the binding of IL-18 to IL-18R, leading to inhibition of IL-18-induced IFN production by human NK cells (Lee et al., 2001).

Receptor expression and regulation Analysis of the promoter regions of IL-18R and b genes are not yet sufficiently detailed. In addition the inducing factors and cell types expressing IL-18R on inflammatory cells are not suitably clarified as well as those in non-immune tissues. IL-18 is strongly expressed at such sites and its role at those sites remains largely uninvestigated. IL-18R/IL-1Rrp mRNA is constitutively expressed in various organs of mice including thymus, spleen, liver, lung, intestine, colon, placenta, prostate and heart, but not in the brain, kidney, skeletal muscle and pancreas (Parnet et al., 1996). The expression pattern of IL-18Rb/AcPL is highly similar to that of IL-18R/IL-1Rrp which is expressed in the lung, spleen, leukocytes, and colon, but not in the heart, brain, kidney, and muscle (Born

THE CYTOKINES AND CHEMOKINES

IL - 18 RECEPTOR AND IL - 18 RECEPTOR BINDING PROTEIN

et al., 1998). It remains to be determined which cell types express IL-18R in the tissues. Weak expression of IL-18R/IL-1Rrp mRNA is also observed in the testis and ovary. Expression of IL-18R in the ovary is upregulated by hormones (Tsuji et al., 2001).

Regulatory mechanisms for IL-18R expression on T cells This has been investigated in association with the differentiation of TH1 type cells. Freshly isolated T cells or naive CD4 T cells express marginal amounts of IL-18R. However, IL-18R is highly expressed on differentiated TH1 cells stimulated with IL-12 or with IL-12 plus antigens, but not on TH2 cells. Therefore, IL-18R can be a selective surface marker of TH1 cells (Xu et al., 1998; Yoshimoto et al., 1998; Tominaga et al., 2000; Chan et al., 2001; Nakanishi et al., 2001a; Shao et al., 2001; Smeltz et al., 2001). T cells stimulated with IL-12 have the capacity to specifically bind IL-18. The importance of IL-12 in the regulation of expression of IL-18R was confirmed in mutant cells deficient in STAT4, which is required for IL-12 signaling (Lawless et al., 2000; Nakahira et al., 2001). Expression of IL-18R in CD4() T cells stimulated with antigen is downregulated by the cytokine IL-4 (Smeltz et al., 2001). IL-4deficient T cells or the downstream IL-4 signalling molecule STAT6 (−/−) T cells express higher levels of IL-18R following TCR stimulation. Thus IL-18R expression may also be an indicator of TH1-predominant tissue injury (Rothe et al., 1997b; Ohkusu et al., 2000). However, recent investigations have revealed that IL-18 can induce production of TH2 type cytokines including IL-4 and IL-13 in T and NK cells (Hoshino et al., 1999c; Yoshimoto et al., 2000). In addition, IL-18 stimulates production of TH2 type cytokines in basophils (Yoshimoto et al., 1999). This suggests that cells producing TH2 type cytokines also express IL-18R and respond to it, although not as strongly as is induced by IL-12 in T cells. IFN and IL-12 are both capable of inducing IL-18R and IL-18Rb in human T cells and NK cells (Sareneva et al., 2000). In addition, IFN remarkedly up-regulates the expression of MyD88. Therefore, type I IFN released following viral infection may be involved in host defense through direct inhibition of viral replication and through activation of NK cells by augmenting IL-18R expression. However, NK cells

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constitutively express IL-18R, as well as IL-12Rb, and both IL-18 and IL-12 augment cytolytic activity independently of each other (Hyodo et al., 1999). It is not known whether both of the IL-18 receptor chains, IL-18R and IL-18Rb, are expressed simultaneously on TH1 cells and NK cells.

Expression of receptor on other cell types Various other types of cells, immune and nonimmune, have been shown to express IL-18R. Hematopoietic cell lines (Nakamura et al., 2000), and fibroblast-like synovial cells (Moller et al., 2002) express IL-18R. Keratinocytes (Koizumi et al., 2001; Mee et al., 2000), neutrophils (Leung et al., 2001; Wyman et al., 2002), and eosinophils (Wang et al., 2001) also express IL-18, but the biological meaning of IL18R expression on these cells is unclear.

Release of soluble receptor (IL-18 binding protein: IL-18BP) A soluble IL-18 receptor (an IL-18 binding protein: IL-18BP) has been purified from human urine (Novick et al., 1999) and from the sera of mice sequentially administered Propionibacterium acnes (P. acnes) and LPS (Aizawa et al., 1999). IL-18BP (38 Kd protein) has six isoforms deriving from differential splicing of the same mRNA (Kim et al., 2000). IL-18BP belongs to the immunoglobulin super family. No exon encoding a transmembrane domain was found in an 8.3 Kb genomic sequence. There seems to be no species specificity when comparing human and murine IL-18BP, as human IL-18BP can also bind and inactivate murine IL-18. IL-18BP binds IL-18 with high affinity, suggesting that IL-18BP may physiologically play a role of a soluble decoy receptor, functionally similar to the membrane associated IL-1R type II. IL18BP abolishes induction of IFN production by IL-18 both in vitro and in vivo. Unlike the soluble decoy receptor for IL-1, IL-18BP has little homology with IL18 or IL-18R (Dinarello, 1996; Aizawa et al., 1999; Novick et al., 1999; Xiang and Moss, 2001). The Ig domain of IL-18BP is homologous to the third Ig domain of the decoy receptor of IL-1, IL-1R type II (Novick et al., 1999). Recently the entire mouse locus encoding all the IL-1 family members has been identified (Taylor et al., 2002; Nicklin et al., 2002).

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Homology with poxvirus proteins Interestingly, IL-18 BP is highly homologous to proteins derived from several poxviruses (Novick et al., 1999; Xiang and Moss, 1999a,b; Born et al., 2000; Smith et al., 2000; Calderara et al., 2001). In fact, Molluscum contagiosum virus, a common human poxvirus, encodes a family of proteins with homology to IL-18BP (Xiang and Moss, 1999). The proteins in this family have high affinity binding activity to IL-18 and have the capacity to inhibit both IL-18 binding and IL-18-induced IFN production (Novick et al., 1999; Reznikov et al., 2000). Since IL-18 plays a role in host defense against various types of microbes including viruses, these proteins may act as an important escape mechanism from the host immune system.

(Adachi et al., 1998). Targeted disruption of the MyD88 gene results in ablation of signaling by both IL-1 and IL-18 (Adachi et al., 1998). T cells from mice deficient in the MyD88 gene are defective in proliferative responses as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in IFN production and NK cell activity in response to IL-18 are also abrogated. Furthermore, IL-18-induced activation of NFB and c-Jun N-terminal kinase (JNK) is absent in MyD88-deficient cells. IRAK-deficient mice have an impaired response of their splenocytes to stimulation with IL-18 as well as IL-1 (Kaanakaray et al., 1999; Swantek et al., 2000). Taken together, MyD88-IRAK-TRAF6-NFB signaling seems to be the major signaling pathway for IL-18 as well as for IL-1.

Second signaling pathway of IL-18 Regulation of IL-18BP production Detailed analysis of the regulatory mechanism for IL-18BP production has not been carried out. IL-18BP is constitutively expressed in the spleen, and its gene expression is up-regulated by IFN (Kim et al., 2000; Paulukat et al., 2001). IL-18BP treatment of animals ameliorates colitis induced by dextran sulfate sodium (Sivakumar et al., 2002). It will be of interest to determine whether IL-18BP administered in vivo can regulate IL-18 activity in inflammatory diseases. Recent data in the setting of brain trauma suggests that it may play such a role (Yatsiv et al., 2002).

SIGNAL TRANSDUCTION IL-1R/IL-18R is homologous to the innate immune receptors, Toll-like receptors (TLRs) in their structure (Medzhitov et al., 1998; O’Neil and Dinarello, 2000; Akira et al., 2001). Their cytoplasmic regions contain six distinct domains that are well conserved (O’Neill and Dinarello, 2000). These homologous domains seem to be important for interactions with signal transducing proteins. Ligand binding to IL-18R does not lead to activation of the JAK/STAT signaling pathway, but rather activates IL-1R-associated kinase (IRAK) and TRAF-6 that affect nuclear translocation of NFB (Matsumoto et al., 1997; Robinson et al., 1997; Kojima et al., 1998). MyD88, an adapter molecule in IL-1 signaling, also participates in signaling by IL-18

IL-18 has been suggested to activate the mitogen activated protein kinase (MAPK). This protein tyrosine kinase, as well as the src kinase LCK, is activated in TH1 cells stimulated with IL-18 (Tsuji-Takayama et al., 1997). Since MAPK pathways are involved in cell growth, IL-18 may exert proliferative actions on both T and NK cells (Tomura et al., 1998a). Possible signal transducing pathways are shown in Figure 29.2.

Role of toll-like receptors Most members of the IL-1R/TLR family are involved in response to infections. Some of them participate in innate immunity as pattern recognition receptors for microorganisms (Medzhitov et al., 1998). To date, 10 TLRs have been identified, and biological functions of five of them TLR-2, TLR-4, TLR-5, TLR-6, and TLR-9, have been clarified (Hoshino et al., 1999b; Takeuchi et al., 1999, 2000a,b; Hayashi et al., 2001; Hemmi et al., 2000). All five TLRs share signaling pathways with IL1R/IL-18R. However, only TLR4 has an additional signal transduction pathway other than the MyD88/IRAK pathway, because MyD88-deficient macrophages show delayed activation of NFB only following stimulation with TLR-4-activating ligands, specifically LPS (Kawai et al., 1999). However, MyD88-deficient cells do not translocate NFB to the nucleus following incubation with ligands stimulating the other TLRs (Akira et al., 2001). Recently, a second adapter molecule of the TLR4-mediated NFB-activating pathway

THE CYTOKINES AND CHEMOKINES

IN VITRO ACTIVITIES

(Kunikata et al., 1998; Lauwerys et al., 1998; Nakanishi et al., 2001a), macrophages (Munder et al., 1998; Schindler et al., 2001), and dendritic cells (Fukao et al., 2000; Stober et al., 2001) also produce IFN- in response to IL-12 and IL-18.

IL-18 IL-18Ra (IL-1Rrp)

IL-18Rb (AcP-L)

IRAK

717

MyD88

Synergistic action of IL-12 and IL-18 induces interferon. IRAK

TRAF-6

MAPK MEKK4

IjK

AP-1

NF-kB 1kB

mRNA NF-kB

NF-kB

NF-kB

NF-kB

Target gene

Cell Proliferation

Gene Transcription

FIGURE 29.2 Signal transducing pathways of IL-18. was identified (Fitzgerald et al., 2001; Horng et al., 2001; Henneke and Golenbock, 2001). However, it is noted that NFB is not activated in MyD88-deficient cells after stimulation with IL-18 (Adachi et al., 1998), suggesting the absence of alternative NFB-activating pathways other than the MyD88-mediated one.

IN VITRO ACTIVITIES

The underlying mechanism for the potent synergy of IL-12 and IL-18 in IFN induction has been analyzed. IL-12 augments expression of the functional IL-18 receptor on CD4 T cells (Ahn et al., 1997, Nakanishi et el., 2001a). The functional IL-18 receptor is strongly expressed on TH1 cells but not on TH2 cells (Xu et al., 1998b). There may be several ways by which IL-18 signaling enhances IFN gene expression. The IFN gene promoter has consensus sequences for NFB that can be activated following IL-18 signaling. STAT-4 is essential for IL-12 signaling, the Cyclosporin A sensitive NFAT binding site, and the C3 intronic enhancer region (Xu et al., 1996; Sica et al., 1997) may all be important for enhancing IFN expression. In addition, IL-18 has also been suggested to directly activate another transcription factor, AP-1, required for IFN gene expression (Barbulescu et al., 1998). The marked synergism between IL-12 and IL-18 for IFN induction in T and NK cells might be partly caused by the combination of these distinct IFN-inducing signals acting in turn on cis-acting elements (Matsumoto et al., 1997; Muzio et al., 1997; Robinson et al., 1997; Adachi et al., 1998; Barbulescu et al., 1998; Tsuji-Takayama et al., 1999; Nakahira et al., 2001; Yang et al., 2001).

Induction of IFN production

Other mechanisms for interferon induction

IL-18 was originally discovered as an IFN-inducing factor. However, IL-18 by itself induces only trace amounts of IFN in purified splenic T cells. This is also the case for another IFN-inducing factor, IL-12. Combined stimulation of IL-18 and IL-12 induces remarkably high levels of IFN in these cells (Okamura et al., 1995, 1998a; Ahn et al., 1997; Nakanishi et al., 2001a). This combination induces IFN production in CD4 (TH1) T cells (Kohno et al., 1997), CD8 T cells (Tomura et al., 1998a), and NK cells (Okamura et al., 1995; Hunter et al., 1997; Tomura et al., 1998b; Fehniger et al., 1999). In addition to these major producer cells, B cells activated with anti-CD40 antibody

Participation of TCR in IFN induction in T cells has been considered, but the signals from TCR stimulated by antigens are not required for the induction of IFN following stimulation with the combination of IL-12 and IL-18 (Yang et al., 2001). Cyclosporin A does not inhibit the IFN induction by this combination. A signal pathway including GADDb, p38 MAPK and MEKK4 may be involved in this induction. Another combination of cytokines, IL-10 plus IL-18 synergistically up-regulates IFN production as well as cytotoxicity in NK cells (Cai et al., 1999; Micallef et al., 1999), but the underlying mechanism for this is not clear at present.

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Induction of TH2 cytokines by IL-18 IL-18 induces both TH1 and TH2 cytokines (Hoshino et al., 1999c, 2000, 2001; Yoshimoto et al., 1999; Nakanishi et al., 2001a). IL-18 amplifies production of TH2 cytokines, IL-4 and IL-13, in T cells, NK cells, and basophils in the presence of IL-2 or IL-3, but never in the presence of IL-12 (Hoshino et al., 1999c; Yoshimoto et al., 2000). The genetic background of the animals from which the cells are taken and the presence of pre-existing cytokines such as IL-4 and IL-12 may affect the induction of TH1 and TH2 cytokines (Xu et al., 2000). NKT cells stimulated by cognate antigens also produce IL-4 in the presence of IL-18 (Leite-De-Moraes et al., 2001). IL-18 induces TH2 responses in vivo, as well. When IL-18 is administered or overexpressed in mice, IgE levels are markedly increased dependent on intact IL-4/STAT6 signaling mechanisms (Hoshino et al., 2000;Yoshimoto et al., 2000). When administered with IL-12, it is strongly suppressed (Wild et al., 2000). Thus a dual regulatory role of IL-18 is seen in the production of IgE.

Mechanism for induction of production of TH2 cytokines by IL-18 Macrophages may play important roles in this regard through production of various monokines or cytokines. There are two different types of macrophage that can be defined, likely consequent to different stages of activation or of differentiation. One type of macrophages responds to LPS and produces TNF, IL-6, IL-12, and nitric oxide. These macrophages do not usually express nuclear retinoic acid receptors (RARE) or PPAR, and induce TH1 development through production of IL-12. The other type of macrophage strongly express RARE and PPAR together with surface scavenger receptors such as CD36 and produces only a marginal amount of TNF, IL-6, IL-12, and nitric oxide following stimulation by LPS (Alleva et al., 2002; Sidell et al., 2002). These latter types of macrophage restrictedly express membrane antigens, F4/80, macrosialin, sialoadhesin, C-type lectin, FIZZ1, and Ym1 (Gordon 1999, Raes et al., 2002). IL-4 induces FIZZ1 and Ym1 (Raes et al., 2002), and it also up-regulates CD36 following engagement of PPAR in the presence of lipoxygenase products

(Ricote et al., 2000). There is no evidence that this type of macrophage is actively involved in the induction of TH2 responses, but at least they do not seem to induce TH1 type cytokines and may be involved in the termination of inflammatory reactions. It is of interest that there are two types of macrophages, ‘reductive’ macrophages with high levels of intracellular glutathione and ‘oxidative’ macrophages with reduced concentrations (Murata et al., 2002). The former is induced by IFN and produces augmented nitric oxide and IL-12, and thereby is able to induce TH1 cytokines. In contrast, oxidative macrophages produce high levels of IL-6 and IL-10. Since IL-18 suppresses production of TNF (Sakao et al., 1999; Reddy et al., 2001), it will be of interest to examine the distribution of IL-18R on macrophages and the relationship between IL-18 and macrophage function.

Effect of IL-18 on cytolytic activity IL-18 augments the cytolytic activity of NK cells (Okamura et al., 1995; Tomura et al., 1998b). Mature NK cells constitutively express the IL-18 receptor and can be activated by IL-18 without participation of IL-12, IFN, or IL-2 (Hyodo et al., 1999) although both IL-2 and IL-12 potently synergize with IL-18 (Son et al., 2001). IL-18 may be required for the development of NK cells, because NK cells from IL-18-deficient mice are defective in cytolytic activity (Takeda et al., 1998). IL-18 enhances the killing activity of liver NK1.1 CD4 T cells (NKT cells) against liver lymphocytes independently of a Fas-mediated mechanism (Tsutsui et al., 1997), but in a perforin-dependent manner (Dao et al., 1998). IL-18 directly activates the cytotoxicity of CD8 T cells (Kohyama et al., 1998). IL-18 is involved in the development of both alloreactive cytotoxic T cells (CTL) and allo-specific TC1 cells in mixed lymphocyte culture (Okamoto, et al, 1999). Memory CD8 T cells characterized by CD44 expression proliferate following stimulation with IL18, IL-12 and type 1 IFN, whereas naive CD8 T cells or CD4 T cells do not (Tough et al., 2001). It has not been clarified whether the signal-transducing system needed for IFN production is shared by that for perforin/granzyme in NK, NKT and CD8 T cells (Walker et al., 1999).

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Effect of IL-18 on Fas ligand expression IL-18 strongly induces Fas ligand expression on NK cells (Tsutsui et al., 1996) and on TH1 cells (Dao et al., 1996). The physiological relevance of this is not known, but it may involve activation-induced cell death (AICD) of lymphocytes (Leite-de-Moraes et al., 2001). Since autoreactive or malignant lymphocytes may appear in the population of lymphocytes expanded in inflammatory reactions, it may be necessary to exclude them after pathogens are eliminated. IL-18 may be involved in such a mechanism, but there is no literature describing autoimmune diseases or lymphoma in IL-18 deficient mice. The effect of IL-18 on Fas ligand expression may be associated with its attenuating effect on graft versus host disease (GVHD) (Reddy et al., 2001). Administration of IL-18 to mice with acute GVHD following transplantation of lymphocytes protects them from death and intestinal pathological changes. This is associated with elevated expression of Fas on donor T cells. IL-18 failed to protect mice when Fas-deficient lpr mice are used as donors. Induction of Fas ligand by IL-18 may mediate its antitumor effect on some cancer cells (Hashimoto et al., 1999). On the other hand, IL-18 may be involved in the development of liver pathology induced by LPS (Faggioni et al., 2001). Therefore it is very probable that IL-18 exerts some of its biologic effects through activation of Fas/Fas ligand signaling.

Other actions; induction of chemokines, cytokines and enzymes by IL-18 IL-18 stimulates production of IL-1, IL-1b, IL-13, GM-CSF (Ushio et al., 1996), IL-8, vascular cell adhesion molecule (VCAM)-1 and TNF (Puren et al., 1998a,b, 1999, Mendoza et al., 2001). IL-18 acts on chondrocytes to induce expression of genes such as IL-6, inducible nitric oxide synthase, inducible cyclooxygenase, and stromelysin (Olee et al., 1999). IL-18 induces histidine decarboxylase in various organs when administered to mice (Yamaguchi et al., 2000). Human circulating neutrophils express IL-18R and are activated by IL-18 to express high levels of CD11b and secrete IL-8, IL-1b and TNF (Leung et al., 2001). IL-18 may be involved in bone metabolism because it inhibits osteoclast formation (Horwood et al., 2001).

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PHYSIOLOGICAL AND PATHOLOGICAL ROLE OF IL-18 Knockout mouse phenotypes IL-18-deficient mice develop normal lymphoid organs. Flow cytometric analysis revealed that these mice contain normal numbers of B cells, T cells, and NK cells in the spleen, and show normal development of T cells in the thymus. Although T-cell responses to anti-CD3 mAb in IL-18-deficient mice are equivalent to those in wild-type littermates, NK activity in knockout mice is slightly reduced (Takeda et al., 1998). However, the decreased NK activities in mutant mice are improved by exogenous stimulation with IL-18. IL-18-null mice have impaired TH1 development, although IL-18 is reported not to be a differentiation factor for TH1 by itself (Takeda et al., 1998). This is supported by the fact that TH1 development is more impaired in double knockout mice for IL-12 and IL-18 genes than in IL-12 single knockout mice. Thus, IL-18 is involved in the functional development of NK cells and also in TH1 development. However, genetically resistant mice lacking IL-18 develop TH1 response, demonstrating that IL-18 is not essential for the differentiation of TH1 cells in vivo (Monteforte et al., 2000). IL-18-deficient mice are resistant to endotoxininduced liver injury but are highly susceptible to endotoxin-induced shock (Sakao et al., 1999). As initially reported, IL-18 is a pivotal factor in acute liver injury induced by P. acnes and LPS (Okamura et al., 1995). As expected, IL-18-null mice do not suffer from P. acnes- nor LPS-induced liver injury. However, the mutant mice die more rapidly and more frequently after LPS challenge in association with extraordinarily higher serum levels of TNF, as compared with wildtype mice (Sakao et al., 1999). This situation can be improved by exogenous administration of IL-18 prior to P. acnes treatment. Similar effects of IL-18 were observed in animal models of GVHD (Reddy et al., 2001). High levels of circulating TNF in mice suffering from acute GVHD are reduced following administration of IL-18. These observations suggest that IL-18 plays a crucial role in the regulation of TNF production. Recent studies suggest that the level of IL-18 is critical with higher levels produced in response to LPS

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limiting antibacterial host defense and lower levels enhancing it (Joshi et al., 2002).

Role of IL-18 in host defense against bacteria, protozoa and fungi IL-18 is involved in the defense against both intracellular and extracellular parasites (Nakanishi et al., 2001a). There are several reports on the suppression of intracellular microbes by IL-18. Following infection with Mycobacteria, higher expression of IL-18 as well as IFN is observed in Mycobacteria-resistant strains of mice than in susceptible strains (Kobayashi et al., 1997,1998).Inresistantmice,IL-18,togetherwithIL-12, may enhance the development of TH1 cells. When IL-18-deficient mice were infected with Mycobacteria, they developed marked granulomatous lesions in the lung and spleen (Sugawara et al., 1999). Augmented IL-18 expression was also observed in human macrophages infected with Mycobacteria (Giacomini et al., 2001). Infection with other intracellular parasites, Trypanosoma cruzi (Meyer zum Buschenfelde et al., 1997), Salmonella typhimurium (Mastroeni et al., 1999), Leishmania monocytogenes (Wei et al., 1999; Ohkusu et al., 2000; Neighbors et al., 2001), and Toxoplasma gondii (Cai et al., 2000; Yap et al., 2001) also induces IL-18 and IFN. Treatment of mice with antiIL-18 antibody impaired resistance to infection with Salmonella, resulting in increases in the number of bacteria in the liver and spleen. In contrast, administration of IL-18 to mice infected with a lethal dose of a virulent strain of Salmonella reduces the bacterial number in the tissues, rescuing them from death. On the other hand, macrophages infected with Salmonella exhibited reduced expression of IL-18, suggesting the ability of this pathogen to suppress production of IL-18 (Elhofy and Bost, 1999; John et al., 2000). IL-18 knockout mice are however resistant to Trichinella spiralis infection, expelling the worms rapidly and only developing low levels of encysted larvae in muscle (Helmby and Grencis, 2002). Normal mice treated with IL-18 have worsened disease and slower expulsion. Endogenous IL-18 is also involved in the defense against infection with extracellular parasites, Yersinia enterocolitica (Bohn et al., 1998), and Legionella pneumophila (Brieland et al., 2000). Administration of anti-IL-18 antibody to mice infected with Yersinia

enterocolitica caused a 100- to 1000-fold increase of bacterial counts in the spleen. IL-18 exerts antifungal activity against Cryptococcus neoformans. Peritoneal exudate cells incubated with IL-18, together with IL-12, suppress the growth of C. neoformans (Zhang et al., 1997; Kawakami et al., 2000). The effect is dependent on both IFN production by NK cells and nitric oxide production by macrophages. This suggests that IL-18 plays a role in innate immunity. In fact, administration of IL-18 to mice strongly suppresses the growth of C. neoformans in the brain and lung, resulting in prolongation of survival (Kawakami et al., 1997). Anti-IFN antibody completely abrogates the suppressive effect of IL-18. Susceptibility of AIDS patients to C. neoformans has been suggested to be associated with the reduced response to IL-18 and IL-12 (Brummer, 1999). However, IL-18 may be involved in the promotion of the development of chronic gastrointestinal helminth infection by suppressing IL-13 production (Helmby et al., 2001) in vivo.

Role of IL-18 in viral infection Following infection with the Epstein-Barr virus (Setsuda et al., 1999), influenza A virus, and Sendai virus (Pirhonen et al., 1999), IL-18 expression was elevated, suggesting participation of IL-18 in the defense against viral infection. In fact, a protective effect of IL-18 against infection with the Herpes simplex virus (HSV) (Fujioka et al., 1999), Vaccinia virus (Tanaka-Kataoka et al., 1999), and Encephalomyocarditis virus (Tovey et al., 1999) has been demonstrated in mouse models. Administration of IL-18 to mice before infection of HSV markedly improves the survival of mice. The effect is also observed in athymic nude mice and SCID mice, supporting the notion that IL-18 augments innate immunity. The effect is dependent on IFN and independent of NK cells and NO, but which cells produce the IFN in this setting is unknown. On the other hand, IL-18 increased production of HIV-type 1 by 5–30-fold in chronically infected U1 monocytic cells (Shapiro et al., 1998). IL-18 may activate HIV via production of TNF and/or IL-6, and activation of NFB, but the detailed mechanism is not known. Murine cytomegalovirus is lethal in IL-12-deficient mice but not in IL-18-deficient mice (Pien et al., 2000). Ectromelia and cowpox

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PHYSIOLOGICAL AD PATHOLOGICAL ROLE OF IL - 18

viruses express IL-18 binding protein homologues whose relative affinities are related to the host range of virus (Calderara et al., 2001)

Antitumor activity of IL-18 The IL-18 gene is located in the human chromosomal region 11q22–q23, which is a target for deletion in the development of several solid tumors including carcinomas of the breast, cervix, stomach, ovary, bladder and melanoma (Baysal et al., 2001). However, as of yet, there are no reports about the deletional mutation or polymorphism of IL-18 gene associated with cancer. Enhanced expression and increased levels of circulating IL-18 have been observed in patients with several cancer types (Lissoni et al., 2000; Amo et al., 2001; Kawabata et al., 2001; Park et al., 2001). The augmented IL-18 is correlated with advanced disease and metastases (Lissoni et al., 2000). However, levels of IFN do not parallel those of IL-18 in these cancers. Serum IL-18 levels decreased after surgical resections of cancer (Kawabata et al., 2001). IL-18 levels are determined by ELISA in these studies, and it is necessary to examine whether the detected IL-18 is processed and in the active form. Normal ovarian epithelial cells release detectable levels of processed IL-18 in the culture supernatant, while ovarian carcinoma cells release only unprocessed precursor IL-18 in spite of abundant intracellular pro-IL-18 (Wang et al., 2002). On the other hand, expression of IL-18R in peripheral blood mononuclear cells from cancer patients is low (Kobashi et al., 2001). Thus, although IL-18 is detected in the serum of some cancer patients, it is not known whether IL-18 plays an effective role in the host defense or rather might promote cancer growth.

Animal models of IL-18 antitumor activity IL-18 mediates antitumor effects in several types of transplantable malignant tumors (Table 29.3). The mechanisms and the factors responsible seem to be different in different tumor types. One mechanism involves activation of NK cells or CTLs. Suppression of the growth of Meth A sarcoma cells and CL8-D1 melanoma cells in mice by IL-18 may be due to this mechanism (Micallef et al., 1997a,b; Osaki et al., 1998). It is probable that these cells are activate

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TABLE 29.3 Antitumor actions of IL-18 Type of tumor cells

Cell lines

Mechanisms

Sarcoma Lymphoma Melanoma Lung cancer cell

Meth A EL-4 B16 RWGT2

Breast cancer cell

MDA-231

Myeloma

ARH-77

Melanoma Mammary carcinoma Fibrosarcoma

CL8–D1 SCK

Bladder cancer cell

MBT2

NK and CTL NK and CTL NK and CTL Inhibition of bone metastasis Inhibition of bone metastasis Inhibition of bone metastasis Fas/Fas ligand Inhibition of angiogenesis Inhibition of angiogenesis Inhibition of angiogenesis

T241

through production of IFN. Tumor cells transfected with the IL-18 gene exhibit reduced tumorigenicity and IL-18 elicits immunoprotective effects against parental tumor cells in a partially IFN-dependent manner (Fukumoto et al., 1997; Tan et al., 1998; Heuer et al., 1999, Ju et al., 2001; Nagai et al., 2002).

Antiangiogenesis Second, inhibition of angiogenesis may be involved in antitumor action by IL-18. Using engineered SCK mammary carcinoma cells and MBT2 bladder cancer cells, it has been shown that IL-12 and IL-18 synergistically induce regression of tumor cell growth (Yamanaka et al., 1999; Coughlin et al., 1998; Ju et al., 2000). It has been suggested that the effect could be due to the inhibition of angiogenesis, probably through production of IFN-inducible protein, IP-10 induced by IFN. Intratumoral co-injection of adenoviral vectors expressing IL-18 and IP-10 into tumor nodules derived from J558 myeloma cells cures established tumors in mice (Liu et al., 2002). Depletion of T cells diminished this effect, indicating the critical role of T cells. On the other hand, IL-18 was shown to act as an angiogenic factor in in vitro experiments (Park et al., 2001). This indicates that IL-18 may exert opposite action depending on the presence or absence of IFN. Recently, IL-18 and VEGF have been

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shown to be increased in the serum of patients with systemic lupus erythematosus and to correlate with endostatin levels (Robak et al., 2002).

Fas/Fas ligand signaling A third mechanism for the antitumor activity of IL-18 may rely on Fas/Fas ligand signaling. The antitumor action of IL-18 against CL8-D1 melanoma cells in mice is dependent on Fas/Fas ligand system, while that of IL-12 against the same CL8-D1 cells depends on perforin/granzyme system (Hashimoto et al., 1999). Canine IL-18 may directly cause apoptosis of canine breast cancer cells by activating Fas/Fas ligand signaling (Okano and Yamada, 2000). Finally, IL-18 may suppress bone metastasis of breast cancer cells and lung cancer cells by inhibiting the osteoclastic bone-resorption (Nakata et al., 1999; Iwasaki et al., 2002).

IL-18 may promote tumor growth The possibility that IL-18 helps tumor cells to grow or to metastase (Cho et al., 2000; Vidal-Vanaclocha et al., 2000) has been raised. Deficiency in caspase-1 reduces hepatic metastasis of murine melanoma cells probably by decreased induction of adhesion molecules on hepatic sinusoidal endothelial cells (VidalVanaclocha et al., 2000), and melanoma cells transfected with IL-18 antisense DNA reduce tumor growth with concomitant infiltration with NK cells (Cho et al., 2000). Higher levels of IL-18 administration in such liver tumor models mediate antitumor effects (Vidal-Vanoclocha, personal communication). IL-18 treatment of patients with cancer at several centers began in the spring of 2002, supported by GlaxoSmithKline and Hayashibara Pharmaceuticals.

Pathological role of IL-18 in animal models of disease IL-18 is expressed in various organs and detected in the circulation in association with active stages of autoimmune and inflammatory diseases in animal models. For example, IL-18 is detected in the pancreas of animal models of autoimmune insulindependent diabetes mellitus (IDDM) (Rothe et al., 1997a,b, 1999, 2001; Iwahashi et al., 1998; Yoon et al.,

1998), in the brain of experimental autoimmune encephalomyelitis (EAE; Jander and Stoll, 1998, 2001; Wildbaum et al., 1998; Furlan et al., 1999; Shi et al., 2000), in mice with collagen-induced arthritis joints (Leung et al., 2000; Wei et al., 2001), in myasthenia gravis (Im et al., 2001), in the liver of LPS-induced hepatitis (Tsutsui et al., 2000), in the kidney in the setting of renal injury following ischemia-reperfusion (Daemen et al., 1999; Melnikov et al., 2001), in various tissues of GVHD model animals (Reddy et al., 2001), in the intestine of mice with TNBS-induced colitis and dextran sulfate sodium-induced colitis (Kanai et al., 2001; Sivakumar et al., 2002) and in the lung in the setting of inflammatory reactions of asthma (Kumano et al., 1999). In autoimmune MRL lpr/lpr mice, the IL18Rb chain is overexpressed and the lymphocytes are hyperresponsive to IL-18. This may lead to IFNdependent autoimmune pathology (Neumann et al., 2001). Atherosclerosis (Mallat et al., 2001) and congestive heart failure(Naito et al., 2002) are associated with elevated serum levels of IL-18. It may not be easy to come up with an overarching concept about the pathological role of IL-18 in these animal models. IL-18 is usually situated upstream of the cascade of cytokines involved with tissue injury. In most of the animal models, IL-18, derived mostly from monocyte-derived cells, plays a role in the destructive pathway through induction of IFN and resulting production of harmful free radicals. Blockade of IL-18 by antibody or by IL-18BP ameliorates disease severity in many disease models (Wildbaum et al., 1998; Tsutsui et al., 2000; Im et al., 2001; Mallat et al., 2001; Sivakumar et al., 2002). In contrast, administration of IL-18 promotes collagen-induced arthritis (Leung et al., 2000). However, IL-18 also inducesanti-inflammatoryorcompensatorycytokines such as IL-4 and IL-13, and may not necessarily be involved in the destructive pathway in these models. In fact, administration of IL-18 to NOD mice suppresses the onset of IDDM (Rothe et al., 1999). Conversely, administration of IL-13, which is one of the downstream cytokines induced by IL-18, prevents IDDM (Zaccone et al., 1999). In a bone marrow transplantation model, blockade of IL-18 accelerates acute GVHD symptoms and, in contrast, administration of IL-18 attenuates the disease (Reddy et al., 2001). As for asthma models, the severity of symptoms varies depending on the stage of disease in which blockade

THE CYTOKINES AND CHEMOKINES

PHYSIOLOGICAL AD PATHOLOGICAL ROLE OF IL - 18

of IL-18 is carried out (Wild et al., 2000; Walter et al., 2001; Blease et al., 2001). Blockade of IL-18 at the stage of sensitization by the antigens attenuates eosinophilia and airway hypersensitivity, and at the stage of exposure to the antigens makes it worse. It seems that the factor crucial for injurious reaction is the presence of both IL-12 and IL-18 as complementary factors. In addition, IL-18 may be involved in the feedback suppression of production of destructive molecules.

IL-18 in disease states in humans and diagnostic utility

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TABLE 29.4 Diseases in which IL-18 is detected Disease

Clinical samples

Rheumatoid arthritis

Synovial fluid, synovial cells, serum Serum Lesion Serum Lesion, serum Serum Serum Serum Serum (abnormally high) Serum (abnormally high) Cord blood Serum Keratinocytes Retro-placental serum

Sjogren syndrome Crohns disease GVHD Multiple sclerosis Hemophagocytic syndrome Sepsis Infectious mononucleosis PNP deficiency Multiple tuberculoarteriosis

IL-18 is detectable in a variety of clinical samples, some of which are correlated with disease severity, making it potentially a useful diagnostic marker (Table 31.4). For example, it is observed in the blood of patients with hemophagocytosis (Takada et al., 1999), systemic lupus erythematosus (Wong et al., 2000a,b),pulmonarysarcoidosis(Shigeharaetal.,2000, 2001), primary biliary cirrhosis (Yamano et al., 2000), minimal-change nephrotic syndrome (Matsumoto and Kanmatsuse, 2001), atopic dermatitis (Tanaka et al., 2001), developing IDDM (Nicoletti et al., 2001a), and Grave’s disease (Miyauchi et al., 2000), in the oral

PVL Diabetes Dermatitis Abruptio placenta delivery

mucosa of patients with primary Sjögren’s syndrome (Kolkowski et al., 1999), in the cartilage of patients with osteoarthritis (Saha et al., 1999), in the synovial fluid of patients with rheumatoid arthritis (Yamamura et al., 2001), in the cerebrospinal fluid of patients with meningoencephalitis (Fassbender et al., 1999), in the

"Compensatory Pathway" (Th2 dominant)

"Destructive Pathway" (Th1 dominant)

IL-18

IL-18



IL-12 Macrophage NO,ROI

IL-3

Macrophage

Basophil Mast Cell

IL-2

IL-2 T cell

NK cell

NK cell

T cell

Fas L Perforin IFN-c

PGE 2

IL-4, 13 Histamine

IL-13

IL-4, 10, 13

NO, ROI, TNF

Wound healing

Allergy

Tissue injury

Antimicrobial

FIGURE 29.3 A possible working hypothesis on the roles of IL-18 in inflammatory responses. THE CYTOKINES AND CHEMOKINES

724

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demyelinating lesions of multiple sclerosis patients (Balashov et al., 1999; Nicoletti et al., 2001b), in the lesions of leprosy patients (Garcia et al., 1999), in the intestinal mucosa of patients with Crohn’s disease (Monteleone et al., 1999; Pizarro et al., 1999), in the serum of patients with multiple sclerosis (Losy and Niezgoda, 2001; Karni et al., 2002), in the serum of patients with myasthenia gravis (Jander and Stoll, 2002), and in the bronchoalveolar lavage fluid of septic patients (Mathiak et al., 2001). Extremely high levels of IL-18 levels are detected in the serum of patients with Still’s disease (Kawashima et al., 2001; Kawaguchi et al., 2001), and high levels in the blood and cerebrospinal fluid of patients suffering from inflammatory demyelinating polyneuropathy (Jander and Stoll, 2001) are detected. Cord blood does not usually contain significant levels of cytokines, however, high levels of IL-18 are detected in great frequency in the cord blood of neonates suffering from paraventricular leucomalacia, many of which subsequently manifest cerebral palsy (Minagawa et al., 2002). Measuring cord blood levels of IL-18 may be of prognostic help in this disease. The pathological significance of these observations, in relationship to the fundamental functional role of IL-18, remains to be clarified. A possible working hypothesis for the multifold actions of IL-18 is summarized in Figure 29.3.

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Yatsiv, I., Morganti-Kossmann, M.C., Perez, D. et al. (2002). Elevated intracranial IL-18 in humans and mice after traumatic brain injury and evidence of neuroprotective effects of IL-18-binding protein after experimental closed head injury. J. Cereb. Blood Flow Metab. 22: 971–978. Yin, K.J., Chen, S.D., Lee, J.M. et al. (2002). ATM gene regulates oxygen-glucose deprivation-induced nuclear factorkappaB DNA-binding activity and downstream apoptotic cascade in mouse cerebrovascular endothelial cells. Stroke 33: 2471–2477. Yoon, J.W., Jun, H.S. and Santamaria, P. (1998). Cellular and molecular mechanisms for the initiation and progression of beta cell destruction resulting from the collaboration between macrophages and T cells. Autoimmunity 27: 109–122. Yoshimoto, T., Takeda, K., Tanaka, T. et al. (1998). IL-12 upregulates IL-18 receptor expression on T cells, TH1 cells, and B cells: Synergism with IL-18 for IFN- production. J. Immunol. 161: 3400–3407. Yoshimoto, T., Tsutsui, H., Tominaga, K. et al. (1999). IL-18, although anti-allergic when administered with IL-12,

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stimulates IL-4 and histamie release by basophils. Proc. Natl. Acad. Sci. USA. 96: 13962–13966. Yoshimoto, T., Mizutani, H., Tsutsui, H. et al. (2000). IL-18 induction of IgE: Dependence on CD4 T cells, IL-4 and STAT6. Nat. Immunol. 1: 132–137. Zaccone, P., Phillips, J., Conget, I. et al. (1999). Interleukin-13 prevents autoimmune diabetes in NOD mice. Diabetes 48: 1522–1528. Zepter, K., Haffner, A., Soohoo, L.F. et al. (1997). Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli. J. Immunol. 159: 6203–6208. Zhang, T., Kawakami, K., Qureshi, M.H. et al. (1997). Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells. Infect. Immun. 65: 3594–3599. Zhou, X.Y., Wang, X., Wang, H. et al. (2002). Ku affects the ATM-dependent S phase checkpoint following ionizing radiation. Oncogene 21: 6377–6381.

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30 Interleukin 1 Family [F5 to F10] Sanjay Kumar GlaxoSmithKline, King of Prussia, PA, USA

Perseverance is a great element of success. If you only knock long enough and loud enough at the gate, you are sure to wake up somebody. Henry Wadsworth Longfellow

INTRODUCTION There are four members in the interleukin-1 (IL-1) family of cytokines. These are the signaling agonists IL-1a and b, the IL-1 receptor antagonist (IL-1RA), and, despite low sequence homology but high structural similarity, IL-18 is now widely accepted as the fourth member of the IL-1 family (Dinarello, 1994; Bazan et al., 1996). IL-1a and b are proinflammatory cytokines that are involved in a variety of biological activities (Dinarello, 1996). IL-1 stimulates innate host immune and repair responses to various forms of insults or infections. IL-1 is known to induce production of adhesion molecules, other cytokines and chemokines, growth factors, and metalloproteinases. IL-1-mediated responses include an increase in acutephase proteins, fever, vasodilation and bone resorption, among others (Dinarello, 1996). IL-1a and b bind to the type I IL-1 receptor (IL-1RI) which forms a heterodimer with an accessory receptor, IL-1Racp (O’Neill and Greene, 1998). The ligand The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

binding domain of IL-1RI is composed of three immunoglobulin-like domains and the cytoplasmic domain contains a toll-like region thought to be involved in signaling (Heguy et al., 1992). The IL-1R complex initiates signal transduction events through TRAF6 and IRAK leading to activation of NFjB and AP1, which in turn induces genes involved in immune and inflammatory responses (O’Neill and Greene, 1998; Auron, 1998). A decoy receptor, IL-1RII, has also been identified that functions to sequester IL-1a and b and prevent signaling (McMahan et al., 1991). The naturally occurring antagonist, IL-1RA, binds to the same receptor complex but is unable to initiate signaling, and thus effectively antagonizes the effect of IL-1s (Eisenberg et al., 1990). IL-18 binds to a distinct high-affinity receptor complex composed of IL-18R and an accessory protein, IL-18Racp (Torigoe et al., 1998; Born et al., 1998). Upon binding to its cognate receptor, IL-18 initiates signaling leading to the production of several cytokines such as IFNc, GM-CSF and IL-13 from

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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lymphoid cells. It also enhances Fas-dependent cellmediated immune responses including cytotoxicity mediated by T and NK cells. With dendritic, T and NK cells, it enhances specific T-cell effectors. This results in a cell-mediated immune response that is involved in cancer immunity and delayed-type hypersensitivity (Okamura et al., 1995; Tanaka et al., 2000). A soluble IL-18 binding protein has also been identified that binds to IL-18 and functions to sequester IL-18 and possibly other cytokines, although its biology is not yet completely understood (Dinarello, 2000; Xiang and Moss, 2001). Given the important roles that IL-1 family cytokines play in both physiological and pathological conditions, it was expected that additional IL-1 family members may reside in the human genome especially because of the existence of a large number of orphan IL-1R-related proteins (IL-1Rrp). Advances in EST sequencing, completion of human genome sequencing and subsequent analysis has led to the identification of several novel genes. In the past two years, six genes encoding novel IL-1 homologs have been identified by several groups. These are IL-1HY1, FIL1d, e, , , IL-1H1, H2, H3, H4, IL-1RP1, RP2, RP3, IL-1H, IL-1L1, IL-1d, e, IL-1Hy2 and FKSG75 (Mulero et al., 1999; Smith et al., 2000; Kumar et al., 2000; Barton et al., 2000; Pan et al., 2001; Lin et al., 2001; Debets et al., 2001). Because similar genes were identified by different investigators, each homolog was given multiple names. Recently, a unified nomenclature has been adopted (Sims et al., 2001). Acknowledging the four IL-1 family members already identified, the six novel IL-1 homologs have been renamed as IL-1 family members, IL-1F5–IL-1F10 in chronological order. Table 30.1 provides a listing of all novel IL-1 homologs including gene symbols, protein, various splice variants and their respective GenBank accession numbers.

CHROMOSOMAL LOCALIZATION AND GENE STRUCTURE All known members of the IL-1 family reside on chromosome 2 with the exception of IL-18, which resides on chromosome 11. A combination of techniques

including cytogenetic analysis, radiation hybrid mapping, human genome sequencing as well as targeted sequencing has resulted in an almost complete reference sequence, map position and exon–intron boundaries for all members of the IL-1 family (Nicklin et al., 1994; Nothwang et al., 1997; Lander et al., 2001; Nicklin et al., 2001). A schematic representation of the order of various genes along chromosome 2 is shown in Figure 30.1. Such analysis indicates that all six novel homologs reside in the same cluster as IL1A, IL1B and IL1RN on human chromosome 2 within an approximately 350 kb span separating IL1A and IL1RN. IL1A is closest to the centromere whereas IL1RN resides closer to the telomere on the q arm of chromosome 2. IL1A, IL1B and IL1F8 are transcribed towards the centromere whereas the rest of the genes are transcribed towards the telomere. The most probable gene structure for all novel homologs and IL-1A is shown in Figure 30.2. There does not appear to be any new member of the IL-1 family in this cluster but several genes are transcribed into two or more splice variants. The gene organization of the various homologs is quite similar with minor differences. As shown in the sequence alignment (Figure 30.3), IL-1 family members exhibit significant homology in the C-terminus half of the protein. The N-terminal regions, which often contain the prodomain are less conserved. Most of the novel homologs, with the exception of IL-1F7, do not contain a prodomain and correspond to mature IL-1. A majority of C-terminal halves of each gene is conserved and encoded by the last three exons. For many of these genes, several variants have been identified suggesting potential use of alternate promoters and exon splicing. A dendrogram of various homologs depicting their evolutionary relationship is shown in Fig. 30.4. IL-18 appears to be the most distant relative of the IL-1 family members, suggesting a very early split. The rest of the members fall into distinct subgroups. IL-1F5 and IL-1F10 are much closer to IL-1RA but distinct from IL-1A and B. IL-1F7 appears to have diverged from a common precursor that gave rise to IL-1A and B. IL-1F6, F8 and F9 form a separate group. The evolutionary relationship is also reflected in the arrangement of these genes along the chromosome such that each subgroup is clustered together (see Figure 30.1). Such clustering of the IL-1

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TABLE 30.1 Nomenclature of novel IL-1 homologs Human gene

Mouse gene

Protein

Accession No.

Old names/ Accession (Ref.)

Variant/ Accession (Ref.)

IL1F5a

Il1f5

IL-1F5

AF186094 (Mulero et al., 1999)

IL-1Hy1/AF186094 (Mulero et al., 1999), FIL1d/AF201830 (Smith et al., 2000), IL-1H3/AF200493 (Kumar et al., 2000), IL-1RP3 (Busfield et al., 2000), IL-1L1 AJ242737–38 (Barton et al., 2000), IL-1d/AF230377 (Debets et al., 2001)

IL-1F5b/AJ242738 (Barton et al., 2000)

IL1F6

Il1f6

IL-1F6

AF201831 (Smith et al., 2000)

FIL1e/AF201831 (Smith et al., 2000)

IL1F7

Il1f7b

IL-1F7

AF201832 Smith, 2000 #68]

FIL1/AF201832 (Smith et al., 2000), IL-1 H4 (Kumar et al., 2000), IL-1RP1 (Busfield et al., 2000), IL-1H AF251118–20 (Pan et al., 2001)

IL-1F7b/AF200496 (Kumar et al., 2000)

IL-1F7c/AF251120 (Pan et al., 2001) IL1F8

Il1f8b

IL-1F8

AF201833 Smith, 2000 #68]

FIL1g/AF201833 (Smith et al., 2000), IL-1 H2/AF200494 (Kumar et al., 2000),

IL1F9

Il1f9

IL-1F9

AF200492 (Kumar et al., 2000)

IL-1 H1 (Kumar et al., 2000), IL-1RP2 (Busfield et al., 2000), IL-1e/AF206696 (Debets et al., 2001)

IL1F10

Il1f10b

IL-1F10

AF334755 (Lin et al., 2001)

IL-1Hy2 (Lin et al., 2001), FKSG75 (unpublished)

IL-1F8b/AF200494 (Kumar et al., 2000)

IL-1F10b AY026753 (unpublished)

a

In this nomenclature IL-1F1, IL-1F2, IL-1F3, and IL-1F4 are reserved for IL-1a, IL-1b, IL-1RA and IL-18, respectively. A corresponding murine ortholog is yet to be identified.

b

family members most likely suggests a stepwise duplication of a common primordial gene leading to several homologs. While these homologs are expressed at low levels in various cells, many are strongly induced in response

to a variety of agents such as LPS, PMA or other cytokines, suggesting tight regulation of expression. However, no regulatory sequences including promoters, enhancers or suppressors for any of these genes have yet been identified.

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FIGURE 30.1 A schematic representation of the arrangement of various IL-1 family genes on human chromosome 2. Solid boxes represent various genes along the chromosome. The arrow represents the direction of transcription of different genes towards centromere or telomere. The physical distance in kb is shown. The sequence of the IL-1 cluster for this analysis was derived from GenBank entries NT_01936 and BN000002.

PROTEIN SEQUENCE AND STRUCTURE The three members of the IL-1 family, IL-1a, IL-1b and IL-1RA share 25% identity with each other but bind to a common signaling receptor (Table 30.2). X-ray crystallographic analysis of IL-1b and IL-1RA revealed remarkable structural similarity. Structural comparison to IL-18, which shares only approximately 18% identity with IL-1s, showed striking similarity that led to the acceptance of IL-18 as a distant member of the IL-1 family even though it binds to a distinct receptor (Bazan et al., 1996). The crystal structure of IL-1b consists of 12 b strands packed in a single domain known as b-trefoil. The limited sequence homology between the IL-1 family members has been suggested as resulting from the extensive use of the backbone elements and relatively

FIGURE 30.2 A schematic representation of exon– intron structure of IL-1A and various novel IL-1 homologs. Introns are represented by lines whereas boxes represent exons. Shaded parts of exons indicate untranslated regions. The numbers above the exons indicate the approximate number of amino acid residues encoded by the exons, whereas the numbers below represent the exon numbers starting from 5 end. The choice of exon splicing and resulting mRNA is shown on the right with their corresponding GenBank accession numbers. few invariable residues to maintain the b-trefoil structure and receptor recognition. In fact, the fibroblast growth factor family also exhibits a similar b-barrel structure but they bind distinct receptors and have functions different from those of IL-1s (Zhu et al., 1991). The structures of IL-1F5, F7 and F8 have been modeled using the X-ray crystal structure of IL-1b and IL-1RA (Smith et al., 2000). The structures of most novel IL-1 members could be easily modeled with minimum energy violation and be superimposed on to the structures of IL-1b and IL-1RA (Plate 30.5 (see plate section)). In addition, NMR data have also been obtained for IL-1F5 and are consistent with the overall b-barrel fold of IL-1RA (Barton et al., 2000).

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FIGURE 30.3 Amino acid sequence alignment of various members of the IL-1 family. The mature region of IL-1a, IL-1b, IL-1RA and IL-18 and full-length IL-1F5–IL-1F10 were aligned using the Clustal algorithm of the MEGALIGN program of Lasergene software. Residues matching the majority are boxed and shaded. The linear sequences of amino acid residues involved in the formation of b-strands of IL-1a and IL-1b that appear to be conserved among various members are indicated by arrow bars below the alignment.

FIGURE 30.4 Phylogenetic tree of various IL-1 family members. The alignment in Figure 30.3 was used to generate a phylogenetic tree showing the most probable evolution of all IL-1 family members based on the sequences corresponding to their respective mature regions. While the core b-trefoil structure of IL-1 made up of 12 b-strands is well conserved, the major differences between various homologs among themselves and with IL-1b lie in the loops connecting b-strands. Interestingly, it is the loops that also differ between IL-1a, b and RA.

CYTOKINE EXPRESSION AND REGULATION IL-1F5 IL-1F5 was first identified as IL-1HY1 by Mulero et al., (1999), by high throughput cDNA clone screening

technology. Subsequently, it was identified as FIL-1d (Smith et al., 2000) IL-1L1 (Barton et al., 2000), IL-1RP3 (Busfield et al., 2000), and IL-1d (Debets et al., 2001). A murine ortholog of this gene was also identified as IL-1H3 (Kumar et al., 2000). The predicted polypeptide sequence of both human and murine IL-1F5 exhibits approximately 42% identity to human IL-1RA protein. The predicted protein does not appear to contain a signal peptide or a propeptide domain at the N-terminus. None the less, a secreted protein of approximately 20 kDa is found in cells transfected with plasmids encoding IL-1F5, suggesting an alternative mechanism for secretion of IL-1F5. Interestingly, several clones corresponding to both human and murine cDNAs were found to have different 5 untranslated regions derived from distinct exons, suggesting potential splice variants that may arise due to activity of at least two promoters. This is further supported by the fact that lung tissue is reported to lack the second exon compared with other tissues (Smith et al., 2000). The IL-1F5 mRNA is predominantly expressed in embryonic and epithelial tissues such as skin, lung and stomach (Mulero et al., 1999; Smith et al., 2000; Debets et al., 2001). Of skin-derived cells, only keratinocytes express IL-1F5 mRNA. Low-level expression is also detected in the placenta, spleen, brain, leukocyte and macrophage cells (Table 30.3). Furthermore, IL-1F5 message is up-regulated by treatment of

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TABLE 30.2 Sequence identity among various IL-1 family members. The sequence alignment in Figure 30.3 was used to generate a pairwise comparison of various IL-1 family members

IL-1a IL-1b IL-1RA IL-18 IL-1F5 IL-1F6 IL-1F7 IL-1F8 IL-1F9 IL-1F10

IL-1a

IL-1b

IL-1RA

IL-18

IL-1F5

IL-1F6

IL-1F7

IL-1F8

IL-1F9

IL-1F10

100

17.0 100

13.2 21.1 100

12.1 13.7 14.5 100

13.0 19.0 42.1 13.6 100

14.6 22.9 26.3 10.8 24.7 100

13.8 18.3 19.7 11.5 23.4 28.7 100

14.7 26.1 24.3 10.9 26.0 45.5 26.3 100

14.5 17.6 25.0 12.1 22.7 54.1 25.0 41.7 100

12.6 18.5 32.5 12.6 37.7 25.2 21.9 25.8 25.8 100

human monocytic cells by phorbol esters and LPS similar to that of IL-1RA. IL-1F5 does not bind IL-1R, instead it antagonizes the NFjB inducing activity of IL-1F9 initiated through IL-1Rrp2 (IL-1R6) (Debets et al., 2001). Interestingly, unlike IL-1RAa which is often used at approximately 1000-fold excess over IL-1 for effective antagonism, IL-1F5 acts as a potent antagonist at a concentration similar to that used for its agonist, IL-1F9. The levels of IL-1F5, IL-1F9 and IL-1Rrp2 were significantly up-regulated in skin from lesional psoriatic patients characterized by chronic cutaneous inflammation compared to skin from healthy individuals. Thus, IL-1F5, IL-1F9, IL-1Rrp2 systems may be involved in skin inflammation and may contribute to the pathogenesis of psoriasis.

IL-1F6 A murine EST corresponding to IL-1F6 was first identified in a search of GenBank. Subsequently, the human IL-1F6 was cloned from human genomic sequence (Smith et al., 2000). The predicted polypeptide of IL-1F6 is approximately 26% identical to IL-1RA and its open reading frame does not contain either a signal peptide or a prodomain. The IL-1F6 mRNA is expressed in lymphoid organs and cell lines, particularly in T cells and in fetal brain (Table 30.3).

IL-1F7 IL-1F7 was identified in a search of GenBank EST database as FIL-1 (Smith et al., 2000). Subsequently, it was also identified as IL-1H4 (Kumar et al., 2000),

IL-1RP1 (Busfield et al., 2000) and IL-1H (Pan et al., 2001). An examination of sequences identified by various investigators reveals the existence of at least two additional isoforms of IL-1F7 (IL-1F7b and IL-1F7c). The predicted polypeptide exhibits approximately 20% identity to IL-1RA. Among various IL-1 homologs, IL-1F7 is the only molecule that contains a prodomain. Furthermore, IL-1F7b also contains a putative caspase cleavage site analogous to IL-1b and IL-18 (Kumar et al., 2000). In contrast, IL-1F7 contains an upstream exon but lacks the exon encoding the caspase cleavage site. IL-1F7c lacks an exon encoding a 40-amino-acid region predicted to encode several b-strands involved in generating the typical b-barrel fold of IL-1. Therefore, IL-1F7c is likely to be a nonfunctional protein. There is some evidence to suggest that the various isoforms are differentially expressed. IL-1F7 mRNA is expressed in B cells, placenta, testes, lymph node and lung. It is also expressed and could be induced by phorbol esters in human peripheral blood mononuclear cells and dendritic cells (Table 30.3). IL-1F7b was demonstrated to bind to the IL-18R in receptor precipitation experiments using IL-18Ra–Fc fusion protein (Pan et al., 2001; Kumar et al., 2002). However, IL-1F7b had no effect on the IFNc-inducing activity of IL-18. Further analysis indicated that IL-1F7b could be cleaved by caspase 1, and to a lesser extent by caspase 4, into a mature form (Kumar et al., 2002). However, the cleavage of IL-1F7b by either caspase 1 or 4 was not as efficient as that of IL-18. Although the transfection of a plasmid encoding IL-1F7 and IL-1F7b results in processing and secretion of a mature form, it is not clear if in vivo process-

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TABLE 30.3 A summary of properties of novel IL-1 homologs Gene

Prodomain

Receptor

Tissue/Cell expression

Signaling pathway

Probable function

Ref.

IL-1F5

No

IL-1Rrp2

Skin, lung, epithelial tissues, keratinocytes, spleen, lymph node, tonsil, bone marrow, fetal brain, leukocytes and various human cell lines

NFjBd

Antagonizes IL-1F9 and possibly involved in psoriasis/ chronic inflammation

Mulero et al., 1999; Smith et al., 2000; Kumar et al., 2000; Barton et al., 2000; Busfield et al., 2000; Debets et al., 2001

IL-1F6

No

Unknown

Bone marrow, heart, placenta, lung, testes, colon

Unknown

Unknown

Smith et al., 2000

IL-1F7

Yesa

IL-18Rb

Skin, placenta, lung, colon tumor, bone marrow, uterus, tonsil, testes, thymus, B and plasma cells and various human cell lines

Unknown

Unknown

Smith et al., 2000; Kumar et al., 2000; Busfield et al., 2000; Pan et al., 2001

Probable modulation of IL-18 activity Anti-tumor activity

Buffer et al., 2002 Gao et al., 2003

IL-1F8

No

Unknown

Tonsil, bone marrow, lung, ovary, heart

Unknown

Unknown

Smith et al., 2000; Kumar et al., 2000

IL-1F9

No

IL-1Rrp2

Skin, epithelial tissues, keratinocytes, lung, lymph node, testes, thymus, tonsil, brain, placenta, prostate, NK cells, parathyroid tumor and various human cell lines

NFjB

Possibly involved in psoriasis/ chronic inflammation

Kumar et al., 2000; Busfield et al., 2000; Debets et al., 2001

IL-1Rc

spleen, skin, tonsil, B cells

Unknown

Unknown

Lin et al., 2001

IL-1F10 No a

Contains a prodomain and a caspase 1 cleavage site. Binds IL-18R but does not appear to agonize or antagonize IL-18 signal at least in terms of IFNc production. c Binds IL-1R but there are no signaling data available. d Antagonizes IL-1F9-mediated NFjB activation. b

ing enzyme is caspase 1 or another enzyme. The cleavage of IL-1F7b appears to be biologically relevant since the pro and mature forms of IL-1F7b bind to IL-18Ra–Fc fusion protein with different affinities. The

pro and mature IL-1F7b bind to IL-18Ra–Fc protein with an affinity of 24 lM and 120 nM, respectively, as assessed by a surface plasmon resonance assay (Kumar et al., 2002). In contrast, IL-18 binds to

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IL-18Ra–Fc protein with an affinity of 1.8 nM in the same assay. Such low affinity of IL-1F7b to IL-18R perhaps explains its inability to affect the IFNc-inducing activity of IL-18. It is also possible that IL-1F7 utilizes an as yet unidentified accessory receptor with IL18Ra. The processing of IL-1F7b also results in increased dimerization of the mature form as compared to the pro form. The mature IL-1F7b forms a dimer with an association constant of 5 nM compared with 4 lM for the pro form (Kumar et al., 2002). It remains to be seen whether IL-1F7 also dimerizes in vivo. None the less, the dimerization of IL-1F7 in vitro appears to be unique among the IL-1 family members. Recently, IL-1F7b has been shown to have antitumor activity (Gao et al., 2003). Gambotto and colleagues constructed recombinant adenovirus encoding IL-1F7b and demonstrated the secretion of IL-1F7b in culture media upon adenovirus infection of A549 cells. A single intratumoral injection of recombinant adenovirus into an established MCA205 fibrosarcoma resulted in significant suppression of tumor growth compared with saline or control adenovirus injection. Furthermore, multiple injections of recombinant adenovirus led to a complete inhibition of tumor growth. The antitumor effect of IL-1F7b was observed only in NKT-deficient mice but not in nude or SCID mice or in IL-12, IFNc and Fas liganddeficient mice. The exact molecular mechanism of the antitumor effect of IL-1F7b is not known but recent evidence suggests a requirement of functional T and B cells and involvement of IL-12-dependent adaptive immunity (Gao et al., 2003).

IL-1F8 IL-1F8b was first identified by sequencing of an osteoclastoma cDNA library (Kumar et al., 2000). The predicted polypeptide sequence of this cDNA matched well only with the N-terminus half of IL-1RA. Subsequently, another cDNA, IL-1F8 was cloned from the human genome which matched with both N- and C-terminal halves of IL-1RA (Smith et al., 2000). It appears that the osteoclastoma version of IL-1F8 is an aberrant splice variant, the likely result of a cloning artifact. Like several other novel homologs, IL-1F8 exhibits approximately 25% identity with IL-1RA but it does not contain either a signal peptide or a

prodomain. Low levels of IL-1F8 mRNA are expressed in tonsil, bone marrow, heart, placenta, lung, testes and colon (Table 30.3).

IL-1F9 IL-1F9 was identified by EST analysis from an epithelial cell cDNA library (Kumar et al., 2000). Subsequently, it was also identified as IL-1RP2 (Busfield et al., 2000) and IL-1e (Debets et al., 2001). The predicted polypeptide sequence of IL-1F9 contains neither a signal peptide nor a prodomain and is approximately 25% identical to IL-1RA. Circular dichroism spectrum of recombinant IL-1F9 matches well with that of IL-1RA but is slightly different to IL-1b (Kumar et al., 2000). IL-1F9 mRNA and protein are expressed in embryonic and epithelial tissues such as skin, lung and stomach (Table 30.3). Among various cell types, keratinocyte and epithelial cells appear to be the predominant cell type, expressing IL-1F9. Furthermore, treatment of keratinocytes with TNF and/or IL-1b stimulates the expression of IL-1F9 (Kumar et al., 2000; Debets et al., 2001). IL-1F9 does not bind IL-1R or IL-18R but has recently been shown to bind IL-1Rrp2 and to activate NFjB in transfected Jurkat cells. As mentioned before, IL-1F5 was shown to act as an antagonist of IL-1F9 (Debets et al., 2001). The levels of IL-1F5, IL-1F9 and IL-1Rrp2 are significantly up-regulated in skin from lesional psoriatic patients characterized by chronic cutaneous inflammation compared with skin from healthy individuals (Debets et al., 2001). Thus, the IL-1F5, IL-1F9, IL-1Rrp2 system may be involved in skin inflammation and may contribute to the pathogenesis of psoriasis. The presence of IL-1F5 and IL-1F7 in epithelial barriers of the body suggests that these novel homologs may function to promote a response to injury or infection. Consistent with this hypothesis, murine IL-1F9 mRNA was shown to be up-regulated in vivo in response to chronic contact hypersensitivity or viral infection (Kumar et al., 2000).

IL-1F10 IL-1F10 was identified by EST analysis as IL-1HY2 (Lin et al., 2001). The predicted polypeptide sequence

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of IL-1F10 exhibits approximately 32% identity to IL-1RA. Like most novel IL-1 homologs, IL-1F10 does not contain either a signal peptide or a prodomain. However, transfection of a plasmid encoding IL-1F10 protein into CHO cells leads to protein being secreted into the media. IL-1F10 is expressed in human skin, spleen and tonsil. IL-1F10 was also shown by immunohistochemical analysis to be expressed in basal epithelia of skin and in proliferating B cells of tonsil by immunohistochemical analysis (Table 30.3). IL-1F10 is the only novel IL-1 homolog that binds to the IL-1RI with affinity comparable to that of IL-1b and IL-1RA (Lin et al., 2001). Using a surface plasmon resonance assay, IL-1F10 was reported to bind to IL-1RI with an affinity of 98 nM compared with 21 and 38 nM for IL-1b and IL-1RA, respectively. It is not clear whether the IL-1R binding activity of IL-1F10 results in any agonistic or antagonistic activity with IL-1.

AREAS OF ACTIVE RESEARCH Prediction of potential functions of novel IL-1 homologs has relied heavily on sequence alignment and structural modeling. An inherent bias with these analyses is that they assume the novel molecules to have IL-1-like functions. Because all novel IL-1 homologs were identified by database searches using an IL-1 signature profile, it was expected that most novel molecules would function similarly to an agonist like IL-1 or an antagonist like IL-1RA. Although a clear function(s) for any of the homologs has yet to be identified, progress has been made with some of the molecules. Identification of these genes is also exciting since there exists a large number of orphan IL-1R-related proteins. Of the six novel homologs, four have been shown to bind receptors in the IL-1 family with limited or no functional relevance. IL-1F7 and IL-1F10 have been shown to bind IL-18R and IL-1RI, respectively, but there does not appear to be a functional consequence of these interactions (Pan et al., 2001; Lin et al., 2001; Kumar et al., 2002). IL-1F9 and IL-1F5 bind to IL-1Rrp2 as an agonist and antagonist, respectively (Debets et al., 2001). There are a total of 10 IL-1R-like molecules in the human genome and a systematic analysis with each ligand and receptor is necessary to pair novel ligands with orphan receptors. It is important to note

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that the affinity of ligands in the IL-1 family for their cognate receptors increases substantially in the presence of an accessory receptor. In the absence of a functional assay it is not clear whether the proteins are folded in an active conformation even though most recombinant proteins have been shown to be soluble. It is entirely possible that these novel homologs bind to receptors partially or entirely different from IL-1Rs, which will have an implication for coupling to a particular signaling pathway. For example, a receptor that might lack the intracellular tolllike receptor region domain may not signal like IL-1 even though it may contain three extracellular IgGlike domains to maintain a binding mode similar to IL-1. Another widely used method to characterize novel molecules functionally is the examination of their expression pattern in various tissue and cells. Tissue expression of several of these novel homologs has been extensively studied and some clues related to their potential functions are beginning to emerge. The expression pattern of IL-1F5 and IL-1F9 appear most interesting as they are highly expressed in epithelial cells such as keratinocytes (Debets et al., 2001). The expression of murine IL-1F9 was shown to be elevated in response to chronic contact hypersensitivity and viral infection. Consistent with this, hIL-1F9 was shown to be significantly elevated in skin from patients with lesional psoriasis. These data suggest a protective and/or reparative role of the IL-1F9 and F5 in response to injury or infection. IL-1F7 has been shown to be expressed in plasma cells of tonsil whereas IL-1F10 is expressed in proliferating B cells, suggesting a potential role of these molecules in immunoglobulin production (Lin et al., 2001; Kumar et al., 2002). Such coexpression in related cell lineages also suggests a potential agonist/antagonist relationship between IL-1F7 and IL-1F10. Identification of genes regulated by novel IL-1 homologs by exploitation of the microarray technique and further pathway predictions based on analysis of coordinately regulated genes will allow accelerated characterization of these novel molecules. Another technique gaining wider use is gene transfer using viral vector systems. These can be used both in vitro and in vivo and have the potential to uncover functional phenotype based on overexpression or underexpression if a dominant negative construct is

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used. Such an analysis is underway with several of these novel homologs and has resulted in identification of an antitumor activity for IL-1F7b (Gao et al., 2002). Although labor-intensive and time-consuming, generation of null or transgenic mice is often one of the most direct approaches to understanding the function of novel genes. No transgenic mice for any of the novel homologs have yet been described; although murine orthologs for most of the genes have either been identified or will be available shortly with the eminent completion of the mouse genome sequencing. It will be exciting to see the phenotype displayed by transgenic mice deficient for novel IL-1 homologs.

with the IL-18Ra chain in response to IL-18 resulting in an inhibition of IL-18-induced response (Bufler, 2002).

ACKNOWLEDGEMENTS I acknowledge Dr Martin Nicklin of the University of Sheffield for sharing of genomic data prior to publication, Dr John Sims of Immunex Corporation for permission to use the homology structural model of IL-1F5 and Drs Janet Kumar, John White and Simon Blake of GSK for critical reading of the manuscript.

REFERENCES SUMMARY With the completion of human genome sequencing and identification of most if not all genes, it is a daunting task to annotate human genes. A systematic search of human genome sequence has resulted in identification of six novel homologs of the IL-1 family. The expression of these genes appears to be quite restricted compared with known IL-1s and few of them bind and/or signal through the known receptors (Table 30.3). It is thus expected that these molecules will bind to unique receptors and will have specialized functions distinct from those of IL-1s. For novel IL-1 homologs, clues obtained by educated predictions combined with various technologies will undoubtedly lead to functional annotation of these genes in the coming years.

ADDENDUM Bufler et al., has recently published new data with IL-1F7b (Bufler, 2002). It appears that while IL-1F7b binds IL-18Ra, unlike IL-18, it fails to recruit the IL-18Rb chain and therefore is unable to functionally activate the IL-18R. In addition, they unexpectedly observed that IL-1F7b also binds to IL-18BP and enhances its ability to inhibit IL-18-induced IFN- production in a human natural killer cell line. The authors proposed that the binding of IL-1F7b to IL-18BP results in recruitment of IL-18Rb chain, which then becomes unavailable to form a complex

Auron, P.E. (1998). The interleukin 1 receptor: ligand interactions and signal transduction. Cytokine Growth Factor Rev. 9, 221–237. Barton, J.L., Herbst, R., Bosisio, D. et al. (2000). A tissue specific IL-1 receptor antagonist homolog from the IL-1 cluster lacks IL-1, IL-1ra, IL-18 and IL-18 antagonist activities. Eur. J. Immunol. 30, 3299–3308. Bazan, J.F., Timans, J.C. and Kastelein, R.A. (1996). A newly identified interleukin-1? Nature 379, 591–591. Born, T.L., Thomassen, E., Bird, T.A. and Sims, J.E. (1998). Cloning of a novel receptor subunit, Acpl, required for interleukin-18 signaling. J. Biol. Chem. 273, 29445–29450. Bufler, P., Azam, T., Gamboni-Robertson, F. et al. (2002). A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. Proc. Nat Acad. Sci., USA, 99, 13723–13728. Busfield, S.J., Comrack, C.A., Yu, G. et al. (2000). Identification and gene organization of three novel members of the IL-1 family on human chromosome 2. Genomics 66, 213–216. Debets, R., Timans, J.C., Homey, B. et al. (2001). Two novel IL-1 family members, IL-1d and IL-1e, function as an antagonist of NF-jB activation through the orphan IL-1 receptor-related protein 2. J. Immunol. 167, 1440–1446. Dinarello, C.A. (1994). The interleukin-1 family: 10 years of discovery. FASEB J. 8, 1314–1325. Dinarello, C.A. (1996). Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147. Dinarello, C.A. (2000). Targeting interleukin 18 with interleukin 18 binding protein. Ann. Rheum. Dis. 59, 17–20. Eisenberg, S.P., Evans, R.J., Arend, W.P. et al. (1990). Primary structure and functional expression from complimentary DNA of a human interleukin-1 receptor antagonist. Nature 343, 341–346. Gao, W., Kumar, S., Lotze, M.T. et al. (2003). Innate immunity mediated by the interleukin-1 homologue 4 (IL-1H4/IL1F7) proinflammatory cytokine induces IL-12-dependent adaptive and profound antitumor immunity. J. Immunol. 170, 107–113. Heguy, A., Baldari, C.T., Macchia, G., Telford, J.L. and Melli, M. (1992). Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential

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for IL-1R signal transduction. J. Biol. Chem. 267, 2605–2609. Kumar, S., McDonnell, P.C., Lehr, R. et al. (2000). Identification and initial characterization of four novel members of the interleukin-1 family. J. Biol. Chem. 275, 10308–10314. Kumar, S., Hanning, C.R., Bringham-Burke, M.R. et al. (2002). Interleukin-1F7b (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7b binds to the IL-18 receptor but does not induce IFN- production. Cytokine 18, 61–71. Lander, E.S., Linton, L.M., Birren, B. et al. (2001). Initial sequencing and analysis of the human genome. [Erratum appears in Nature 2001 Jun 7;411(6838):720]. Nature 409, 860–921. Lin, H.S., Ho, A.S., Haley-Vicente, D. et al. (2001). Cloning and characterization of IL-1HY2, a novel interleukin-1 family member. J. Biol. Chem. 276, 20597–20602. McMahan, C.J., Slack, J.L., Mosley, B. et al. (1991). A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types. EMBO J. 10, 2821–2832. Mulero, J.J., Pace, A.M., Nelkin, S.T. et al. (1999). IL1HY1: A novel interleukin-1 receptor antagonist gene. Biochem. Biophys. Res. Commun. 263, 702–706. Nicklin, M.J., Weith, A. and Duff, G.W. (1994). A physical map of the region encompassing the human interleukin-1 alpha, interleukin-1 beta, and interleukin-1 receptor antagonist genes. Genomics 19, 382–384. Nicklin, M.J.H., Barton, J.L., Nguyen, M. et al. (2001). A sequence-based map of the nine genes of the human interleukin-1 cluster. Genomics 79, 718–725. Nothwang, H.G., Strahm, B., Denich, D. et al. (1997). Molecular cloning of the interleukin-1 gene cluster: construction of an integrated YAC/PAC contig and a partial

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transcriptional map in the region of chromosome 2q13. Genomics 41, 370–378. Okamura, H., Tsutsui, H., Komatsu, T. et al. (1995). Cloning of a new cytokine that induces IFN-c production by T cells. Nature 378, 88–91. O’Neill, L.A. and Greene, C. (1998). Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J. Leukocyte Biol. 63, 650–657. Pan, G., Risser, P., Mao, W. et al. (2001). IL-1H, an interleukin 1-related protein that binds IL-18 receptor/IL-1Rrp. Cytokine 13, 1–7. Sims, J.E., Pan, Y., Smith, D.E. et al. (2001). A new nomeclature for IL-1-family genes. Trends Immunol. 10, 536–537. Smith, D.E., Renshaw, B.R., Ketchem, R.R. et al. (2000). Four new members expand the interleukin-1 superfamily. J. Biol. Chem. 275, 1169–1175. Tanaka, F., Hashimoto, W., Okamura, H. et al. (2000). Rapid generation of potent and tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells. Cancer Res. 60, 4838–4844. Torigoe, K., Ushio, S., Okura, T. et al. (1998). Purification and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272, 25737–25742. Xiang, Y. and Moss, B. (2001). Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis. J. Biol. Chem. 276, 17380–17386. Zhu, X., Komiya, H., Chirino, A. et al. (1991). Threedimensional structures of acidic and basic fibroblast growth factors. Science 251, 90–93

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31 The fibroblast growth factors Barbara Ensoli, Cecilia Sgadari, Giovanni Barillari and Paolo Monini Istituto Superiore di Sanità, Rome, Italy

The mind cannot possibly grasp the full meaning of the term of a hundred million years; it cannot add up and perceive the full effects of many slight variations, accumulated during an almost infinite number of generations Charles Darwin

INTRODUCTION Fibroblast growth factors (FGFs) constitute a large family of factors regulating complex biologic processes that, in most cases, involve extensive tissue remodeling, such as embryonic development, neoangiogenesis, wound healing, nerve regeneration, chronic inflammation and cancer (reviewed in Ornitz and Itoh, 2001). These processes require spatial and temporal integration of several cell responses, including cell survival, proliferation, migration and invasion, and cell differentiation. All these responses or functions are induced or modulated by the interactions of FGFs with low- and high-affinity receptors that are present inside the cells, at the cell surface and/or in the extracellular matrix (ECM). These include heparan sulphate proteoglycans (HSPGs), tyrosine kinase receptors (FGFRs) and other molecules that are required for specific FGF functions. The interaction of FGFs with these receptors not only triggers signaling pathways inducing or modulating The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

cell responses to FGFs, but also mediates FGFR cell trafficking. In fact, most FGFs are imported or exported in and out of cells and are translocated to the cell nucleus complexed with their receptors. Internalization and nuclear translocation of receptors results in specific signaling pathways that appear to be different from those elicited at the cell surface (reviewed in Jans and Hassan, 1998; Keresztes and Boonstra, 1999). Thus, FGFs act on cells through autocrine and paracrine effects that are modulated by both receptor activation and trafficking. However, FGFs also possess intracrine actions (reviewed in Delrieu, 2000). In fact, due to the presence of strong nuclear localization signals, several FGF isoforms are not exported but localize exclusively in the nucleus and/or nucleolus, probably in association with an intracellular truncated version of membrane receptors. Although the interaction of FGF with molecules present outside cells (particularly ECM molecules) is the key for specificity, modulation and integration of FGF responses, intracrine actions

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appear to be fundamental for some FGFs endowed with intracellular isoforms, or for FGFs that are not released out of cells. Most, if not all, FGFs are expressed in embryos and play a key role in invertebrate or vertebrate embryonic development. It is thus tempting to speculate that the extraordinary large number of FGF family members stems from the necessity to regulate the extraordinary complexity of developmental processes. Wound healing, neoangiogenesis or nerve regeneration are also developmental processes, in that they require disruption and reconstruction of old and new tissue with new architecture and functions. The distinct bent of FGFs to interact with ECM molecules appears to originate from the necessity to take control of ECM degradation and remodeling during these processes. For example, by binding to ECM HSPG, FGFs are capable of creating and stabilizing long-lasting local concentration gradients that are likely to be required for specific local morphogenetic or remodeling processes. In turn, these interactions result in several regulatory mechanisms such as FGF stabilization, local storage of FGF and selection of heparan sulfates (HS) and HSPG protein core sequences that, released upon ECM degradation, confer distinctive receptor affinity and specificity to FGFs (reviewed in Powers et al., 2000). Given the multiple effects of FGFs in several biologic processes, changes in FGF production or release are associated with several pathologic conditions, particularly developmental syndromes, chronic inflammation, tumor angiogenesis and growth, all requiring extensive tissue remodeling. In recent years, the study of the role of FGFs and FGFRs in these pathological conditions has increased our knowledge of FGF actions and, most importantly, has led to the identification of therapeutic targets. For example, non-toxic heparin-mimicking polyanionic compounds or porphyrin analogs capable of interfering with FGF binding and dimerization, as well as new classes of inhibitors of the catalytic domain of FGFRs which are inactive against other tyrosine kinase receptors, are now available. These compounds have been shown to be capable of blocking vascular cell proliferation, angiogenesis, tumor growth, and to revert FGF-transformed cells to a non-transformed phenotype. Hopefully, these findings will lead to the identifica-

tion of other molecular targets and inhibitors with lower toxicity for the prevention and therapy of FGFassociated diseases.

THE FGF FAMILY MEMBERS FGFs are a large family of heparin-binding factors that appear to have originated from a common ancestor upon a complex process of gene duplication coincident with the evolutionary emergence of vertebrates (Coulier et al., 1997). FGFs are, in fact, present in invertebrate and FGF-like genes have also been identified in baculovirus, Drosophila and Caenorhabditis elegans. Four FGF genes have been identified in zebrafish and up to seven FGF-like genes in Xenopus or chicken, whereas mammalian FGFs form a large family of growth factors consisting of at least 22 members. In vertebrate, FGFs share up to 70% homology and show a highly conserved gene structure. Certain vertebrate members can be grouped into subfamilies. Members of the same subfamily show increased homology, similar tissue distribution and biochemical properties and/or functions. Examples are the subfamily grouping FGF8, FGF17 and FGF18, that are characterized by high sequence homology (70–80%), similar receptor specificity and expression in the same tissue compartments, or the group of FGF11–14, also named FGF homology factors 1–4 (FHF1–4), a group of closely related members showing 54% to 71% amino-acid identity that, unlike all other FGFs, are not found in the extracellular environment. A database of FGF sequences is available at http://cytokine.medic.kumamoto-u.a.c.jp/CFC/FGF/ FGF.html.

Gene structure and chromosomal localization The prototypical structure of vertebrate FGF genes ( fgf ) includes three coding exons separated by large stretches of non-coding DNA sequences. In some FGF members, alternative in frame splicing divides exon 1 in up to four alternative subexons. The ATG initiation codon is always located in exon 1 or at the beginning of the first 5 subexon, however, FGF2 (basic FGF, bFGF) and FGF3 (int-2), have additional alternative 5

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translated regions that initiate from upstream CUG codons (Plate 31.1A). These members, as well as FHF1–4/ FGF11–14 that use alternative 5 exons have multiple amino termini resulting in protein isoforms of different size (reviewed in Powers et al., 2000). Positional mapping of FGF genes in the human genome indicates that the FGF family originated both through multiple gene duplication and chromosomal duplication and translocation events (Coulier et al., 1997; Yamashita et al., 2000). In fact, although most human FGF genes are scattered on chromosomes 3, 4, 5, 8, 10, 13, 15, 17, 19 and X, seven of the 22 members are clustered on chromosome 8p21–p22 (fgf17, fgf20), chromosome 11q13 (fgf3, fgf4, fgf19) and chromosome 12p13 (fgf6, fgf23).

Protein sequence and structure Vertebrate FGFs have a molecular weight ranging from 17 to 34 kDa and share 13% to 70% homology, however, FGF proteins from related (orthologous) chromosomal regions across species are highly conserved and present 90% amino-acid sequence identity. Vertebrate FGFs show a large internal ‘core’ region of about 140 amino acids containing most of the amino-acid residues involved in binding to membrane tyrosine kinase FGFRs and HS binding domains (Plate 32.1A and B). The core region contains a highly homologous sequence of 28 conserved and six identical amino acids, 10 of which bind to FGFRs. The amino-terminal sequence preceding the core region also contains amino-acid residues interacting with FGFRs and, in many but not all FGFs, leader sequences and nuclear localization sequences (NLS) for FGF secretion and nuclear import (see below and Plate 31.1A) (Ornitz, 2000; Plotnikov et al., 2000). FGFs have similar structural features, with a highly compact internal region, constituted by the core, and less structured amino (N) and carboxy (C) termini. The FGF core region folds in 12 antiparallel b strands (numbered from b1 to b12, Plate 31.1A) that are organized in three four-stranded b sheets arranged in a characteristic trefoil structure (the FGF b-trefoil fold) (Plotnikov et al., 2001 and references therein). This structure, that is remarkably similar to the interleukin-1b (IL-1b) barrel (Zhang et al., 1991), is closed by the N- and C- terminal sequences that show variable length (Plotnikov et al., 2001). In particular,

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the N and C termini are very short in FGF1 (acidic FGF, aFGF), FGF2, FGF4 and FGF7 (keratinocyte growth factor, KGF) and almost coincident with the end of the b-trefoil core, whereas they are extended in other family members including FGF3, FGF5, FGF8, FGF9 and FGF16–19. The different lengths of the N and C termini appear to have important functional implications that have been extensively investigated in FGF1, FGF2 and FGF9. As compared with FGF1 and FGF2, the FGF9 N- and C-terminal sequences are longer and, as a consequence, they can fold into stable a helices (Plotnikov et al., 2001). The N a helix plays a key role in FGF9 receptor specificity, as its sterical hindrance prevents binding to FGFR1 that, by contrast, is recognized with high affinity by both FGF1 and FGF2 (Plotnikov et al., 2001). This may explain the preferential binding of FGF9 to FGFR3, its mitogenic activity for glia cells, smooth muscle cells and fibroblasts, and the lack of effects on endothelial cells (Naruo et al., 1993). Thus, the variable N and C termini of FGF proteins are likely to modulate and regulate part of the receptor and HS binding activity by the more conserved core region.

Protein production and mechanisms of protein secretion and export Most FGFs, with the exception of the FHF subfamily (see below), are secretory proteins that act extracellularly, where they bind membrane-associated and ECM HSPG. Most FGFs are secreted from cells through the classical N-terminal signal peptide (leader sequences)-dependent pathway (Plate 31.1A). However, many family members show unusual secretion pathways. For example, some family members, namely the homologous FGF9, FGF16 and FGF20, lack canonical signal peptides/leader sequences, but are nevertheless secreted through the Golgi pathway (Miyake et al., 1998; Ohmachi et al., 2000). In particular, FGF9 uses a hydrophobic leader sequence that, unlike classical signal peptides, is not cleaved in the endoplasmic reticulum (ER) (Miyakawa et al., 1999; Revest et al., 2000). FGF3 secretion is unusually inefficient as compared with the other FGFs endowed with classic leader sequences. This is due to the action of a intracellular cysteine-rich FGF receptor (CFR) that binds and retains FGF3 in the Golgi, where the receptor is located (Burrus et al., 1992; Zhou et al., 1997;

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Kohl et al., 2000). Part of the receptor is then cleaved at the C terminal and released in the extracellular environment leading to secretion of CFR-bound FGF3 (Kohl et al., 2000). The angiogenic FGF1 and FGF2 lack leader sequences (Plate 31.1A) and are released from cells through non-classical protein export pathways (Mignatti et al., 1992; Samaniego et al., 1995; Samaniego et al., 1997). Release of FGF1 appears to occur in response to stress stimuli leading to inflammation, such as heat shock (Jackson et al., 1992). FGF1 export is blocked by protein synthesis inhibitors and is potentiated by Golgi inhibitors or inhibitors of the classic secretory pathway (Jackson et al., 1995). Upon heat shock, FGF1 is exported as a latent homodimer which is stabilized by intermolecular disulfide bonds and appears to be bound to chaperon proteins, including synaptotagmin-1 and the calciumbinding protein S100A13, that are required for heat shock-dependent FGF1 release (Carreira et al., 1998; Tarantini et al., 1998; LaVallee et al., 1998b). In this form, FGF1 does not bind HSPG and is not mitogenic (Jackson et al., 1992; Jackson et al., 1995; Tarantini et al., 1995). However, chaperon-bound inactive FGF1 is readily activated upon exposure to reduced glutathione, which is typically found in anoxic tissue (Jackson et al., 1995). Since anoxia is a known angiogenesis inducer, these data point to release of FGF1 as a highly regulated proangiogenic event elicited in response to stress conditions and tissue damage. FGF2 is present in large quantities in the extracellular matrix (DiMario et al., 1989) and, like FGF1, is also released through a Golgi-independent pathway (Plate 31.1A) (Mignatti et al., 1992; Samaniego et al., 1995, 1997), however, evidence indicates that FGF2 is not exported in response to temperature stress (Shi et al., 1997). Mechanical cell damage, cell membrane mechanical distortion or sarcolemma stress are all potent stimuli for FGF2 cell exit and localization in the extracellular compartment, probably in response to Ca mobilization due to increased membrane permeability (Yu et al., 2001). This most likely plays a key role during regenerative or developmental processes including wound healing (McNeil et al., 1989), morphogenesis of cranial sutures (Yu et al., 2001), skeletal muscle growth in response to mechanical load (Clarke and Feeback, 1996), or smooth muscle cell proliferation in injured arteries (Calara

et al., 1996). In addition, in the absence of cell damage or membrane permeability changes, FGF2 is released through a specific energy-dependent export pathway involving the catalytic subunit of the Na,K ATPase (Samaniego et al., 1995, 1997; Florkiewicz et al., 1998; Dahl et al., 2000). These data serve to indicate the existence of a specific export mechanism activated in response to non-mechanical forces in physiologic or pathologic conditions as, for example, FGF2 release from endothelial cells and Kaposi’s sarcoma (KS) cells in response to inflammatory cytokines (see below) (Ensoli et al., 1989, 1994b; Samaniego et al., 1995, 1997, 1998; Barillari et al., 1999b). Upon export, FGF2 localizes at the cell surface or in the ECM, where it binds HSPG, however, upon HSPG saturation or degradation it can be mobilized and released as a soluble form (see below) (Barillari et al., 1999a; Trudel et al., 2000).

Protein subcellular localization and intracellular trafficking Several members of the FGF family are imported or exported in and out of cells, and are translocated to the cell nucleus. However, trafficking and nuclear import of endogenous or exogenous FGF molecules occurs through different pathways. Subcellular localization of endogenous proteins is largely modulated by the opposite action of protein export signals and NLS, whereas the intracellular fate of exogenous FGF molecules is determined by their association with high- or low-affinity receptors (Kiefer et al., 1994; reviewed in Keresztes and Boonstra, 1999; Delrieu, 2000). For example, despite the presence of NLS at the N termini of the protein, nuclear import of endogenous FGF1 is poor, and cytoplasmic FGF1 is efficiently released owing to protein export mechanisms (Zhan et al., 1992). However, exogenous FGF1, like other FGFs including FGF2, is efficiently taken up and translocated to the cell nucleus by target cells (Zhan et al., 1992). As discussed below, this occurs through unique receptor-mediated pathways involving membrane tyrosine kinase FGFRs, HSPG or CFR (Zuber et al., 1997; Keresztes and Boonstra, 1999).

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Intracellular and nuclear trafficking of endogenous FGF proteins Intracellular trafficking of endogenous FGF molecules is believed to result in intracrine signaling. This is best exemplified by the members of the FHF subfamily, which show a relatively low homology with other family members, lack leader sequences and, unlike all other FGFs, are not exported in the extracellular milieu (Smallwood et al., 1996). FHFs are expressed during craniofacial and limb development as several isoforms which show different subcellular distributions, including cytoplasmic, nuclear, or both nuclear and cytoplasmic localization (Wang et al., 2000; Munoz-Sanjuan et al., 2000, 2001; and references therein). In addition, different FHF isoforms can show nucleoli exclusion or may target the nucleolus in a highly specific way (Munoz-Sanjuan et al., 2000). The different localization of the FHF isoforms is likely to be associated with different intracrine actions and functions in the developing and adult nervous system (Wang et al., 2000; Munoz-Sanjuan et al., 2000). Subcellular localization of endogenous FGFs that are exported out of the cell can be modulated by the action of NLS. For example, although, as discussed above, a large part of FGF3 localizes in cytoplasm due to the action of leader sequences or is sequestered in the Golgi upon CRF binding, a large part of the endogenous protein localizes in the cell nucleus owing to N- and C-terminal NLS (Plate 31.1A). Additional sequences with nuclear import activity are acquired upon initiation at an upstream CUG codon leading to the production of a high-molecular weight (HMW) isoform, which is the most frequent pathway for protein translation (Plate 31.1A). In addition, nucleolar localization signals are present at the C termini of the protein (Kiefer and Dickson, 1995) (Plate 31.1A). The fate of endogenous FGF3 is determined by finely tuned antagonistic actions of nuclear import signals and leader sequences (Antoine et al., 1997). In fact, deletion of leader sequences causes FGF3 to be no longer secreted and, as a consequence, the protein localizes exclusively in the cell nucleus, particularly in the nucleolar compartment. Secreted FGF3 is known to have strong autocrine and paracrine mitogenic effects for epithelial lineage cells (Mathieu et al., 1995). In contrast, nucleolar FGF3 inhibits cell growth, probably due to the inactivation of NoBP, a

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protein which is expressed in the nucleolus at the G1–S transition and, in the absence of FGF3, promotes cell proliferation (Kiefer and Dickson, 1995; Reimers et al., 2001). Thus, the modulation of subcellular FGF3 localization can regulate the autocrine mitogenic effects of the protein through intracrine actions delivered to the cell nucleus. Similarly to FGF3, FGF2 can also be initiated at alternative upstream CUG codons, particularly in response to cell stress conditions, or in the presence of inflammatory cytokines. This results in the synthesis of four HMW protein isoforms of 34, 24, 22.5 and 22 kDa (Plate 31.1A). In addition, a low-molecular weight (LMW) species of 18 kDa is originated upon usage of the AUG codon (Plate 31.1A) (Florkiewicz and Sommer, 1989; Prats et al., 1989; Ensoli et al., 1989; Arnaud et al., 1999). All these isoforms are colinear variants differing only for their N-terminal extensions. As for FGF3, the FGF2 HMW isoforms acquire NLS-like signals (Plate 31.1A) and, unlike the LMW species that shows a prominent cytoplasmic localization and is exported, they are mostly nuclear and are not released by producing cells (Renko et al., 1990; Powell and Klagsbrun, 1991; Arnaud et al., 1999). The intracrine actions of the FGF2 HMW isoforms and the autocrine or paracrine effects of LMW FGF2 show distinctive differences (reviewed in Delrieu, 2000). For example, unlike the 18 kDa species, the HMW isoforms stimulate the growth of fibroblasts maintained in low-serum conditions, and promote seruminduced proliferation of quiescent smooth muscle cells (Davis et al., 1997; Arese et al., 1999). Furthermore, the 34 kDa species acts as a survival factor for fibroblasts grown in low-serum conditions (Arnaud et al., 1999). In this context, it has been shown that the HMW isoforms are part of a 320 kDa complex incorporating the FGF2-interacting factor (FIF), a nuclear antiapoptotic factor. By contrast, the 18 kDa species is present in a complex of 130 kDa, that lacks detectable FIF (Van den Berghe et al., 2000). Moreover, as compared with the 18 kDa species, the HMW isoforms show a different modulation of protein kinase C (PKC) and ERK1/2 activity, and stimulate fibroblast motility and growth even in the presence of mutated surface FGFRs lacking the tyrosine kinase domain (Gaubert et al., 2001 and references therein). Thus, these data point to specific intracrine actions of FGF2 HMW

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isoforms that may have important effects on growth and survival of FGF2 producing cells, as also indicated by the restricted expression of intranuclear FGF2 isoforms in specific cell populations during embryogenesis (Dono and Zeller, 1994).

Intracellular trafficking of exogenous FGF proteins Upon release in the extracellular compartment, FGF1 and FGF2 are internalized and translocated to the cell nucleus of target cells. An increasing body of evidence indicates that FGF internalization and nuclear translocation are coupled with the import of cognate membrane tyrosine kinase FGFRs and HSPGs, that are all internalized or translocated with FGF to the nucleus or to a perinuclear compartment (reviewed in Keresztes and Boonstra, 1999). Furthermore, nuclear translocation of intracrine FGF isoforms may involve the concomitant translocation of truncated FGFRs that, due to the lack of transmembrane or leader sequences, are retained intracellularly (Givol and Yayon, 1992; Johnston et al., 1995; Kilkenny and Hill, 1996; Ezzat et al., 2002). Translocation of exogenous FGF1 and FGF2 and their receptors to the nucleus appears to occur with several cell types and during specific phases of the cell cycle. In fibroblasts, FGF1 and FGFR1 trafficking occurs during the transition between G0 and G1 and, upon internalization, FGFR1 is translocated to a perinuclear compartment, whereas FGF1 accumulates in the nucleus (Imamura et al., 1994; Prudovsky et al., 1994, 1996). In addition to fibroblasts, nuclear accumulation of FGF1 and/or FGFR1 has been described in endothelial cells, kidney cells, rat uterus cells, myoblast or astrocytes (Prudovsky et al., 1994, 1996; Hu et al., 2000; reviewed in Keresztes and Boonstra, 1999). Exogenous FGF2 is also translocated to the cell nucleus and nucleolus of primary vascular endothelial cells at the transition between G0 and G1 (Bouche et al., 1987; Baldin et al., 1990). However, in condrocytes FGF2 and FGFR1 nuclear translocation occurs in late G1 and, upon internalization, FGFR1 localizes in a juxtanuclear position (Kilkenny and Hill, 1996). In condrocytes, an intracellular truncated FGFR1 is also relocated in a juxtanuclear compartment in late G1, even in the absence of exogenous FGF2 (Kilkenny and

Hill, 1996). These data indicate a synchronized nuclear translocation pathway for exogenous FGF2 and intracrine FGF2 HMW isoforms. Nuclear translocation of exogenous FGF2 and/or FGFR1 has also been described in astrocytes, Chinese hamster ovary cells and fibroblasts. However, in these cells FGFR1 appears to localize directly inside the nucleus, probably due to the action of importin b, a component of multiple nuclear import pathways (Maher, 1996; Clarke et al., 2001; Reilly and Maher, 2001 and references therein). FGF internalization is also mediated and regulated by low-affinity HSPG receptors, as indicated by the down-regulation of membrane HSPG upon exposure of cells to FGF (cell desensitization) (Rusnati et al., 1993). Studies with HSPG-depleted or -deficient cells showed that FGF2 can be internalized by cells even in the absence of HSPGs. However, HSPGs are required for the translocation to the cell nucleus of the internalized protein (Amalric et al., 1994; Fannon and Nugent, 1996). These findings most likely reflect the existence of two different internalization pathways, one mediated by FGFRs and HSPG, the other by HSPG (Gannoun-Zaki et al., 1991; Roghani and Moscatelli, 1992; Rusnati et al., 1993; Reiland and Rapraeger, 1993; Murono et al., 1993). The FGFR-mediated pathway occurs at an early time-point upon cell exposure to FGF, is very fast, drives FGF directly into the cytosol and, subsequently, into the cell nucleus. However, nuclear import requires a protective action of cointernalized HSPGs, as in their absence imported FGF2 is degraded, most probably in the lysosmal compartment (Rusnati et al., 1993; Reiland and Rapraeger, 1993). This is likely to be a major pathway for FGF nuclear import, as both FGFR and membrane and ECM HSPG are present in vivo, and HSPG are known to be part of the high-affinity FGF–FGFR complex (see below). (The protective effect of HSPGs may explain recent data indicating that nuclear translocation of both FGF1 and FGF2 in vascular endothelial cells is insensitive to lysosomal inhibitors (Hu et al., 2000).) By contrast, the pathway driven by HSPG is slow, occurs at later time-points, and does not target FGF2 to the cytosol (Rusnati et al., 1993; Reiland and Rapraeger, 1993; Rusnati and Presta, 1996). Probably, however, HSPGs also bring FGF to the cell nucleus.

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THE FGF FAMILY MEMBERS

In fact, HSPGs have been found to be present in a complex with FGF2 in the nucleus of cells exposed to exogenous FGF2 protein (Amalric et al., 1994). Furthermore, HSPG present in the ECM are known to possess NLS, and are internalized by cells and translocated to the cell nucleus in specific phases of the cell cycle (Fedarko and Conrad, 1986; Liang et al., 1997). In addition, recent data indicated that the protein core of both heparan and chondroitin sulfate proteoglycans behave as high-affinity receptors for FGFs, and potentiate their mitogenic activity (Milev et al., 1998; Goretzki et al., 1999; Mongiat et al., 2000). Thus, taken together, these data indicate that FGF is translocated to the cell nucleus in the form of a FGF/FGFR/HSPG molecular complex, although HSPGs can drive FGF translocation to the nuclear compartment even in the absence of FGFR. Unlike FGFRs, CFR that, as discussed above, mediates FGF3 secretion, appears to decrease the intracellular accumulation of exogenous FGF1 and FGF2. This FGF receptor shows a prominent association with the Golgi membranes but it is also present on the cell surface, particularly in human cells, and, as a secreted form, in the ECM. CFR may act by sequestering FGF at the cell surface or in the ECM, or may promote rapid transport of internalized FGF out of the cells. Since CFR is highly conserved in chicken, rat and humans and, in addition to FGF1, FGF2 and FGF3, is known also to bind FGF4, it is conceivable that it may mediate intracellular trafficking of other FGF family members (Burrus et al., 1992; Gonatas et al., 1995; Steegmaier et al., 1997; Zuber et al., 1997).

Binding of FGF proteins to the extracellular matrix Binding of FGFs to ECM HSPGs has several important functional implications such as protecting them from degrading agents, creating a reservoir of inactive factors that can be mobilized upon relevant biological processes, limiting the rate of FGF diffusion to favor local specific responses, or stabilizing long-lasting FGF concentration gradients required for tissue remodeling processes. As discussed below, these include tissue growth and differentiation during embryogenesis, wound healing, vasculogenesis or neoangiogenesis and also pathological processes

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such as chronic inflammation or tumors. During these processes, ECM is degraded by the concerted action of proteases, including metalloproteinases (MMPs), plasmin, elastin and heparanases, and FGFs are released in the form of molecular complexes with HS or HSPG protein core fragments (Whitelock et al., 1996; Milev et al., 1998; Nagase and Woessner, Jr., 1999; Goretzki et al., 1999; Ornitz, 2000; Mongiat et al., 2000; Iozzo and San Antonio, 2001). Although FGF/HS complexes are known to bind with high affinity to FGFRs (see below), different HS, HS fragments or HS sugar moieties show different activities, and can also be inhibitory for FGFs (Ornitz, 2000). For example, the HS chains of perlecan (a major component of ECM and most basement membranes) isolated from primary endothelial cells, endothelial cell lines or colon carcinoma cells, show different modulatory effects on FGF2 receptor affinity, binding and signaling (Knox et al., 2002). Different fragments of HS from smooth muscle cells mediate signaling of bound FGF2 through different FGFRs, and a similar differential receptor preference is shown by HSPG undergoing different posttranslational modifications that, moreover, appear to be cell specific (Berry et al., 2001; Fiore, 2001). Both the HS chains and the protein core of perlecan induce highaffinity binding of FGF2 or FGF7 to FGFR in HSdeficient cells (Aviezer et al., 1994; Mongiat et al., 2000). By contrast, glypican, a membrane-bound HSPG, inhibits FGF7 binding to FGFR (BonnehBarkay et al., 1997). Furthermore, resident cells are not the only source of ECM molecules, as cells infiltrating tissue may bring specific HSPGs in loco. For example, activated macrophages overexpress syndecan-2 and bear specific HS chains on CD44 that stimulate the binding of FGF2 to specific FGFRs (Clasper et al., 1999; Jones et al., 2000). All these mechanisms indicate that HSPGs are likely to play a fundamental role in tissue-regulated affinity of binding, specificity and activity of both FGFs and FGFRs (Ornitz, 2000). Different concentrations of soluble heparin or HS have dual effects on FGFs: high concentrations are inhibitory, whereas low concentrations are stimulatory, and the relative concentrations of FGF and HS appear to modulate the final response (Padera et al., 1999; Nugent and Iozzo, 2000; Fannon et al., 2000). Notably, ECM degradation is generally initiated by

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stimuli which affect both FGF and HSPG production, thus affecting their relative concentrations. For example, angiogenic stimuli induce endothelial cells to increase synthesis and pericellular deposition of perlecan (reviewed in Iozzo and San Antonio, 2001) and to produce angiogenic factors including FGF2 (see below). FGF2, in turn, up-regulates the expression of MMP2, which degrades basement membrane and ECM molecules (Ensoli et al., 1994a; Toschi et al., 2001). Degradation of ECM molecules, particularly perlecan, causes the release of additional sequestered factors, including hepatocyte growth factors or platelet factors, and inflammatory cytokines, such as interleukins and interferon (IFN)c (Iozzo, 1998). These factors, in turn, play a fundamental role in the expression and production of both ECM molecules and FGFs, as well as MMPs and other degradative enzymes (Ito et al., 1990; Ensoli et al., 1994a; Samaniego et al., 1995, 1997, 1998; Rutter et al., 1997; Sharma and Iozzo, 1998; Pilcher et al., 1999; Toschi et al., 2001). HSPGs themselves can be a reservoir of MMPs, namely MMP7, which is also released upon HSPG degradation (Yu and Woessner, Jr., 2000). Perlecan degradation also causes the release of the FGF-binding protein (FGF-BP), which mobilizes FGF1 and FGF2 with modulatory effects on angiogenesis (see below). HS chains released upon ECM degradation, or the HSPG ectodomains shed by cells, are very stable and, in turn, protect FGFs from degradation and oxidation, and stabilize the correct folding of bound FGF molecules (Lobb, 1988; Burke et al., 1993; reviewed in Rusnati and Presta, 1996; Ornitz, 2000). Thus, they ensure a long-lasting activity not only of pre-stored FGFs, but also of FGFs produced in response to local stimuli during the process leading to ECM degradation and tissue remodeling. In the absence of ECM degradation, binding of FGFs to ECM HSPGs severely limits their diffusion rate, and maintains them in a limited microenvironment close to the site of production. This creates a localized concentration gradient that is stabilized by HSPGs. This has important implications for FGFs signaling in development or tissue remodeling. For example, during limb development, FGF10 expressed by the mesoderm induces the formation of the apical ectodermal ridge, that signals back to the mesoderm by secreting FGF8 (Martin, 1998). These processes require long-lasting FGF gradients that are achieved

through binding of FGFs to ECM HSPGs (Cohn et al., 1995). Thus, by these actions ECM regulates the action of FGFs and integrates the several steps that are involved in FGF-dependent biological responses.

EXPRESSION, REGULATION, FUNCTIONS OF FGFs, AND DISEASE STATES Expression of FGFs and their regulation Most FGFs are expressed in several tissues and cell types in development (see below) and many of them are overexpressed in tumors. In fact, most of the FGF genes have been identified by screening tumor genes for a mitogenic effect on 3T3 fibroblasts. FGF3 and FGF8, for example, have been isolated from mammary tumor cells, FGF4 from stomach tumors and KS lesions and FGF9 from glial cells. FGF7, isolated as an epithelial cell growth factor from a fibroblast cell line, is expressed, like FGF10, in fibroblasts, especially of muscle origin. The other FGFs, such as FHF1–4/FGF11–14 and FGF16–20, have been more recently identified by homology-based molecular techniques and are mainly expressed during embryonic development (reviewed in Powers et al., 2000). FGF2 expression has been detected in all organs and tissues examined (Baird et al., 1986) and in endothelial cells of some, but not all, blood vessels (Hanneken et al., 1989; Cordon-Cardo et al., 1990). FGF2 is also synthesized by cultured fibroblasts, endothelial cells, glial cells and smooth-muscle cells (Schweigerer et al., 1987; Gospodarowicz et al., 1988; Hatten et al., 1988; Ensoli et al., 1989, 1994a; Samaniego et al., 1995, 1997; Faris et al., 1998; Barillari et al., 1999b). Because these cell types are ubiquitous, the expression of FGF2 may be widespread in vivo. FGF1 appears to have a more limited distribution than FGF2. It has been found in neural tissue, kidney, prostate and cardiac muscle (Thomas et al., 1984; Crabb et al., 1986; Gautschi-Sova et al., 1987; Casscells et al., 1990; Grothe and Nikkhah, 2001), and has also been identified in cultured vascular smooth cells (Winkles et al., 1987).

THE CYTOKINES AND CHEMOKINES

EXPRESSION OF FGF S , REGULATION , FUNCTIONS , AND DISEASE STATES

Various signals have been shown to trigger the expression of angiogenic factors during adulthood. These include metabolic stress (i.e. low pO2, low pH, or hypocalcemia), mechanical stress (e.g. pressure generated by proliferating cells), immune/inflammatory response (including immune/inflammatory cells infiltrating the tissue) and genetic mutations (e.g. activation of oncogenes or deletions of tumorsuppressor genes that control production of angiogenesis modulators) (reviewed in Carmeliet and Jain, 2000). Angiogenic FGFs undergo a similar modulation. It has been demonstrated that hypoxia enhances FGF2 expression in several xenograft tumor models (Le and Corry, 1999; Ishibashi et al., 2001). Hyperglycemia is also associated with increased levels of FGF2 in several conditions. For example, high FGF2 levels have been detected in pregnancy complicated by diabetes and it has been suggested that it may be responsible for the placental alterations characteristic of this complication (Di Blasio et al., 1997). In experimental models of atherogenesis, chronic exposure of endothelial cells to high levels of shear stress promotes FGF2 expression (Malek et al., 1993; Gosgnach et al., 2000). Similarly, increased tensile wall stress has been shown to strongly induce FGF2 production in vascular smooth muscle cells (Cheng et al., 1997). Inflammatory cytokines produced by accessory cells at sites of active neovascularization also promote expression of FGF2. This has been shown for IFNc alone or combined with IL-1b and tumor necrosis factor (TNF)a in cultured endothelial cells, KS cells (see below) and KS-like angioproliferative lesions induced in mice by these inflammatory cytokines (Ensoli et al., 1989; Samaniego et al., 1995, 1997, 1998; Fiorelli et al., 1998; Faris et al., 1998; Barillari et al., 1999b). Transforming growth factor (TGF)b has been shown to promote FGF2 expression and autocrine proliferation of renal and corneal stromal fibroblasts (Strutz et al., 2001), suggesting a role for FGF2 in the pathogenesis of renal tubulointerstitial and corneal fibrosis. Evidence from various experimental systems demonstrated that expression of FGF2 is down-regulated by IFNa and IFNb in vitro and that systemic administration of IFNa inhibits FGF2 expression and tumor growth in several murine xenograft tumor models (Singh et al., 1995; Dinney et al., 1998). In addition,

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systemic administration of IFNa has been shown to reduce body fluid FGF2 levels and to be an effective and well tolerated therapy for several diseases, such as infantile hemangiomas (Chang, E. et al., 1997) and KS (Mihalcea et al., 1999).

Functions of FGFs Functions in development Most FGFs are expressed in invertebrate or vertebrate embryos, indicating that they have important roles in embryogenesis. In vertebrates, some FGF genes, including fgf3, 4, 15, 17 and 19, are mainly or exclusively expressed during embryonic life, whereas others are expressed in both embryonic and adult tissues (i.e. fgf1, 2, 5–7, 9–14, 16, 18 and 20–23) (reviewed in Ornitz and Itoh, 2001). The gene-knockout mouse approach has been used to study the function(s) of several members of the FGF family in development. Most frequently, the phenotypes obtained range from early embryonic to postnatal lethality, as in fgf4, 8, 9, 10, 15 and 18 null mice. However, fgf-knockout mice also present with subtle abnormalities, as for example, the angora mutation or mild cerebellar defects in adult fgf5 or fgf17 null animals (reviewed in Ornitz and Itoh, 2001). These findings most likely reflect the unique tissuespecific expression of some FGFs, as in the case of lethal phenotypes, or FGF functional redundancy in milder phenotypes, probably generated by overlapping patterns of expression of FGF family members with similar receptor-binding properties and activity. Indirect evidence indicates, however, that functional redundancy may involve even distant FGF family members, as indicated by studies with fgf1- and fgf2knockout mice. Homozygous gene deletions of fgf2 lead to viable and fertile mice indistinguishable from fgf2/ littermates by gross examination. However, FGF2-deficient mice present with subtle but significant reductions in the number and organization of cortical neurons, vascular hypotension resulting from an impaired neural control of the vascular tone, and a delayed healing of epithelial wounds (Dono et al., 1998; Ortega et al., 1998). These data indicated compensation of fgf2 deletion by other FGF genes, most likely fgf1, as FGF1 shares with FGF2 many structural and functional similarities. However, fgf1 null mice do

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not show any of the abnormalities associated with deletion of fgf2 and exhibit normal brain structure and normal rates of wound healing; furthermore, fgf1–fgf2 double knockout mice show defects similar to those observed in fgf2 null mice (Miller et al., 2000). Thus, compensation of fgf2 deletion is likely to occur through the activity of other FGFs, an observation indicating extensive functional overlapping between family members with a variable degree of homology. Evidence indicates that some FGFs play a fundamental role in very early stages of development, as they promote the division of embryonic and extraembryonic cells in pre-implantation mouse embryos, probably upon the fifth cell division (Chai et al., 1998). In addition, FGF3 and FGF4 have been shown to induce mesoderm differentiation during the formation of the three germinal layers in chick gastrulation (Paterno et al., 1989). Furthermore, FGF8 is required for the correct migration of mesodermic and endodermic cells, and in fgf8 null mice this results in embryonic death on day 7 (Paterno et al., 1989; Sun et al., 1999) (Table 31.1). FGFs have also been shown to play a significant role in organogenesis, including the development of the nervous system, lungs and limbs (reviewed in Powers et al., 2000; Ford-Perriss et al., 2001). For example, FGF8, FGF17 and FGF2 intervene in midbrain and cerebellar development (Crossley et al., 1996; Ortega et al., 1998; Ornitz and Itoh, 2001) whereas FGF10

plays a key role in lung development by promoting the branching and differentiation of lung epithelium (Sekine et al., 1999) (Table 31.1). FGF1, FGF2 and FGF4 are critical in limb bud induction and development, as demonstrated both in chick and mouse models (Table 31.1). In fact, the implant of beads soaked with these FGFs in the flanks of chick embryos results in the induction of ectopic limb buds which can develop in almost normal structures (Cohn et al., 1995). Moreover, removal of limb bud areas critical for correct limb development can be replaced by the administration of recombinant FGF2 or FGF4 (Niswander et al., 1993; Fallon et al., 1994). Interestingly, it has been demonstrated that thalidomide (a drug causing phocomelia, or congenital defects of the limbs) not only blocks limb buds (Stephens, 1988) but also interferes with FGF2-induced angiogenesis in vitro and in vivo (D’Amato et al., 1994). These data suggest that the teratogenic effects of thalidomide may be due to modulation of FGF signaling, although the precise nature of this interference is uncertain (Powers et al., 2000).

Functions in vasculogenesis Recent studies have demonstrated that FGF signaling plays a crucial role in the earliest stages of vasculogenesis, including differentiation of mesodermal cells in blood islands and appearance of the hemangioblasts,

TABLE 31.1 FGF roles in development Tissue/Organ

FGF member(s) mainly involved

Role/Activity

Mesoderm induction

FGF3, FGF4, FGF8, FGF2

Epithelial to mesenchymal transition of epiblastic cells, formation of 3 germinal layers during gastrulation

Vascular system

FGF2

Formation of blood islands in yolk sac, induction and differentiation of VEGFR-2 hemangioblast

Limb

FGF1, FGF2, FGF4, FGF5, FGF8, FGF10

Limb induction and development, myogenesis, chondrogenesis

CNS

FGF2, FGF8, FGF15, FGF17, FHF1–4

Midbrain and cerebellar development, neuronal differentiation, proliferation and survival

Lung

FGF10, FGF9, FGF2

Branching and differentiation of lung epithelium, modulation of elastin synthesis

Ear

FGF8

Development of middle ear (3 ossicle chain) and of structures deriving from the first branchial arch (jaws, teeth)

VEGFR-2, vascular endothelial growth factor receptor 2; CNS, central nervous system. THE CYTOKINES AND CHEMOKINES

EXPRESSION OF FGF S , REGULATION , FUNCTIONS , AND DISEASE STATES

the putative common precursors of endothelial cells and hematopoietic cells (Amaya et al., 1991, 1993; Choi et al., 1998; Poole et al., 2001). Moreover, upon exposure to FGF2, these pluripotent precursors express the vascular endothelial growth factor receptor 2 (VEGFR-2), a marker specifically associated with the hemangioblast lineage (Flamme et al., 1997) (Table 31.2). In addition, FGF2 is capable of inducing vasculogenesis in quail blastodisc-derived embryoid bodies, and dissociated epiblasts (Krah et al., 1994; Poole et al., 2001). In contrast, FGF2 and FGFRs do not seem to play a major role in the subsequent morphogenesis of the vascular system, in which, instead, VEGF becomes essential (Risau and Flamme, 1995).

Functions in angiogenesis Angiogenesis is the process through which new capillaries are formed from pre-existing vessels (Klein et al., 1997). This process involves a number of temporally and spatially tightly controlled steps. Stimulation of endothelial cells with a variety of molecules, the best characterized being growth factors such as

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VEGFs and FGFs, induces endothelial cells to produce and export proteases, such as MMPs and plasminogen activators (Folkman and Shing, 1992). The proteases digest the basement membrane surrounding the vessel, allowing endothelial cells to invade the surrounding tissue, where they proliferate and migrate to form a sprout. The sprout elongates and the endothelial cells differentiate to form a lumen. Eventually, the endothelial cells in the newly formed vessels produce growth factors such as plateletderived growth factor (PDGF)-BB, which attracts supporting cells, pericytes and smooth muscle cells to the vessel (Hirschi and D’Amore, 1997). The supporting cells and the basement membrane that surround the endothelial cells are critical for vessel stability. In physiologic conditions angiogenesis is strictly regulated; the vessels develop in an organized fashion and once the need to supply the tissue with nutrients and oxygen is met, production of the stimulatory factor ceases and endothelial cells become quiescent. FGF2 and FGF1 were the first angiogenic growth factors to be purified and sequenced in the early 1980s by means of their heparin-binding capacity,

TABLE 31.2 FGF functions in adulthood Tissue/Organ

FGF member(s) mainly involved

Role/Activity

Angiogenesis

FGF1, FGF2, FGF3, FGF4, FGF7

Proliferation and migration of EC, induction of pericellular proteolysis (uPA, MMPs) and EC invasion, differentiation of capillary-like tubules, deposition of ECM components, expression of integrins, synergy with other angiogenic factors (VEGF), down-regulation of GBP-1

Wound healing

FGF1, FGF2, FGF7, FGF10

Proliferation and chemotaxis of EC, epithelial cells, fibroblasts and keratinocytes, induction of pericellular proteolysis (uPA, MMPs), induction of angiogenesis, deposition of ECM components, expression of integrins

Central and peripheral nervous system

FGF2

Survival of cultured neurons, protection from toxic and mechanical injuries, neurite growth and branching, neurotransmission or neuromodulation

Hematopoiesis

FGF2, FGF4

Synergize with hematopoietic cytokines in inducing early and committed precursors proliferation and differentiation (erythroid and myeloid), proliferation of stromal cells, promote survival of hematopoietic precursors, antagonize downmodulating effects of TGFb

EC, endothelial cell; uPA, urokinase-like plasminogen activator; MMPs, matrix metalloproteases; ECM, extra-cellular matrix; VEGF, vascular endothelial growth factor; GBP-1, guanylate binding protein-1; TGFb, transforming growth factor b. THE CYTOKINES AND CHEMOKINES

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using heparin-affinity chromatography (Shing et al., 1984; Maciag et al., 1984; Esch et al., 1985). These two prototypes of FGFs are preferentially implicated in angiogenesis as demonstrated in a variety of in vivo models (Folkman and Shing, 1992). In addition to these factors, FGF3 and FGF4 have also been shown to promote neovascularization in the chick chorioallantoic membrane assay (Costa et al., 1994; Yoshida et al., 1994), whereas FGF7, although widely thought of as an epithelial cell mitogen, was found to be active in the rat cornea assay (Gillis et al., 1999). To date, the role of the other FGFs as angiogenic factors remains undefined (Tables 31.3 and 31.4).

FGF1 and FGF2 have been shown to be autocrine growth factors for a variety of cell types involved in the angiogenic process, including endothelial cells and smooth muscle cells (Shing et al., 1984; Winkles et al., 1987; Ensoli et al., 1989; Samaniego et al., 1995, 1997). FGF2 also stimulates EC to migrate and to form capillary-like structures in vitro (Montesano et al., 1986; Tsuboi et al., 1990) (Table 31.2). FGFs can induce endothelial cells either to proliferate or to migrate by signaling through the same receptor. In addition, receptors other than FGFRs or HS, such as integrins (Miyamoto et al., 1996; Kanda et al., 1999), as well as mechanical properties of the

TABLE 31.3 Roles of FGFs in diseases Function

FGF member(s) mainly involved

Disease/Activity

Rheumatoid arthritis

FGF2, FGF1

Cartilage disruption upon neovascularization, synovia hyperplasia, inhibition of OCIF production by synovial cells

Eye neovascular diseases

FGF2

Retinal neovascularization in PDR, choroidal neovascularization in AMD

Neurodegenerative diseases

FGF2

Reduced or sequestered FGF in Parkinson’s and Alzheimer’s diseases, increased in Huntington’s disease

Atherosclerosis

FGF1, FGF2

Neointimal cell proliferation, neovascularization of atheromatous lesions

Tissutal fibrosis

FGF2

Renal tubulointerstital and corneal fibrosis

Early hyperplastic Kaposi’s sarcoma

FGF2

Expressed by KS cells and resident endothelial cells, increased in KS tissues and sera, released from ECM storage by HIV-1 Tat, induction of neoangiogenesis, GBP-1 down-regulation, growth, migration and invasion of KS and endothelial cells, KS and endothelial cell survival through Bcl-2 up-regulation in synergy with HIV-1 Tat, induction of MMP expression and activity in synergy with HIV-1 Tat KS inhibited by antisense oligonucleotides against FGF2 FGF2-mediated angiogenesis and KS cell invasion inhibited by HIV protease inhibitors

Tumor angiogenesis

FGF2, FGF4, FGF5

Increased in tissue and/or body fluids of many tumors, correlates with tumor burden and prognosis, released by tumor cells themselves, by accessory cells or by tumor cells in response to signals from accessory cells, released by ECM storage by enzymatic (proteases, heparanases) and non-enzymatic (FGF-BP) mechanisms

OCIF, osteoclastogenesis inhibitory factor; PDR, proliferative diabetic retinopathy; AMD, age-related macular degeneration; KS, Kaposi’s sarcoma; ECM extra-cellular matrix molecules; GBP-1, guanylate binding protein-1; MMP, matrix metalloproteinase; FGF-BP, fibroblast growth factor binding protein. THE CYTOKINES AND CHEMOKINES

TABLE 31.4 Main features of fibroblast growth factors Name

Synonym

Functions

Associated diseases

FGF1

Acidic FGF, Single form, no signal sequence, aFGF NLS, mainly exported, nuclear translocation in target cells; autocrine and paracrine

Limb development, angiogenesis, wound healing

Rheumatoid arthritis, atherosclerosis

FGF2

Basic FGF, bFGF

Four CUG isoforms with NLS, one ATG form lacking NLS, no signal sequence, exported (ATG form) or nuclear/nucleolar (CUG forms), intracrine activity (CUG forms), translocated in the nucleus of target cells (ATG form); autocrine and paracrine (ATG form)

Mesoderm induction, vasculogenesis (development of blood islands, induction of (VEGFR-2) hemangioblasts), development of limb, SNC, angiogenesis, wound healing, neuron survival, neurite branching, hematopoiesis (synergy with hematopoietic cytokines)

Rheumatoid arthritis, atherosclerosis, neovascular and neurodegenerative diseases, tissutal fibrosis, Kaposi’s sarcoma, tumor angiogenesis, tumor growth and tumor invasion

FGF3

Int-2

One CUG form, one ATG form, signal sequence, multiple NLS, secreted (ATG form), secreted and nuclear/nuclear (CUG form), autocrine, paracrine and intracrine, secretion modulated by CFR

Mesoderm induction, angiogenesis

Mammary carcinogenesis (mouse)

FGF4

Kaposi FGF Signal sequence hst-1

Mesoderm induction, limb development, angiogenesis, hematopoiesis

Gastric cancer, Kaposi’s sarcoma, tumor angiogenesis

FGF5 FGF6

hst-2

Structure and trafficking

Signal sequence

Limb development

Pancreatic cancer

Signal sequence

Skeletal muscle regeneration

Leukemia

TABLE 31.4 Continued Name

Synonym

Structure and trafficking

Functions

Associated diseases

FGF7

KGF

Signal sequence

Keratinocyte growth factor, kidney and lung development, angiogenesis, wound healing

Breast cancer, prostatic hyperplasia

Signal sequence, 7 isoforms

Limb and CNS development

Prostatic and ovarian cancer

FGF8 FGF9

GAF

Non-canonical leader sequence, secreted

Glia-activating factor, motoneuron survival, lung development

Synovial chondromatosis

FGF10

KGF-2

Signal sequence, similar to FGF7

Limb and lung development, wound healing

?

FGF11–14 FHF1–4

No signal sequences, several isoforms with NLS, localization in nucleus/nucleolus, intracrine actions

Limb and SNC development

?

FGF15

Mouse gene, no human homolog identified

CNS development

?

FGF16–19

Signal sequence (FGF17–FG19), no signal sequence in FGF16 that is, however, secreted

CNS development (FGF17)

?

Secreted despite absence of signal sequence, homologous to FGF9

Limb development

Potential oncogene?

FGF21

Signal sequence

Expressed in liver

?

FGF22

Signal sequence

Heart development

?

FGF23

Signal sequence

Expressed in brain and thymus

Renal phosphate wasting disorder (autosomal-dominant hypophosphatemic rickets)

FGF20

XFGF-20

NLS, nuclear localization sequences; VEGFR-2, vascular endothelial growth factor receptor 2; CNS, central nervous system; CFR, cysteine-rich FGF receptor; KGF, keratinocyte growth factor.

EXPRESSION OF FGF S , REGULATION , FUNCTIONS , AND DISEASE STATES

extracellular microenvironment (Ingber and Folkman, 1989) and activation of molecules involved in cell adhesion, such as focal adhesion kinase (FAK) (Hatai et al., 1994), can also modulate the biologic response of cells to FGF2 (see below). Finally, it has been recently demonstrated that FGF2 down-regulates in endothelial cells the expression of the guanylate binding protein-1 (GBP-1), which, in contrast, is upregulated by inflammatory cytokines (Guenzi et al., 2001) (Table 31.2). This modulatory mechanism is responsible for FGF-mediated angiogenic switch. In fact, when GBP-1 is expressed, endothelial cells cease to divide and become activated, whereas GBP-1 down-regulation by FGF induces endothelial cells to proliferate and to acquire the angiogenic phenotype (Guenzi et al., 2001). As discussed above, proteolytic activity is of pivotal importance during the first phase of angiogenesis, as invasive endothelial cells must degrade the perivascular ECM in order to migrate into tissue to be vascularized. FGF1 and FGF2 are key modulators of the proteolytic response of endothelial cells during this process (Table 31.2). In particular, FGF2 has been shown to increase in endothelial cell production and/or activation of plasminogen activators (PAs), including urokinase-type PA (uPA), that converts the zymogen plasminogen into active plasmin, a protease capable of degrading several ECM components and of activating MMPs (Presta et al., 1986; Ensoli et al., 1994b; Barillari et al., 1999a; Toschi et al., 2001). FGF2 also up-regulates uPA receptor (uPAR) expression in endothelial cells (Mignatti et al., 1991), enhancing their capability to synthesize and bind uPA on the cell membrane. In contrast, plasmin activates TGFb1 which counteracts the stimulatory effects of FGF2 on endothelial cells by down-regulating uPA and MMPs expression, and by stimulating the synthesis of protease inhibitors, such as plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitors of MMPs (TIMPs) (Klein et al., 1997). Expression and activity of MMPs are also modulated by FGFs (Table 31.2). FGF2, in particular, has been shown to induce both MMP-2 mRNA expression (Ensoli et al., 1994a; Barillari et al., 1999a) and MMP-2 protein secretion and activation in endothelial cells (Toschi et al., 2001). However, FGF2 promotes the expression of specific MMP inhibitors such as tissue inhibitors of MMP-1 and MMP-2, thus promoting a

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negative regulation of MMP activity (Andersen et al., 1998; Toschi et al., 2001). FGF2 also modulates angiogenesis by promoting the expression of integrins (Table 31.2). Integrins are membrane heterodimeric receptors that mediate cell interaction with ECM proteins such as vitronectin, fibronectin, laminin and collagens, thus transducing ECM information to the interior of the cell. These integrins are highly expressed at the sites of active angiogenesis and provide adhesion signals which are required for endothelial cell survival during proliferation, migration and invasion (Barillari et al., 1993, 1999a, 1999b; Sepp et al., 1994; Ensoli et al., 1994b). Ligation of avb3 integrin has been found to be necessary for FGF2-mediated angiogenesis in the chicken chorioallantoic membrane, quail embryo, rabbit cornea, mouse retina or human skin grafts in immunodeficient mice (Brooks et al., 1994; Friedlander et al., 1995; Hammes et al., 1996). In fact, avb3 integrin ligation promoted by FGF2 is essential for the sustained activation of MAPK in response to angiogenic factors, and for endothelial cell proliferation (Eliceiri et al., 1998). In agreement with these data, block of avb3 with specific antagonists promotes apoptotic death in endothelial cells through induction of both the p53-inducible cell cycle inhibitor p21WAF/CPP1 and the bax cell-death pathway (Stromblad et al., 1996). These findings indicate, therefore, that binding of ligands to avb3 suppresses this apoptotic pathway and promotes cell survival and differentiation during angiogenesis. In addition, FGF2 has been shown to promote the expression of the antiapoptotic oncogene Bcl-2 in endothelial cell undergoing an apoptotic stimulus (Karsan et al., 1997). Another mechanism by which FGFs promote angiogenesis is the synergy with other angiogenic growth factors, particularly VEGF (Asahara et al., 1995; Samaniego et al., 1998).

Functions in wound healing Wound healing is a complex process that involves chemotaxis and proliferation of cells, including inflammatory cells, neovascularization, synthesis of extracellular matrix proteins and remodeling of the scar. Local application of FGF1, FGF2 and FGF10 accelerates wound healing in several animal models (Mellin et al., 1995; Okumura et al., 1996; Xia et al.,

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1999). It is likely that these FGFs and FGF7 play a role in several steps of this physiologic process, in particular in the formation of the granulation tissue (repair phase) and in remodeling of collagen fibers and organization of the neo-formed vessels (regeneration phase) (Powers et al., 2000). This is in large part due to their capability of promoting chemotaxis and proliferation of many cell types, including endothelial cells, fibroblasts, epithelial cells and keratinocytes (Table 31.2) (Rubin et al., 1989; Bennett and Schultz, 1993). Moreover, induction of uPA in endothelial cells and fibroblasts (Besser et al., 1995) promotes a pericellular zone of fibrinolisis facilitating the migration of endothelial cells through the fibrin clot present at the wound site (Table 31.2) (Werb, 1997). FGF2 also facilitates endothelial cell migration during the repair phase by promoting the expression of the avb3 integrin, that mediates the binding to ECM components such as vitronectin and fibrinogen (Barillari et al., 1999b). This integrin complex can also bind MMPs, which are activated by FGF2 (Ensoli et al., 1994a; Toschi et al., 2001), thus providing another mechanism for the formation of a pericellular zone of fibrinolisis during the repair phase (Brooks et al., 1996) (Table 31.2).

Functions in the nervous system FGFs are well recognized for their actions on the nervous system not only as neural morphogenetic factors (as summarized above) but also as neurotrophic factors. In particular, FGF2 has been recognized as a potent survival factor for a wide range of brain neurons in culture, such as cortical and hippocampal neurons (Morrison et al., 1986; Walicke et al., 1986) (Table 31.2). FGF2 has been shown to protect neurons from toxic substances such as glutamate and amyloid b proteins (Mattson et al., 1993 and references therein), independently of its mitogenic activity for glial cells (Walicke and Baird, 1988) (Table 31.2). The effect of FGF2 on neuronal survival has also been examined in several models in vivo. For example, implantation of FGF2-soaked gel foam rescued medial septal neurons after fimbria fornix transection (Otto et al., 1989) and anotomized neurons in the dorsal lateral geniculate nucleus (Agarwala and Kalil, 1998). In addition, exogenously applied FGF2 rescues

injured sensory neurons and supports neurite outgrowth of transectioned peripheral nerves, suggesting that FGFs may also have a role in peripheral nerve regeneration (Grothe and Nikkhah, 2001) (Table 31.2). The molecular mechanisms of FGF2 neurotrophic activity are not fully understood, but recent data indicate that the MEK/ERK signal transduction cascade is involved in this response to FGF2 (Abe and Saito, 2000). FGF2 also affects neurite growth and regeneration through pathways independent of its survivalpromoting activity. In particular, FGF2 has been recognized as a branching factor, promoting bifurcation and growth of axonal branches but not axon elongation, which is modulated by other growth factors (Aoyagi et al., 1994). Finally, since FGF2 modulates synaptic transmission in the hippocampus, it has been hypothesized that it may act like a neurotransmitter or a neuromodulator.

Functions in hematopoiesis Evidence indicates that FGF2 is expressed and produced by bone marrow stromal cells, which include a high proportion of fibroblast as well as mesenchimaltype cells (Oliver et al., 1990; Brunner et al., 1993; Allouche, 1995). FGF2 is also expressed and produced by cells from several mature cell lineages, such as megakaryocytes and platelet, macrophages, granulocytes and lymphocytes (Brunner et al., 1993; Blotnick et al., 1994). These cells produce and respond to FGF2 in an autocrine/paracrine fashion as they express FGFR1 and FGFR2 (Bikfalvi et al., 1992). Furthermore, FGFR4 is expressed in erythroid progenitors and is down-regulated upon cell differentiation (Koritschoner et al., 1999b). FGF2 is released and stored in bone marrow ECM, which serves as a reservoir of growth factors and hematopoietic cytokines for hematopoietic processes (Roberts et al., 1988). Growing evidence indicates that FGF2 positively regulates hematopoiesis by acting on various cellular targets including stromal cells, early and committed hematopoietic progenitors, and probably also mature blood cells (Table 31.2). However, they do not appear to be hematopoietic cytokines per se, as they cannot stimulate proliferation of hematopoietic stem cells or committed progenitors alone. For example, addition

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of FGF4 to human long-term bone marrow cultures increases in a dose-dependent manner the cell density of the stromal layer and the number of hematopoietic colony-forming cells (Quito et al., 1996), however, the primary effects of FGF2 appear to be directed on stromal cells and not hematopoietic cell progenitors. FGF2 synergizes with stem cell factor to promote both erythroid proliferation and differentiation (Koritschoner et al., 1999a), and with granulocyte–monocyte colony-stimulating factor (GM-CSF) to induce myeloid progenitor cell growth (Gabrilove et al., 1994) (Table 31.2). In addition, it counteracts the suppressive effects of TGFb (Gabrilove et al., 1993). Moreover, in association with IL-3, GM-CSF and erythropoietin it induces committed precursors to form granulocyte–monocyte (GM) colonies and erythroid bursts (Gabbianelli et al., 1990; Gallicchio et al., 1991) (Table 31.2). However, there are no data indicating that FGF2 can act alone in these processes. Thus, FGFs action in hematopoiesis consist mostly in the modulation of the complex cytokine network regulating hematopoiesis, including positive and negative regulators (Table 31.2).

FGFs in diseases Owing to the multiple effects of FGFs in several biologic processes, changes in FGF production or release are associated with pathologic conditions of different nature and origin, including chronic inflammation, neovascular diseases, neurodegenerative diseases and tumor angiogenesis (Table 31.3). FGF1 and FGF2 have been shown to be associated with the synovia hyperplasia and with the neovascularization that invades the joints and destroys the cartilage in rheumatoid arthritis. In addition, it has been demonstrated that FGF2 synovial levels correlate with disease severity (Manabe et al., 1999) and that it may contribute to bone destruction by inhibiting the production of the osteoclastogenesis inhibitory factor (Table 31.3) (Yano et al., 2001). Evidence for involvement of FGFs in neovascular diseases comes from the observation that vitreous samples or choroidal neovascular membranes from patients with proliferative diabetic retinopathy (PDR, Table 31.3) or patients with age-related macular degeneration (AMD, Table 31.3) have higher levels of FGF2 as compared with normal individuals (Frank

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et al., 1996; Boulton et al., 1997; Neely and Gardner, 1998). Changes in FGF2 levels are also associated with Alzheimer’s, Huntington’s, and Parkinson’s diseases (Bikfalvi et al., 1997). In Huntington’s disease, an increase in FGF2 expression is correlated with the severity of the disease (Tooyama et al., 1993b), whereas a loss of FGF2 in the substantia nigra is associated with Parkinson’s disease (Tooyama et al., 1993a) (Table 31.3). In addition, several observations suggest that FGF1, FGFR1 and, to a lesser extent, FGF2, may be implicated in the pathogenesis of atherosclerotic lesions (Brogi et al., 1993; Hughes et al., 1993) (Table 31.3). However, cancer development is the disease process most closely related to the actions of FGFs, which mediate hyperplastic reactive proliferative diseases like KS and promote tumor growth and metastasis through effects on both endothelial cells and tumor cells (Table 31.3). These effects are due to both a deregulated expression of FGFs (Folkman et al., 1989; Folkman and Shing, 1992) or, as discussed below, FGFR signaling. In this regard, tumor cells are known to release large amounts of angiogenic factors. In particular, FGF2 plays a key role in tumor pathogenesis by direct effects or in synergy with other angiogenic factors (Shing et al., 1984; Folkman and Klagsbrun, 1987). In addition, circulating cells and inflammatory cells infiltrating tumor lesions can also produce large quantities of FGF2 (Table 31.3). As a consequence, increased FGF2 levels are found in body fluids of patients with a wide spectrum of tumors including solid tumors, lymphomas or leukemias (Nanus et al., 1993; Nguyen et al., 1994; Takahashi et al., 1994; Ascherl et al., 2001) (Table 31.3). The local and systemic increase of FGF2 promotes tumor growth and metastasis not only through angiogenic effects but, as discussed below, by direct effects on tumor cell survival, proliferation and invasion. FGFs produced and released by tumor cells, stromal cells and cells infiltrating tumor lesions exert their activity through autocrine, paracrine and, probably, intracrine effects on all these cell types (Klein et al., 1997). For example, human gliomas constitutively release FGF2 with autocrine mitogenic effects for tumor cells and paracrine angiogenic effects for endothelial cells present in the surrounding stroma (Takahashi et al., 1990; Takahashi et al., 1992). By

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contrast, in pancreatic cancer FGF5 is produced only by accessory cells including macrophages and fibroblasts, most likely in response to epidermal growth factor (EGF), PDGF and TGFa that are overexpressed in pancreatic tumors (Werner et al., 1991; Kornmann et al., 1997). Similarly, although FGF2 is constitutively released by the spindle cells of KS, TH1-type cytokines produced by inflammatory cells infiltrating KS lesions increase in KS spindle cells or induce in endothelial cells FGF2 production and release, indicating a role for an autocrine/paracrine cytokine loop in tumor development and/or progression (Samaniego et al., 1995, 1997, 1998). In fact, evidence indicates that KS lesions are initiated by inflammatory cytokines produced by activated peripheral blood mononuclear cells (PBMC) and lympho–monocytes infiltrating tissues, probably in response to infection by the human herpesvirus 8 (HHV8) (Ensoli et al., 2001; Sturzl et al., 2001). The same inflammatory cytokines, particularly TNFa, IL-1 and IFNc, induce the expression of chemokines that recruit in tissue activated and HHV8-infected circulating cells that exacerbate the reactive processes, and angiogenic molecules (FGF2, VEGF), which mediate lesion formation. Several lines of evidence indicate that, among these factors, FGF2 plays a major role in KS pathogenesis (Table 31.3). In fact, FGF2 is highly expressed by KS spindle cells and promotes, in an autocrine and paracrine fashion, KS and endothelial cell proliferation, migration and invasion, and angiogenesis, by down-regulating GBP-1 expression (that is induced by inflammatory cytokines and blocks endothelial cell growth) and by up-regulating MMP-2 expression and activity in synergy with the HIV-1 Tat protein (Ensoli et al., 1989, 1994a, 1994b; Barillari et al., 1992, 1994b, 1999a, 1999b; Samaniego et al., 1995, 1997, 1998; Fiorelli et al., 1995; Toschi et al., 2001; Guenzi et al., 2001) (Table 31.3). Although FGF2 acts in a synergic way with VEGF in KS and angiogenesis, injection of recombinant purified FGF2 in mice is capable of inducing KS-like lesions alone that are indistinguishable from lesions obtained upon the inoculation of FGF2 and VEGF combined, or KS cells, which produce high levels of both FGF2 and VEGF (Ensoli et al., 1994a; Barillari et al., 1999b; Sgadari et al., 2002). In addition, block of FGF2 but not VEGF expression by antisense oligonucleotides or specific antibodies inhibits KS cell growth, angiogenesis and formation of experimental

KS lesions (Ensoli et al., 1994b). Furthermore, indinavir and saquinavir, two antiretroviral drugs directed against the human immunodeficiency virus (HIV) protease, are capable of blocking KS and mouse KSlike lesions through potent antiangiogenic effects inhibiting FGF2-mediated KS and endothelial cell invasion (Sgadari et al., 2002) (Table 31.2). FGF activity can also be up-regulated in tumors upon ECM remodeling due to enzymatic mobilization of FGF molecules bound to HSPG (Table 31.3). In fact, expression of MMPs and heparanases by tumor cells and accessory cells is associated with tumor aggressiveness and metastatic potential. For example, MMPs have been found to be significantly increased in malignant melanoma (Ray and Stetler-Stevenson, 1995), renal cell carcinoma (Slaton et al., 2001), ovarian carcinoma (Yoneda et al., 1998) and KS lesions (Ensoli et al., 1994a; Barillari et al., 1999a; Toschi et al., 2001), and increased levels of heparanases have been detected in breast, colon and liver carcinomas (Hulett et al., 1999; Vlodavsky et al., 1999). Thus, by modulating the expression of degradative enzymes, tumors mobilize FGFs from the ECM making them available in active form for tumor cell survival, growth and invasion. In addition, a non-enzymatic displacement of FGF2 from ECM has been demonstrated for the HIV-1 Tat protein in acquired immunodeficiency syndrome (AIDS)-associated KS. Like FGFs, Tat has heparinbinding activity and is released by HIV-1-infected cells even in the absence of leader sequences (Ensoli et al., 1990, 1993; Chang, H.C. et al., 1997). Several lines of evidence indicate that extracellular Tat retrieves preformed HSPG-bound FGF2 into a soluble, biologically active form that mediates the growth of endothelial and KS cells (Ensoli et al., 1994a; Barillari et al., 1999a; reviewed in Ensoli et al., 2001). Furthermore, Tat synergizes with FGF2 in increasing MMP-2 secretion and activation (Ensoli et al., 1994a; Toschi et al., 2001). Thus, Tat has the capability of increasing the bio-availability of FGF2 by direct mobilization or by activating ECM degradation enzymes, and by these methods it acts as a progression factor in AIDS–KS (Table 31.3). FGFs are also released from the ECM through the activity of a carrier protein, the FGF–BP, that shuttles FGFs from their sites of storage to FGFRs. Originally isolated from an epidermoid carcinoma cell line, FGF–BP is expressed in the skin

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and intestine during development, is rather downregulated in adult tissues, and binds both FGF1 and FGF2 in a non-covalent reversible manner (Wu et al., 1991). FGF–BP-coding gene is, however, up-regulated in carcinogen-induced skin tumors as well as in squamous cell carcinoma and colon carcinoma (Czubayko et al., 1997; Kurtz et al., 1997) (Table 31.3). Depletion of FGF–BP in these cell lines by specific ribozymes reduced tumor growth and angiogenesis in a xenograft tumor model, suggesting that FGF–BP regulation may be of fundamental importance for the control of FGF activity (Czubayko et al., 1997). FGFs can also promote tumor growth through antiapoptotic effects. In particular, FGF2 can regulate cell apoptosis by modulating the expression of the antiapoptotic oncogene Bcl-2. Neutralizing antibodies to FGF2 were found to induce apoptosis of human glioma cells overexpressing FGF2 and this effect was overcome by overexpression of Bcl-2 (Murai et al., 1996). Moreover, FGF2 was shown to up-regulate Bcl-2 expression in B-cell chronic lymphocytic leukemia (Konig et al., 1997) and neuroblastoma (Raguenez et al., 1999), protecting tumor cells from apoptotic stimuli. Finally, elevated levels of Bcl-2 protein have been described in advanced KS lesions (Morris et al., 1996) (Table 31.3). Moreover, FGF2 synergizes with the HIV-1 Tat protein in up-regulating Bcl-2 expression in endothelial cells and mouse KS-like lesions, suggesting a further mechanism by which FGF2 and Tat may contribute to AIDS–KS pathogenesis (Ensoli, submitted) (Table 31.3). Finally, when combined with VEGF, FGF2 can induce vascular permeability and edema in the guinea pig model (Samaniego et al., 1998).

RECEPTOR EXPRESSION, REGULATION AND DISEASE STATES Receptor expression and regulation Four distinct membrane FGFRs, belonging to the receptor tyrosine kinase superfamily, have been identified on the basis of their capability of binding the different members of the FGFs family with high affinity (reviewed in Klint and Claesson-Welsh, 1999). All known FGFRs (FGFR1 to FGFR4) show the same

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structure composed of an extracellular binding portion, a single transmembrane helix, and a cytoplasmic domain with tyrosine kinase activity (Plate 31.1B). The extracellular part of the receptor contains three immunoglobulin (Ig)-like domains, namely D1, D2 and D3, with a stretch of acidic amino acids (the acidic box) between D1 and D2. Interactions with FGFs are mediated by D2 and D3 as well as by the linker region connecting them (Plotnikov et al., 2000) (Plate 31.1B). All these elements are preceded by the signal peptide at the protein N terminal (Powers et al., 2000). The cytoplasmic domain is composed of a juxtamembrane domain interacting with docking molecules (see below), two kinase lobes, and a nonstructured C tail (Plate 31.1B). Owing to the large numbers of FGFs, FGFR specificity requires mechanisms to generate a broader receptor repertoire. This is achieved by alternative splicing and, as discussed below, by the activity of HSPGs of various cell origin that bind to both FGFs and FGFRs. Isoforms lacking D1 or the acidic box are generated upon splicing (Plate 31.1B). Although these isoforms have similar binding properties, they have higher affinity for some FGFs. Another and more important mechanism for receptor diversity is the splicing within D3, as this dramatically changes the specificity of the receptor for FGFs (Ornitz, 2000; Plotnikov et al., 2000). Alternative splicing in D3 generates three different forms, namely D3a, D3b and D3c (Plate 31.1B) (Powers et al., 2000). The D3a splice variant codes for a truncated, soluble receptor that may act by sequestering extracellular FGFs, whereas the D3b and D3c forms are membrane variants that are key in determining signaling specificity and appear to be differentially expressed in different tissues. For example, while FGFR2 D3b is expressed by epithelial cells, the FGF2 D3c variant is produced by cells of mesenchymal origin, and this most likely explains the specificity of FGF7/KGF for keratinocytes (Powers et al., 2000). As summarized above, different HSPGs can confer selective or preferential receptor affinity or activity to different FGFs. This is due to sequence variability in the FGF heparin-binding sites (Faham et al., 1998). Moreover, heparin-binding sites have been identified in FGFR1 and FGFR3 (Plate 31.1B), and evidence indicates that different HS sugar moieties or size, tissue origin of HSPG, and HS concentration may modulate

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not only receptor binding and affinity but also receptor specificity. This plays a key role in FGF tissuespecific responses (reviewed in Ornitz, 2000; Iozzo and San Antonio, 2001; Loo et al., 2001).

Givol, 2000). Mutated phenotypes are characterized by gain-of-function due to receptor dimerization, kinase activation, and increased affinity for FGFs, that act by inhibiting chondrocyte cell growth (Kannan and Givol, 2000).

Receptor in disease Since FGF-mediated signaling has profound effects on cell proliferation, migration and differentiation, changes in FGFR expression levels or activity represent a potent mechanism for tumor development and/or progression, or developmental syndromes (Naski and Ornitz, 1998). Alterations of FGFR activity has been found to be associated with aggressive tumors, although their role in certain types of cancers is controversial. Tumors associated with FGFR alterations include prostate hyperplasia and cancer (Giri et al., 1999; Boget et al., 2001), monoclonal gammopathy, myeloproliferative syndromes or multiple myeloma (Sohal et al., 2001; Fonesca et al., 2001; Chesi et al., 2001), breast cancer (Dickson et al., 2000), pituitary tumours (Ezzat et al., 2002) and melanoma (Ahmed et al., 1997). Changes in FGFRs or FGF expression are sometimes associated with a less malignant tumor phenotype, most likely due to the induction of cell differentiation (Korah et al., 2000; van Rhijn et al., 2001). Mechanisms leading to a deregulated FGFR activity in cancer include chromosomal rearrangements and formation of fusion proteins between FGFRs and other gene products (Sohal et al., 2001), generation of truncated receptors with a cytoplasmic localization (Ezzat et al., 2002) or expression of FGFRs with specific splicing patterns (Yamaguchi et al., 1994a; Wang et al., 1995). In addition, tumor HSPGs can promote or inhibit tumor growth and metastasis by regulating FGF2 activity, and can have proangiogenic effects for endothelial cells. These effects are most likely due to activation of FGF2-dependent Erk-1/2 and FAK signaling pathways by tumor HSPG (Liu et al., 2002). Mutations of FGFR1, 2 and 3 are the cause of skeleton development abnormalities such as short-limb skeletal dysplasias, non-syndromic craniosynostosis, and craniosynostosis syndromes including achondroplasia, hypochondroplasia, thanatophoric dysplasia, Apert syndrome, Crouzon syndrome, Pfeiffer syndrome and Jackson-Weiss syndrome (Bellus et al., 1996; Cohen, Jr., 1997; Renier et al., 2000; Kannan and

RECEPTOR SIGNALING AND INHIBITION A low-affinity complex can form between one molecule of FGF and one molecule of FGFR in solution, however, this complex is unstable and does not result in receptor dimerization and activation (Plotnikov et al., 1999). Evidence indicates that heparin or HS are required for complex stabilization and for FGF signaling in vivo. For example, soluble heparin is required for FGF-mediated signaling in cells treated with HS-degrading enzymes (Rapraeger et al., 1991) and FGF-mediated developmental processes are substantially impaired in animals deficient in HS (reviewed in Ornitz, 2000). Heparin or HSPGs act by mediating the formation of FGF dimers and ordered oligomers that are linearly arranged along the HS sugar moiety (Kan et al., 1993; Herr et al., 1997; Loo et al., 2001) (Plate 31.2A). This indicates that FGF dimers/oligomers bound to HS may be able to dimerize or aggregate FGFR, leading to tyrosine phosphorylation. In this context, FGF dimers stabilized by heparin have been shown to be the minimal biologically active form of FGF (Moy et al., 1997; DiGabriele et al., 1998). These findings indicate that FGFR signaling requires the synchronous assembly of FGF, HS and FGFR, as also suggested by data showing that such a complex forms in solution and is active in vivo. The nature of the FGFR active complex has been addressed in several molecular structural studies and different models have been proposed on the basis of the structural features of the FGF–FGFR complex in the presence or absence of heparin. Although these models differ as to the exact nature of the interactions stabilizing the complex, they all suggest that the functional receptor form consists of two FGFR–FGF heterocomplexes that are thermodynamically brought together and stabilized by one molecule of HS (Plate 31.2A) (DiGabriele et al., 1998; Plotnikov et al., 1999; Venkataraman et al., 1999; Stauber et al., 2000). The

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most recent structural studies indicate that in both FGF2–FGFR1 and FGF1–FGFR2 complexes HS binds to a ‘canyon’ formed by the juxtaposition of the heparin-binding sites of both receptor and ligand (Plotnikov et al., 1999; Stauber et al., 2000). It is noteworthy that membrane-associated HSPG are far more effective in catalyzing the formation of active FGFR dimers as compared with soluble HSPG that, in turn, acquire a similar activity only upon digestion with heparitinases (Zhang et al., 2001). Thus, these data highlight the role of HSPG degradation and ECM remodeling in biologic responses triggered by FGFR crosslinking and activation. Binding of HS and FGF to FGFR triggers receptor dimerization and both cis or trans FGFR autophosphorylation of up to seven tyrosines in each cytoplasmic C tails of assembled FGFR homo- or heterodimers (Klint and Claesson-Welsh, 1999) (Plates 31.1B and 31.2A). This results in the activation of several signal transduction pathways eliciting several different cell responses that, as discussed above, include cell survival, proliferation, migration and differentiation (Plate 31.2B). The most important FGF-dependent signaling pathway for cell proliferation is mediated by the Ras and MAP kinases. This pathway is activated by FRS2/SNT-1, a docking membrane-anchored protein that is constitutively bound to the juxtamembrane domain of FGFR (Wang et al., 1996; Faris et al., 1996; Kouhara et al., 1997; Ong et al., 2000) (Plate 31.2B). Upon FGFR activation, FRS2/SNT-1 becomes phosphorylated and docks to the membrane the adaptor protein complex Grb2/Sos1 that, in turn, binds and recruits Ras to the FGFR complex (Kouhara et al., 1997). This leads to MPAK activation and cell proliferation (Plate 31.2B). However, recent data indicate that FRS2 activation triggers the assembly of a large multidocking protein complex transducing FGF-dependent proliferation, survival and chemotactic signals (Ong et al., 2001; Hadari et al., 2001). In fact, in addition to Sos1, Grb2 also interacts with the docking protein Gab1, leading to the formation of a FRS2–Sos1–Grb2–Gab1 complex, where Gab1 becomes phosphorylated (Plate 31.2B). Phosphorylated Gab1 present in the docking complex recruits and activates the phosphatidylinositol-3 kinase (P-I3 kinase) that, in turn, activates the Act kinase pathway (Ong et al., 2001; Hadari et al., 2001). This pathway is known to promote cell survival

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through many targets including NFjB and Bad, a proapoptotic member of the Bcl-2 family (Datta et al., 1999) (Plate 31.2B). Moreover, since different FGFinduced biologic responses are mediated by different tyrosine phosphorylation sites on FRS2 (Hadari et al., 2001), these data point to FRS2 as a pivotal regulator of the multiple actions of FGFs, as also indicated by FRS2-mediated regulation of several FGF-dependent developmental processes (Hadari et al., 2001; Kusakabe et al., 2001). Other major signaling pathways triggered by FGFR are the Src and p38 MAP kinase that, unlike the Ras pathway, appear to be preferentially involved in FGFdependent cell migration or differentiation (LaVallee et al., 1998a; Klint et al., 1999; Boilly et al., 2000) (Plate 31.2B). FAK is also known to be present in a complex with FGFRs, that may thus integrate signal transduction by FGFs and integrins, two pathways that are known to be closely coupled (Klint and ClaessonWelsh, 1999) (Plate 31.2B). In addition, FGFRs are known to activate several phospholipases (PLCs) including PLA, PLCc, which activates PKC, and PLD. Although the role of this FGF-dependent response is not completely defined, a mutant receptor incapable of PLCc phosphorylation shows a lower internalization and elicits decreased mitogenic responses (Plate 31.2B) (Klint and Claesson-Welsh, 1999; Powers et al., 2000; Boilly et al., 2000; Cross et al., 2000). All the above signal transduction pathways are initiated by FGFs at the level of the cell membrane. However, as discussed above, FGF-bound membrane tyrosine-kinase receptors are internalized and can be translocated to the cell nucleus. Growing evidence indicates that this is another mechanism for FGF signaling, that is also common to other growth factors or cytokines (Jans and Hassan, 1998; Keresztes and Boonstra, 1999). Although nuclear localization of exogenous FGFs has specific effects on the cell phenotype even in the absence of the cognate receptor, FGFR cotranslocation is required to elicit a complete biologic response. For example, import of a diphtheria toxin– FGF1 fusion protein via a surface toxin receptor induced de novo DNA synthesis in FGFR-negative cells, a response that was associated with the localization of the chimeric protein in the cell nucleus. However, despite nuclear FGF1 stimulation of DNA synthesis, cell division was obtained only upon

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transfection of the cells with FGFR-expressing vectors, indicating that FGFRs are required for full FGF1 mitogenic activity (Wiedlocha et al., 1996). Vice versa, fibroblast variants unresponsive to the mitogenic effects of FGF1 show a substantial impairment of FGF1 translocation to the cell nucleus despite normal membrane FGFR signaling. These data suggest that complete FGF1 biologic responses require nuclear translocation of both FGF1 and FGFR, that are likely to be coupled. In agreement with these data, deletion of FGF1 NLS abrogates FGF1 mitogenic activity that, in turn, can be fully restored by NLS from other proteins (Imamura et al., 1994), suggesting that nuclear translocation of the FGF–FGFR complex may be driven by FGF NLS. By contrast, the exported isoform of FGF2 (i.e. the 18 kDa species) has only a cryptic NLS that is unlikely to play a role in FGFR nuclear translocation. As discussed above, however, FGF2 induces cell internalization of FGFR that, in turn, is translocated to the cell nucleus by importin b. Once localized in the nucleus, FGFR1 tyrosine kinase activity induces the expression of c-jun, that become phosphorylated upon signaling by membrane FGFR1, thus contributing to the modulation of cell proliferation (Reilly and Maher, 2001). This mechanism is also likely to be active for the intracrine forms of FGF2; in fact, both FGFR1 and endogenous FGF2 isoforms co-localize in the cell nucleus of proliferating astrocytes and glioma cells, where they trigger signals stimulating the transition from the G0/G1 to the S phase of the cell cycle (Stachowiak et al., 1997; Joy et al., 1997). Like other tyrosine kinase receptors, FGFR possesses autoinhibitory mechanisms preventing receptor dimerization or kinase activity in the absence of ligand binding. In particular, divalent cations or HS fragments have been shown to inhibit a crucial FGFR dimer-promoting region located in the linker connecting D2 and D3 (Kan et al., 1996; Wang et al., 1997). Furthermore, in most spliced variants the acidic box present in the linker separating D1 and D2 can interact with the receptor D2 heparin-binding sequences, preventing receptor dimerization in the absence of HS-bound FGF (Plotnikov et al., 1999). Finally, FGFRs can autoinhibit tyrosine phosphorylation by preventing binding of peptide substrates to the cytoplasmic catalytic domain of ligand-free receptor molecules (Mohammadi et al., 1996).

As discussed below, on the basis of these data several attempts have been made to identify synthetic inhibitors of FGFR activity for prevention and therapy of FGF-associated diseases.

AREAS OF ACTIVE RESEARCH AND INQUIRY The expression of many FGF types and isoforms, spliced or truncated versions of FGFRs, and HSPG variants in many organs and tissue types poses an extraordinary challenge for future research on FGF biologic actions. Nevertheless, several studies are presently aimed at understanding the molecular mechanisms of FGF specificity of action, the interaction of FGFs with other biologic effectors, and how these pathways integrate in complex biologic processes like embryonic development or angiogenesis. In this regard, increased interest has been recently focused on the study of the mechanisms determining FGF and FGFR specificity in vitro and in vivo. As discussed above, recent genetic, biochemical and structural studies have addressed the role of key amino-acid residues and interactions responsible for recognition and activation of specific FGFRs by the FGF family members, as well as the role of HS and HS sugar moieties in determining FGFR responses. In this context, an area of increasing interest is the dissection of FGF-mediated signaling. In particular, several recent studies have attempted to identify co-stimuli and molecular players involved in the large numbers of different responses that are elicited in a spatial and time-dependent fashion upon FGFR triggering by their FGF ligands. These approaches have been complemented by in vivo studies with knockout mice, leading to inactivation of several FGF family members resulting in viable animals with mild defects that are the object of intense research. Hopefully, this will allow identification of specific processes affected by FGF family members and, at the same time, will shed light on the mechanisms of FGF functional redundancy. As discussed above, however, inactivation of several family members resulted in early lethal phenotypes, underlying the role of FGFs in fundamental pathways that are difficult to dissect by these approaches. Furthermore, as indicated by previous studies, inactivation of

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receptors including FGFR1 or FGFR2 also results in early lethal phenotypes (Deng et al., 1994; Yamaguchi et al., 1994b; Arman et al., 1998). However, recent data show that conditional mutagenesis and targeted expression of dominant-negative transgenes or soluble receptors at specific developmental stages can be used to circumvent some of these problems (Meyers et al., 1998; Sun et al., 1999; Moon and Capecchi, 2000). Hopefully, the complexity of these studies will be reduced by recent advances in conditional mutagenesis technologies (Peitz et al., 2002). A second area of recent, intense research is aimed at the identification of novel FGF members and receptors by random cDNA sequencing, positional cloning and/or homology-based or degenerate polymerase chain reaction techniques. This has recently led to the identification of many FGF family members and, more recently, a pancreatic tyrosine kinasedeficient version of a putative novel FGFR5 (Kim et al., 2001). It is anticipated that the identification of other FGFs and FGFRs will unveil mechanisms responsible for as yet uncharacterized FGF-dependent biological processes. An active area of research focuses on the identification of natural or synthetic compounds to target FGF or FGFRs for prevention or therapy of FGF-associated pathologic conditions, including developmental syndromes, angiogenic diseases and, in particular, cancer. As discussed above, type I IFNs are known to inhibit FGF2 expression and are currently employed in the therapy of FGF2-associated diseases including infantile hemangioma and KS. More recently, nontoxic heparin-mimicking synthetic non-sulfated polyanionic compounds have been obtained through acid-catalyzed polymerization of phenols and formaldehyde (Miao et al., 1997). The resulting compounds have been shown to bind FGF2 but, unlike heparin, they do not induce the formation of active FGF2 dimers or oligomers, thereby inhibiting FGF2 binding to FGFR and FGFR signaling. Owing to these properties, these compounds are able to block FGF2induced proliferation of vascular smooth muscle and endothelial cells, inhibit the formation of new vessels by rat aortic rings, and revert the transformed phenotype of FGF2-transfected cells (Miao et al., 1997). Similarly, other recent studies have led to the identifi-

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cation of a porphyrin analog (TMPP) that, by a molecular mimicry of heparin, inhibits FGF2 smooth muscle cell proliferation by blocking binding to HS of both FGF and FGFR. On the basis of these studies, other porphyrin analogs have been identified that show improved inhibitory properties and may be useful in inhibiting angiogenesis and growth of FGF or VEGFassociated tumors. Since porphyrin derivatives are widely used in the therapy of cancer, it is likely that these new compounds will be the object of further development and specific clinical applications (Aviezer et al., 1994; Segev et al., 2002). Screening of libraries of synthetic compounds for their ability to inhibit tyrosine kinase receptors has recently led to the identification of novel classes of inhibitors that are active against FGFRs but not other tyrosine kinases, including PDGF, EGF and insulin receptors, c-Src, MEK and PKC (Mohammadi et al., 1997, 1998; Panek et al., 1998). Some of these inhibitors, particularly SU5402 and PD 173074, are currently widely used in in vitro and in vivo studies to identify FGF-dependent biologic processes, or in preclinical studies for cancer therapy (see, for example Dimitroff et al., 1999; Demiroglu et al., 2001; Shinya et al., 2001). Recent studies have been aimed at the identification of key molecules involved in FGF responses. In particular, as discussed above, GBP-1 was identified as a key modulator switching endothelial cells from an activated to a proliferative, angiogenic phenotype. In fact, inflammatory cytokines up-regulate GBP-1 that, in turn, induces endothelial cell activation and growth arrest. By contrast, FGF2 down-regulates GBP-1, and this induces them to proliferate and to become angiogenic (Guenzi et al., 2001). Thus, GBP-1 may be targeted for both the therapy of FGFassociated angiogenic diseases or tumors, and to enhance the physiologic effects of FGFs. Several recent studies have shown that the HIV-1 Tat protein shares several properties with FGFs, including heparin-binding activity, the capability of being exported from cells despite the lack of leader sequences (Ensoli et al., 1990, 1993; Chang, H.C. et al., 1997), the internalization through both receptor and HSPG-mediated pathways (Fanales-Belasio et al., 2002), the translocation to the cell nucleus and nucleolus (reviewed in Ensoli et al., 2001; Barillari and Ensoli, 2002). Moreover, Tat shares several properties

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with ECM molecules including fibronectin and vitronectin, such as binding to membrane integrins (a5b1 and avb3) that leads to adhesion-dependent survival signals required for cell proliferation. Notably, Tat is known to act through the FAK pathway (Ensoli and Barillari, 2002) that, as discussed above, is activated by both FGFs and molecules binding to integrins. Finally, by displacing HSPG-bound FGF2 Tat can induce angiogenesis (Ensoli et al., 1994a). Thus, studies aimed at the analysis of these properties of Tat may unveil important features of FGF trafficking and integration of signal transduction pathways involved in angiogenic responses. Hopefully, the present efforts will lead to the identification of novel therapeutic targets and/or generation of FGF/FGFR inhibitors with low toxicity for the therapy of FGF-associated diseases.

SUMMARY We have reviewed data showing that FGFs are a family of related factors playing fundamental roles in several biologic processes involving tissue remodeling such as embryonic development, angiogenesis, wound healing, nerve regeneration, and chronic inflammation and cancer (summarized in Table 31.4). These processes are dependent on several biologic responses mediated by FGFs, including cell survival, proliferation, migration, invasion and differentiation. The capability of FGFs to elicit many different responses through intracrine, autocrine and paracrine actions confers on them a notable plasticity. It is not surprising, therefore, that FGFs also play a key role in hematopoiesis and have neurotrophic properties. In the past 30 years, intensive research on FGFs and their receptors has shed light on the fundamental mechanisms by which these factors exert their biologic functions. These studies have significantly increased our knowledge about the structure of both FGF and FGFR proteins, their stability in vivo, the mechanisms of FGF production, export, import and cell trafficking, the regulation of their functions by ECM molecules, the structure of FGFR in complex with their ligands, and the role and mechanisms of FGFR signaling in several biologic models. However, many important aspects of FGF actions are still poorly

understood. In particular, it has not yet been determined how crosslinking of the same receptor leads, in the same cell, to different responses and, most importantly, how these responses are integrated during the accomplishment of more complex biologic tasks. For example, although it is known that angiogenic FGFs can induce endothelial cells to invade the basement membrane, to migrate within interstitial tissue, to proliferate, and differentiate to originate new blood vessels, the molecular mechanisms modulating these FGF responses in a spatial and temporal-ordered fashion remain poorly understood. Recent studies have attempted to identify specific FGF signaling pathways underlying different responses. This has led to the concept that certain responses are preferentially driven through specific signal transduction pathways elicited by different modes of cell exposure to FGF ligands. For example, in in vitro systems shortterm treatment of endothelial cells with angiogenic FGF results in cell migration, yet, prolonged exposure to FGF appears to be coupled with cell proliferation. Furthermore, FGF-dependent cell migration appears to be preferentially dependent on Src and p38 MAP kinase activation, whereas cell proliferation would be more dependent on ERKs. Most likely, such different responses are in part related to the different timecourse of high-affinity (FGFR) and low-affinity (HSPG) FGF receptor down-modulation upon ligand binding. In particular, down-modulation of FGFR and import of FGF–HS–FGFR complexes appear to be rapid processes, whereas down-regulation of membrane HSPGs and internalization of FGF–HS complexes occur later and with slower kinetics. In addition, dynamic changes of ECM during tissue remodeling play a key role in regulating responses to FGFs by releasing FGFs that, due to their association with different HS or HSPG protein core fragments, can have different activating or inhibitory effects on different FGF responses in different cell types. Moreover, ECM remodeling leads to the release of many biologic effectors that participate in FGF actions. How exactly the activity of these different factors integrate with FGF responses during tissue remodeling is unknown. Thus, it is clear that the complexity of FGFmediated processes represents an extraordinary challenge to future research. Nevertheless, the study of the role of FGFs and FGFRs in pathologic conditions including developmental disorders and syndromes,

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angiogenic diseases, chronic inflammation, neurologic syndromes, hyperplastic, reactive diseases and cancer, has greatly increased our knowledge of FGF actions and led to the identification of new therapeutic molecular targets. Furthermore, these studies have indicated that it is possible to hit FGFs and FGFRs with compounds showing remarkable specificity and low toxicity. Hopefully, in the near future, these findings will be translated into the clinic for the therapy of FGF-associated diseases. Note: The cytokine family cDNA database. (2002). Available at http://cytokine.medic.kumamoto-u.a.c. jp/CFC/FGF/FGF.html.

ACKNOWLEDGEMENTS We thank Annamaria Carinci and Angela Lippa for editorial assistance. Recent experimental work by the authors of this chapter was supported by grants from the Italian Ministry of Health (IX AIDS project) and the Associazione Italiana per la Ricerca sul Cancro (AIRC) to B.E., and from the Italian Ministry of Education, University and Research (MIUR) to G.B.

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32 Hepatocyte growth factor and its receptor, MET Wendy M. Mars, Youhua Liu and Satdarshan P. Singh Monga University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

The difficulty lies, not in the new ideas, but in escaping the old ones, which ramify, for those brought up as most of us have been, into every corner of our minds. John Maynard Keynes

INTRODUCTION The existence of a circulating hepatocyte growth factor (HGF) induced in response to liver injury was first implied by experiments performed in the 1960s. Moolten and Bucher (1967) observed that crosscirculation of blood from rats with regenerating livers could induce a response in the resting livers of surgically conjoined animals. Subsequently, Michalopoulos et al. (1982) showed that a novel, serum-derived factor (at the time referred to as hepatopoietin) from hepatectomized animals could induce cultured hepatocytes to replicate. In the middle to late 1980s, several groups independently isolated and purified the HGF protein to homogeneity from rat platelets, rabbit serum and human plasma (Nakamura et al., 1986, 1987; Gohda et al., 1988; Zarnegar and Michalopoulos, 1989; Zarnegar et al., 1989). Sequencing led to the realization that HGF is identical to scatter factor (Furlong et al., 1991; Weidner et al., 1991), a fibroblast-

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

secreted protein named for its ability to disrupt or ‘scatter’ epithelial cells (Stoker and Perryman, 1985), and to tumor cytotoxic factor (Higashio et al., 1990), a protein which exhibits cytotoxic effects on certain types of cells in culture. With the realization that HGF, scatter factor and tumor cytotoxic factor were the same, it became evident that despite the implications of its more commonly accepted name, HGF is really a pleiotrophic cytokine, capable of inducing a variety of responses on non-hepatocytes, including mitosis, motility, cytoxicity and branching morphogenesis (Montesano et al., 1991). Human HGF is produced as a latent, single-chain glycoprotein (scHGF), which migrates at approximately 97 kDa (including posttranslational processing) under reducing conditions (Nakamura et al., 1989). Biochemically, the protein is both heat- and acid-labile. Mitogenic activity can be abolished by either heating to 100 C for 1.5 minutes or by treating with 1 M acetic acid. Similarly, trypsin digestion or

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reduction using dithiothreitol will destroy its function. Although HGF can bind to its receptor as a zymogen, it must be cleaved between amino acids Arg494 and Val495 in order to induce full biologic activity. The resulting heterodimer consists of an a (approximately 64 kDa) and b (approximately 33 kDa) chain linked by a disulfide bond (Lokker et al., 1992). Interestingly, although the individual subunits are larger, under non-reducing conditions HGF migrates as a single band of approximately 70 kDa, implying the existence of a strong secondary structure. Two full-length forms have been identified that are identical, with the exception of a five-amino-acid deletion in the amino terminus (Seki et al., 1990; Weidner et al., 1991). While the activities are very similar, the two HGFs are biologically distinguishable, displaying different solubilities and dose–response curves when tested on various cell lines (Shima et al., 1994). Additionally, two naturally occurring truncated forms known as NK1 (Cioce et al., 1996) and NK2 (Chan et al., 1991; Miyazawa et al., 1991b) are known which have been reported to be either agonistic or antagonistic, depending upon the system that was under investigation. Historically, because HGF is produced by fibroblasts and can exert its effects upon epithelial cells, HGF has been described as a paracrine cytokine for a variety of cell and tissue types. These include, but are not limited to, liver (Kinoshita et al., 1989), kidney (Montesano et al., 1991; Ishibashi et al., 1992), endothelia (Rosen et al., 1993), placenta (Stewart, 1996), tissue B cells (van der Voort et al., 1997), melanocytes (Imokawa et al., 1998), mammary gland (Soriano et al., 1998) and hair follicles (Lindner et al., 2000). Despite its reputation as a paracrine effector, autocrine activities have also been reported in both normal and malignant tissues. Current data suggest autocrine functions during development (Andermarcher et al., 1996) and in a diverse array of tissue types such as lung (Tsao et al., 1993), muscle (Cortner et al., 1995; Anastasi et al., 1997; Sheehan et al., 2000), transformed breast (Tuck et al., 1996), normal axons (Yang et al., 1998), transformed liver epithelia (Presnell et al., 1998; Yokomuro et al., 2000), and developing dental papilla (Kajihara et al., 1999). The high-affinity receptor for HGF is the protooncogene gene product known as MET (Bottaro et al.,

1991; Naldini et al., 1991a). c-met was originally isolated from the DNA of a carcinogen-treated osteosarcoma cell line because of its ability to transform NIH 3T3 cells in a DNA transfection assay (Cooper et al., 1984). HGF also displays low-affinity binding to heparin and heparin-like molecules (Zarnegar et al., 1990b; Naka et al., 1993) and can interact with dermatan sulfate (Lyon et al., 1998). Thus, it is presumed to interact with cell surface proteoglycans. Structurally, the HGF molecule resembles plasminogen, containing a hairpin loop and four kringle domains as well as a light chain that is structurally similar to the serine protease portion of the molecule (Miyazawa et al., 1989; Tashiro et al., 1990). Nevertheless, no inherent enzymatic activity has been detected within the HGF protein since two of the three amino acids in the classic catalytic triad common to serine proteases have been altered (Plate 32.1). Studies with mutated and truncated forms of HGF have shown that binding to the high-affinity receptor occurs within the first two kringles of the amino terminus (Matsumoto et al., 1991; Lokker et al., 1992), and especially kringle 1 (Lokker and Godowski, 1993; Rubin et al., 2001). The heparin binding site is formed by a small number of amino acids, located within the amino terminus in a region spanning amino acids 60–80 (Zhou et al., 1999). Restoration of a functional catalytic triad has no adverse effects on stimulating hepatocyte growth and in fact, may be stimulatory (Matsumoto et al., 1991). As the latent form of HGF is inactive, much interest has centered on discerning the molecules that lead to the production of active HGF. Foremost among the various molecules that have been shown to cleave latent HGF to its active form are the urokinase-type and tissue-type plasminogen activators (u-PA and t-PA, Mars et al., 1993) as well as a Factor XII-like protein known as HGF activator (Miyazawa et al., 1993). Although in vitro evidence suggests that u-PA alone can cleave HGF, most in vivo studies have implicated a role for the u-PA receptor as well (Mars et al., 1995; Naldini et al., 1995; Powell et al., 2001). The ability to activate HGF has also been ascribed to plasmin (Gak et al., 1992), Factor XIIa (Shimomura et al., 1995) and matriptase (Lee et al., 2000); however, it is less clear as to whether or not these molecules have a significant biological role.

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HGF HGF protein sequence Several laboratories independently cloned the HGF cDNA from human liver, placenta, leukocytes and embryonic lung fibroblast cDNA libraries (Miyazawa et al., 1989; Nakamura et al., 1989; Seki et al., 1990; Rubin et al., 1991; Weidner et al., 1991). The deduced amino-acid sequence from the cDNA nucleotide sequence analyses revealed that HGF is encoded in a single open reading frame and is synthesized as a single-chain pre-pro HGF precursor of 728 amino acids with a calculated molecular weight of 83 kDa. The first 31-amino-acid residues at the N-terminus of pre-pro HGF are hydrophobic and typical of signal peptide sequences found in secretory proteins, including other growth factors (Nakamura et al., 1989). After the co-translational removal of the N-terminal presequence (signal peptide), single chain HGF is proteolytically processed to a mature, heterodimeric, biologically active form consisting of an a and b chain. The proteolytic cleavage site, located between residues 494 and 495 (Arg–Val), is a sequence typically recognized by serine proteases. Thus, it is not surprising that all identified HGF activators (u-PA, t-PA, HGFa, Factor XIIa, plasmin and matriptase) are members of the serine protease family of proteins. The a chain of HGF extends from amino acids 32 to 494 with a predicted molecular weight of 54 kDa, while the b chain consists of 234 amino acid residues (from 495 to 728) with a calculated weight of 26 kDa. The cysteine residues that form the interchain disulfide bond between the a and b chains in HGF are at positions 487 and 604, respectively. HGF also contains four putative glycosylation sites with the canonical Asn–X–Ser/Thr sequence located at positions 294 and 402 of the a chain, and at positions 566 and 653 of the b chain (Miyazawa et al., 1989; Nakamura et al., 1989). The addition of the carbohydrate residues at these sites presumably accounts for the difference between the calculated and observed molecular weights. The a chain of HGF consists of an N terminus (His32–Asn121) followed by four tandem regions that are called ‘kringles’. These ‘kringle’ structures, although not identical, share 40–50% amino acid homology among themselves (Nakamura et al., 1989;

Mizuno and Nakamura, 1993). The kringle domain is a looped, disulfide-linked structure consisting of approximately 78-amino-acid residues and is present in varying numbers (i.e. two in prothrombin and five in plasminogen) in several proteins involved in blood coagulation and fibrinolysis (Ny et al., 1984). Although the function of these kringle domains remains largely unknown, kringles are hypothesized to play a role in the protein–protein interactions necessary for the biologic functions of their resident proteins. The b chain of HGF shows a high degree of sequence and structural similarity to the serine protease domains of several enzymes, especially plasmin (Ny et al., 1984; Miyazawa et al., 1989; Nakamura et al., 1989; Petersen et al., 1990); however, the histidine and serine residues of the catalytic triad (His534–Asp578– Ser673) are replaced by glutamine and tyrosine, respectively (Nakamura et al., 1989). Hence, HGF has no detectable serine protease activity. In addition to human HGF, cDNAs have been cloned from other species including rat, mouse, Xenopus and cat (Okajima et al., 1990; Tashiro et al., 1990; Liu et al., 1993; Nakamura et al., 1995; Kobayashi et al., 2001). Comparison of the overall amino acid and nucleotide sequences between mouse, rat and human indicates that their primary structures share a high degree of conservation (more than 90%) (Okajima et al., 1990; Tashiro et al., 1990; Liu et al., 1993). This explains why little species specificity has been observed in the biologic activities of HGF; the major characteristic features of HGF are conserved in the various species. For example, the lengths of the four individual kringle structures of HGF and the lengths of their a chains (463 amino acids) are exactly the same among human, rat and mouse. Furthermore, the cysteine residues in the kringle structures are all located at positions exactly equivalent (K1  Cys128/Cys206, K2  Cys211/Cys288, K3  Cys305/ Cys383, K4  Cys391/469) to each other (Liu et al., 1993). The cleavage site for proteolytic processing that leads to the formation of the a and b chains, as well as the amino-acid sequences surrounding this site, are identical among mouse, rat and human HGF. Finally, replacements of the histidine and serine residues by glutamine and tyrosine in the catalytic triad of the b chain are exactly retained in these three species (Nakamura et al., 1989; Tashiro et al., 1990; Liu et al., 1993).

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Variant forms of HGF The major HGF transcript in human and rat consists of a 6 kb species (Miyazawa et al., 1989; Nakamura et al., 1989; Rubin et al., 1991) that includes 134 nucleotides of 5-untranslated region, 2184 nucleotides of the HGF coding region and 3.6 kb of the 3-untranslated region. There are several naturally occurring variants of HGF which result from alternative mRNA splicing. Most notably, a variant form of HGF with a five-amino-acid deletion in the first kringle domain (dHGF) has been cloned from both human leukocyte and embryonic lung fibroblast cDNA libraries (Seki et al., 1990; Rubin et al., 1991). The 15 deleted nucleotides are located at the 5-end of the fifth exon and contain a conserved 3 acceptor sequence for splice junctions. Hence, this variant is attributed to an alternative splice event. Functional studies reveal few differences in the overall biologic activities between HGF and the deleted variant although their solubility in solution differs and different dosages are needed to produce similar effects on the same cell types (Shima et al., 1994). Two additional, truncated variants of HGF have also been identified. Each contains the N-terminal region and either the first kringle (HGF/NK1) or the first and second kringles (HGF/NK2) of HGF (Chan et al., 1991; Miyazawa et al., 1991b). These variants are also naturally occurring and generated by alternative splicing. HGF/NK2 is the product of the 1.2-kb isoform of HGF mRNA whereas HGF/NK1 is derived from a 2.2-kb transcript. HGF/NK2 competes with HGF for binding to the HGF receptor, as confirmed by affinity crosslinking studies, and can inhibit its mitogenic activity in vitro (Chan et al., 1991). Furthermore, HGF/NK2 also behaves as an antagonist to HGF in transgenic animals (Otsuka et al., 2000). HGF/NK1 is able to exhibit both agonistic and antagonistic actions, depending upon its cellular context (Miyazawa et al., 1991b; Cioce et al., 1996). In cultured hepatocytes, HGF/NK1 acts mostly as an HGF antagonist but it can also elicit an agonistic response in the presence of heparin (Cioce et al., 1996; Schwall et al., 1996). On the other hand, HGF/NK1 transgenic mice show a phenotype that is similar to HGF transgenic mice (Takayama et al., 1996; Jakubszak et al., 1998), indicating that HGF/NK1 has agonistic actions in vivo. An artificially generated HGF variant, HGF/NK4, con-

taining the N-terminal region and four kringles, can act as a specific antagonist for the pleiotrophic actions of HGF (Date et al., 1997).

HGF crystallographic structure Recently, the crystal structure of the NK1 fragment of HGF was independently solved by two groups (Ultsch et al., 1998; Chirgadze et al., 1999). NK1 assembles as a homodimer in the crystallographic unit in a headto-tail fashion, with the N domain of one monomer packing against the kringle domain of the other. Each NK1 molecule comprises two globular subunits: the N and the K domains connected by a short linker. The overall structure of the N domain is identical to the nuclear magnetic resonance spectroscopic structure of the domain by itself (Zhou et al., 1998). It displays a structurally unique, central five-stranded antiparallel b sheet that is flanked by a short a helix on one side and by a two-stranded antiparallel b sheet on the other. Three exposed, positively charged residues in or near the hairpin-loop region coordinate a sulfate ion in the crystal structure, suggesting that these residues are probably involved in heparin binding (Ultsch et al., 1998; Chirgadze et al., 1999; Zhou et al., 1999). The K domain shows high structural homology to the kringle 4 domain of plasminogen, with a characteristic pattern of three disulfide bridges and an antiparallel two-stranded b sheet, but is different in the region corresponding to the lysine-binding pocket (Ultsch et al., 1998; Chirgadze et al., 1999). The receptor-binding determinants are located near this pocket on the surface of the kringle domain opposite the N domain, revealing a distinctive topology of the putative receptor-binding site (Ultsch et al., 1998; Chirgadze et al., 1999).

HGF-related polypeptides Through cDNA cloning and sequence analysis, Degen et al. (1991) identified another kringle-containing protein known as the HGF-like protein (Han et al., 1991). Based upon the amino-acid sequence, the HGF-like protein was found to be identical to a plasma protein that was isolated as a macrophagestimulating protein (MSP). MSP’s name reflects the protein’s ability to stimulate resident peritoneal

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macrophages to become more responsive to chemoattractants, causing the formation of long cytoplasmic processes and pinocytotic vesicles when introduced on to freshly plated macrophages (Skeel et al., 1991). The mouse and human pre-pro MSP proteins consist of 716 and 711 amino acids, respectively. Similar to HGF, they contain an a chain with four kringle domains and a b chain that possesses a nonfunctional serine protease-like domain (Degen et al., 1991; Han et al., 1991). The overall amino-acid sequence homology between human and mouse HGF and human and mouse MSP is approximately 50% (Degen et al., 1991; Han et al., 1991). Recent studies show that MSP shares some biologic activities with HGF and also plays an important role in regulating cell adhesion and motility, growth and survival (Danilkovitch and Leonard, 1999; Danilkovitch et al., 2000). The receptor for MSP is the tyrosine kinase protein known as RON, a structurally related cousin to the HGF receptor, MET (Gaudino et al., 1994).

HGF gene organization The human HGF gene has been assigned to the long arm of chromosome 7 at 7q21.1 (Weidner et al., 1991; Saccone et al., 1992; Szpirer et al., 1992; Zarnegar et al., 1992). In the mouse, the HGF gene is located at the proximal region of chromosome 5 whereas it is located on chromosome 4 in the rat (Weidner et al., 1991; Szpirer et al., 1992; Wallenius et al., 1997). The structural organizations of both the human and mouse HGF genes were originally determined by analyzing several overlapping clones isolated from genomic libraries (Miyazawa et al., 1991a; Seki et al., 1991; Liu et al., 1994a). These data were recently validated by the sequencing of the human genome (Lander et al., 2001; Venter et al., 2001). The human HGF gene is approximately 70 kb in size (Miyazawa et al., 1991a; Seki et al., 1991), whereas the mouse gene has a length of 65 kb (Liu et al., 1994a). In both species, the HGF gene is composed of 18 exons separated by 17 introns. Structural domains of the HGF protein, such as the signal peptide, amino terminus region, four kringles, and serine protease-like region, are encoded in separate exons (Miyazawa et al., 1991a; Seki et al., 1991; Liu et al., 1994a). Exon 1 contains the 5-non-coding region as well as a typical signal peptide with a hydrophobic core. The N-terminal

region of the a chain that precedes the kringle structures is encoded by exons 2 and 3, with the heparinbinding region localized to exon 2. The next eight exons encode the kringles of the a chain. Similar to other kringle-containing proteins, each of the four kringle structures is encoded by two exons. Exon 13 encodes the proteolytic processing site that separates the a and b chains. The serine protease-like sequence of the b chain is encoded within the remaining five exons (exons 14–18). All intron–exon junctions follow the GT–AG rule established for eukaryotic genes, with both the 5 donor and the 3 acceptor splice sequences closely related to the overall splice consensus sequence (Mount, 1982). Although the mouse HGF gene is approximately 65 kb in size, the gene for the closely related protein, MSP (50% homologous), is only 4.6 kb in length (Degen et al., 1991; Liu et al., 1994a). Nevertheless, the genes encoding both HGF and MSP contain the same number of exons and introns (18 exons and 17 introns). The exons in both genes are similar in size, however, the HGF introns are much larger. Nevertheless, the placement of introns with respect to the amino acid sequence of the two proteins is almost identical. All splice junctions are of the same type (0, I or II) for the corresponding introns and all introns interrupting the kringle domains are in similar positions. The gene structure of another closely related gene, plasminogen (38% homologous), has also been characterized (Petersen et al., 1990). Not surprisingly, the genomic organizations of these two genes are very similar. Plasminogen spans about 52.5 kb of DNA and consists of 19 exons separated by 18 introns.

HGF promoter structure and function To understand the mechanism underlying HGF gene regulation, various groups have cloned the 5 promoter region of the HGF gene from human, mouse and rat genomic libraries (Miyazawa et al., 1991a; Seki et al., 1991; Okajima et al., 1993; Liu et al., 1994c). The promoter regions of the mouse, rat and human HGF genes are 100% identical from the TATA box to 110 bp upstream (Liu et al., 1994c). Transient transfection assays with HGF promoter–reporter gene constructs demonstrate that, in different cell types, this 110-bp region is sufficient to elicit HGF gene expression (Liu et al., 1994c). When a larger upstream region 5 to

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the transcription site is examined, a high degree of homology is observed between the various species until 400 bp; comparisons of the mouse and rat, mouse and human and rat and human sequences show 96%, 88.8% and 86.9% homologies, respectively. Sequence homology then begins to diminish beyond 400 bp (Liu et al., 1994c); however, the regulation patterns of the various HGF genes are believed to be similar across the different species. The promoter region in the 5-flanking region of the HGF gene contains various regulatory elements (Seki et al., 1991; Okajima et al., 1993; Liu et al., 1994c) including a non-canonical TATA box (ATAAA) at position 30. In the mouse gene, four copies of the interleukin 6 (IL-6)-response element, a positive transcription element in several acute-phase protein genes, have been identified (Liu et al., 1994c). Other response elements in the mouse promoter include two liver-specific transcription factor-binding elements (C/EBP), with one site located in the first intron at position 554. At 2539 a sequence homologous to the consensus site for the Pu.1/ETS binding element (a B cell- and macrophage-specific transcription factor belonging to the Ets proto-oncogene family) is present whereas, a TGFb inhibitory element (TIE) (which acts as a negative element in several genes) is found at position 364. Sequences homologous to the consensus sequence of the AP-1 transcription factor are located at positions 341 and 2249, respectively. Two putative estrogen-responsive elements (ERE) have also been identified, with one in the first intron at position 511 and another in the region around –860 bp. Finally, one cAMP-responsive element (CRE) is found at position 965 (Liu et al., 1994b; Jiang et al., 1997). Such diverse regulatory elements suggest that HGF expression is likely to be regulated by a variety of cytokines and steroid hormones.

HGF expression The expression characteristics as well as the diverse activities of HGF make it an important paracrine and/or endocrine mediator of stromal–mesenchymal interactions, significantly contributing towards development, angiogenesis, tissue regeneration, carcinogenesis and other pathologic processes (Zarnegar and Michalopoulos, 1995; Jiang and Hiscox, 1997; Laterra

et al., 1997; Michalopoulos and DeFrances, 1997; Birchmeier and Gherardi, 1998). The HGF mRNA and protein are present in multiple organs in both embryonic and adult tissues including blood, brain, liver, kidney, lung, placenta, skin, spleen and others (DeFrances et al., 1992; Zarnegar and DeFrances, 1993). Although HGF is localized to multiple epithelial organs in the developing embryo, expression is highest in the liver and placenta (Sonnenberg et al., 1993). Embryonic expression is related to specific functions and its developmental relevance is demonstrated by gene knockout studies; loss of HGF results in embryonic lethality due to placental and liver defects (Schmidt et al., 1995; Uehara et al., 1995). In the liver, HGF gene ablation compromises organ development, resulting in diminished liver size and suggesting a role in cell proliferation and survival. Similarly, placental impairment involves an absence in the formation of the labyrinth layer which is responsible for supporting oxygen and nutrient exchange between the maternal and fetal circulation. This is imperative for sustenance of advanced pregnancy as the diffusion process itself is not sufficient to maintain adequate materno–fetal exchange. In unstimulated adult liver, production of HGF is limited to the non-parenchymal cells, specifically the stellate cells (Ito cells or the fat storing cells of liver) and Kupffer cells (resident macrophages) (Wolf et al., 1991a). In placenta, HGF mRNA and protein can be detected in villus syncitium, extravillus trophoblast, amniotic epithelium, endothelial cells and villus mesenchyme (Wolf et al., 1991b). Generation of skeletal muscle from the long-range migration of precursor cells is one of the more definitive and interesting roles of MET in response to HGF. The requirement of HGF for the generation of migrating myogenic progenitors implies an important role in epithelial–mesenchymal transition (EMT) during embryogenesis (Bladt et al., 1995; Birchmeier and Gherardi, 1998). Findings using MET knockout mice have been confirmed in studies where ectopic expression of HGF was able to promote EMT in the chick embryo (Brand-Saberi et al., 1996; Heymann et al., 1996). Another in vivo model discerns a regulatory role for HGF in the migration and differentiation of premyogenic and neural crest cells during normal mammalian embryogenesis (Takayama et al., 1996). HGF transgenic mice (under a metallothionein pro-

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moter) show an inappropriate expression of HGF which influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, as well as patterned hyperpigmentation of the skin. This migration pattern is likely to involve integrins as HGF is able to restore liver defects present in heterozygous SMAD 2/3 knockouts in a manner involving b 1-integrin (Weinstein et al., 2001). Other tissue types also appear to have specific roles for HGF during development and beyond. The participation of HGF in mammary, renal and lung morphogenesis has been extensively studied (Matsumoto and Nakamura, 1997, 2001; Lail-Trecker et al., 1998; Niemann et al., 2000). Tubulogenesis, one of the classic functions of HGF, is an important event during the development of kidney, lung and breast. As HGF and MET knockouts are embryonic lethal at a very early age, no in vivo studies have been able to demonstrate directly a role for HGF in these tissues. Nevertheless, organ culture models have been able to implicate HGF in kidney, lung and mammary morphogenesis and differentiation (Woolf et al., 1995; Yang et al., 1995; Tabata et al., 1996; Ohmichi et al., 1998). For example, successful inhibition of HGF by antisense oligonucleotides or by neutralizing antibodies in embryonic organ cultures results in the abnormal development of kidney, lung, mammary gland and tooth. In the adult respiratory system, HGF localizes to the epithelium of trachea, bronchi and bronchioli, endothelial cells and macrophages of the alveolar walls (Noji et al., 1990; Wolf et al., 1991a). In mature kidney, it is seen in the distal and proximal collecting tubules (Okajima et al., 1990; Tashiro et al., 1990; Wolf et al., 1991b). HGF is abundantly expressed in the nervous system during development (Yang and Park, 1993; Maina et al., 1997, 1998; Yamamoto et al., 1997; Birchmeier and Gherardi, 1998; Maina and Klein, 1999). Numerous in vitro studies have shown a stimulatory effect for HGF on sensory and sympathogenic neurons. HGF can stimulate Schwann cell and axon growth. The somatotropic effect of HGF is limited by the expression of c-met in these cells. Again, lack of a viable knockout model has precluded an in-depth in vivo analysis of the importance of HGF in nervous system development. In the adult nervous system, HGF has been shown to be present in the large neu-

rons of the brain, spinal cord and peripheral ganglia, Purkinje cell epithelia of the choroid plexus, ependyma of the ventricles and many endothelial cells (Zarnegar et al., 1990a). In blood, where the HGF was originally detected, the protein is found in the platelets (Nakamura et al., 1987). The normal human serum levels of HGF as measured by ELISA range between 0.12 and 0.5 ng/ml (Tsubouchi et al., 1991a, 1991b). HGF has also been detected in pancreas (acini and ductal epithelium), skin (epidermis), mammary glands (ductal epithelium) and gastrointestinal tract (epithelium). Several cell lines also express HGF, including cultures from embryonic lung fibroblasts, human epidermal keratinocytes, human iliac artery smooth muscle and human chorionic villi (Stoker et al., 1987; Rosen et al., 1990; Adams et al., 1991). Thus, HGF is ubiquitously present in embryonic and adult tissues indicating a significant role in a variety of cell types.

HGF in disease states HGF has been the subject of intense research regarding its role in tumorigenesis, tumor invasion and metastases (Zarnegar and DeFrances, 1993; Birchmeier and Birchmeier, 1995; Michalopoulos, 1995; Sakata et al., 1996; Vande Woude et al., 1997; Jiang et al., 1999; Comoglio and Boccaccio, 2001). This stems from the fact that HGF has potent organotrophic function for the normal regeneration of tissues such as liver, kidney and lung (Matsumoto and Nakamura, 1997, 2001; Michalopoulos and DeFrances, 1997). Several animal models, cell lines and pathologic tissue studies have implicated HGF in disease initiation as well as progression. In carcinogenesis, there appears to be a clear role for HGF in tumor formation with studies indicating either overexpression or aberrant expression. Other significant changes include inappropriate production of HGF in tumor cells, increased production in stromal cells, increased circulating levels in the blood, elevated levels in other biologic fluids, reduced clearance from blood (if there is primary or secondary hepatic involvement) and increased levels of pertinent HGF activators in tumors. Characteristic changes observed in HGF expression are listed in Table 32.1. In vitro studies have determined that HGF has a proliferative role in several cell lines derived from

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TABLE 32.1 Expression and production of HGF/SF in cancers Tumor

HGF expression and production

References

Bladder cancer

HGF overexpressed and active HGF produced

Joseph et al., 1995

Breast cancer

Elevated expression in malignancy Increase in invasive ductal carcinoma Association with disease state and prognosis

Wang et al., 1994; Tuck et al., 1996; Yao et al., 1996

Colorectal cancer

HGF expression not detected

Hiscox et al., 1997; Di Renzo et al., 1995

Endometrial cancer

Expressed HGF

Wagatsuma et al., 1998

Gastric cancer

HGF blood levels markedly elevated

Glioma

Express and produce activated form of HGF

Taniguchi et al., 1997; Laterra et al., 1997; Moriyama et al., 1996; Moriyama et al., 1995; Yamamoto et al., 1997

Glioblastoma

HGF produced

Krasnoselsky et al., 1994; Yamamoto et al., 1997

Hepatocellular cancer

HGF overexpressed, Low expression of HGF Mixed expression

Ljubimova et al., 1997; Selden et al., 1994; Shiota et al., 1994 Kiss et al., 1997

Kaposi’s sarcoma

High expression

Naidu et al., 1994; Maier et al., 1996

Leukemia

Expressed in malignant cells Increased HGF in blood

Jucker et al., 1994; Hino et al, 1996; Nakamura et al., 1994

Lung cancer Adenoma SCC NSCLC

Positive in tumor Highly expressed Highly expressed Highly expressed and produced Express HGF and elevated serum levels of HGF

Takanami et al., 1996 Singh-Kaw et al., 1995 Harvey et al., 1996 Olivero et al., 1996; Siegfried et al., 1997 Seckl et al., 1994; Itakura et al., 1994

Multiple myeloma

HGF overexpressed and overproduced in myeloma cells

Borset et al., 1996a, 1996b; Seidel et al., 1998

Pancreatic cancer

HGF expressed and produced

Furukawa et al., 1995; Ebert et al., 1994; Ueda et al., 1996

Prostate cancer

HGF produced in stromal cells

Pisters et al., 1995; Humphrey et al., 1995

Rhabdomyosarcoma

HGF expressed

Ferracini et al., 1995

Squamous cell carcinoma

High expression

Takada et al., 1995

Transitional cell carcinoma

Increase levels in tissue and urine

Rosen et al., 1997

SCLC

cancerous tissues of the prostate, pancreas, liver, stomach, ovary, skin and blood (leukemia). HGF can also induce increased motility/migration in cells derived from these tumors (Jiang et al., 1993; Corps et al., 1997). Transgenic mice overexpressing mouse HGF under the metallothionein promoter display an increased propensity to develop diverse types of

tumors (Takayama et al., 1997). These mice reveal a functional link between mechanisms regulating development and cancer as tumors were detected in abnormally developed organs and tissues including mammary gland, skeletal muscle and melanocytes. Tumors observed in these mice included hepatocellular adenomas and hepatic hemangiosarcomas,

THE CYTOKINES AND CHEMOKINES

THE HGF RECEPTOR , MET

melanomas, rhabdomyosarcomas, fibrosarcomas, skin hair follicle tumors, salivary gland adenocarcinomas, adenosquamous carcinomas and adenocarcinomas of the breast. In a separate study, overexpression of the five-amino-acid deleted isoform of human HGF was targeted to the liver. These animals exhibited enhanced formation of hepatocellular carcinomas (Bell et al., 1999). Abnormal expression of HGF displays a positive correlation with disease progression in several types of tumors such as carcinomas of lung, skin, bladder and brain (Natali et al., 1993; Koochekpour et al., 1997; Rosen et al., 1997; Siegfried et al., 1997). This corresponds with the fact that a definite augmentation of tumor cell motility and invasion has been shown using HGF both in vitro and in vivo. For example, addition of HGF to cultures causes the release of matrix degrading proteins (Bennett et al., 2000). HGF can also influence collagenase-1 and stromelysin-1 production in a dose- and matrixdependent manner (Dunsmore et al., 1996) and can affect the stability of cell adhesion molecules (adherens junctions) (Pasdar et al., 1997), thereby providing a means for it to regulate tumor invasion and metastasis. The levels of HGF in tumor extracts and/or serum are increasingly being studied as prognostic indicators of tumor progression and invasion. Increased levels in breast tumors as well as serum from breast cancer patients have been shown to correlate with prognosis and relapse (Yamashita et al., 1994; Tanaguchi et al., 1995). Similar studies in prostate and gastric cancer have also shown a positive correlation of elevated serum HGF and metastases (Taniguchi et al., 1997; Naughton et al., 2001). Recent evidence suggests that the serum elevation detected in recurrent cancers may be due, at least in part, to production by the peripheral blood mononuclear cells, rather than solely by the tumors themselves (Beppu et al., 2001). HGF also has a role in angiogenesis that may contribute to the development of tumors (Grant et al., 1993; Corps et al., 1997;) and can induce neovascularization in several ischemic pathologies such as limb and myocardial ischemias (Aoki et al., 2000; Tomita et al., 2000). Recently, administration of HGF was shown to improve the blood flow in diabetic peripheral artery disease (Taniyama et al., 2001). Other non-tumor-related disease states are also

791

known to display altered HGF expression. Only minimal expression of HGF mRNA is found within intact oxyntic rat mucosa. Immunoblot and Northern blot analyses demonstrate elevated levels of activated HGF following experimentally induced gastric mucosal damage (Milani and Calabro, 2001) and HGF may be responsible for stimulating the activation of COX-2 in gastric epithelium via MET phosphorylation and subsequent ERK2 signaling. Topical and systemic introduction of HGF has ulcer-healing properties (Brzozowski et al., 2001); some anti-ulcer drugs may act by stimulating expression of HGF in the mesenchymal cells of the granulation tissue during the mucosal healing response. The role of HGF in inflammatory bowel disease is increasingly being evaluated (Beck and Podolsky, 1999). As an angiogenic cytokine, elevated serum levels of HGF have been demonstrated in autoimmune conditions like systemic lupus erythematosus and it is thought to play a role in its pathogenesis (Robak et al., 2001). Thus, HGF appears to be abnormally expressed in a multitude of disorders and a careful interpretation of the studies for its ‘cause or effect’ relationship is mandatory.

THE HGF RECEPTOR, MET MET protein sequence The receptor for HGF was found to be the product of the c-met proto-oncogene, a protein that belongs to the receptor tyrosine kinase superfamily (Bottaro et al., 1991; Naldini et al., 1991a, 1991b). The c-met gene was originally isolated from a human osteogenic sarcoma cell line (HOS) treated in vitro with the chemical carcinogen N-methyl-N -nitro-Nnitrosoguanidine (MNNG) (Cooper et al., 1984). The MET protein is a 190-kDa, disulfide-linked heterodimer consisting of an a and a b subunit (Park et al., 1987). The a subunit is heavily glycosylated and is completely extracellular. The b subunit also has an extracellular portion that is involved in ligand binding, as well as a transmembrane segment and a cytoplasmic tyrosine kinase domain containing multiple phosphorylation sites. Both subunits originate from the furin-mediated proteolytic cleavage of a common precursor that is 170 kDa (Mark et al., 1992; Komada

THE CYTOKINES AND CHEMOKINES

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HEPATOCYTE GROWTH FACTOR AND ITS RECEPTOR , MET

et al., 1993) and is glycosylated to produce the mature 190-kDa protein. The nucleotide sequence of the overlapping cDNA clones corresponding to human c-met reveals a single open reading frame of 4224 nucleotide residues encoding a 1408-amino-acid protein that has the characteristics of a tyrosine kinase receptor (Park et al., 1987). The first 24-aminoacid residues at the amino-terminal end of MET are highly hydrophobic and probably serve as a signal peptide to facilitate transport of the protein into the lumen of the endoplasmic reticulum. The aminoterminal extracellular domain consists of 926 amino acids (residues 25–950) and contains many cysteine residues, as well as 10 consensus sites for asparaginelinked N-glycosylation (N–X–S/T). A second hydrophobic domain is found at residues 950–973. This domain has the characteristics of a membranespanning region and is followed immediately by a cluster of hydrophilic amino acids (residues 974–977) that are likely to serve as a cytoplasmic anchor. This putative transmembrane domain divides the MET protein into two regions which correspond to extracellular and intracellular portions of the receptor. The cytoplasmic domain of 435 amino acids (residues 1101–1351) contains a consensus ATP-binding domain and a kinase domain. Comparison of the overall amino acid and nucleotide sequences of c-met for the rat, mouse and human genes reveals a high degree of conservation for the MET primary structure among the three species (Park et al., 1987; Chan et al., 1988; Liu et al., 1996). The overall homology of the amino-acid sequences of the rat and mouse versus the rat and human are 96% and 89%, respectively (Liu et al., 1996). The lengths of the rat and mouse MET proteins are similar, whereas the human counterpart is 26 and 29 residues longer than the rat and mouse, respectively (Park et al., 1987; Chan et al., 1988; Liu et al., 1996). This is largely due to an 18-amino-acid deletion in the extracellular domain of rat and mouse MET. In addition, both the rat and mouse MET proteins are 9-amino-acid residues shorter than their human counterpart at the carboxy terminus. Of particular interest is the high degree of conservation found in the protein tyrosine kinase domain, where the percentage of identity between the rat and mouse, and the rat and human proteins are 99.6% and 97.6%, respectively (Liu et al., 1996). In addition, all of

the putative N-glycosylation sites except one are conserved among the three species. Finally, all the cysteine residues are 100% conserved among the three species and are located in exactly the same positions. The high degree of conservation of the amino acid sequence of MET among the different species is consistent with an important role for the HGF/c-met axis in biologic functions.

MET-related receptors The tyrosine kinase receptor superfamily includes a number of diverse proteins that are important regulators of cell growth, differentiation and development (Ullrich and Schlessinger, 1990; Comoglio, 1993). Based upon overall structural similarities, four subclasses of tyrosine kinase receptors have been defined. The HGF receptor belongs to a new subclass, IV, that includes MET, RON and SEA (Comoglio, 1993). Characteristic structural features of the HGF receptor subclass include a disulfide-linked heterodimer with one cysteine-rich sequence in the extracellular domain of the b subunit. This structure is quite distinct from the two cysteine-rich repeat sequences found in the extracellular domain of the monomeric type I receptors, the cysteine-rich sequences in the disulfide-linked heterotetrameric type II receptors, or the immunoglobulin-like repeats in the extracellular domain of the type III receptors (Figure 32.2) (Ullrich and Schlessinger, 1990; Comoglio, 1993). It should be pointed out that with the exception of the other members of the HGF receptor subclass, the amino-acid sequence in the tyrosine kinase domain of human MET is most closely related to that of the human insulin receptor, sharing 44% identity (Park et al., 1987). These data suggest that the HGF receptor gene family exhibits a close evolutionary relationship with the gene for the insulin receptor and suggests that these receptors may have evolved from a common ancestral gene harboring the basic tyrosine kinase domain.

MET gene organization The c-met gene is located at human chromosome 7q31, on the same chromosome as its ligand gene, HGF (Mizuno and Nakamura, 1993; Lin et al., 1996). Detailed restriction mapping, Southern hybridization

THE CYTOKINES AND CHEMOKINES

THE HGF RECEPTOR , MET

I

II

and DNA sequencing have revealed that the gene encoding MET spans more than 120 kb in length and consists of 21 exons separated by 20 introns (Lin et al., 1998; Liu, 1998). The sizes of the exons range from 81 bp (exon 16) to approximately 4 kb (exon 21), while the sizes of the introns are more heterogeneous, ranging from 0.1 kb to about 26 kb. The sequences of all the intron–exon splice junctions follow the GT–AG rule established for eukaryotic genes, and both the 5 splice donor and the 3 acceptor sequences are closely related to the consensus splice sequence. Ten of the splice junctions are type 0, where the intervening sequence is located between two codons, while the rest of the junctions are types I and II, with the intervening sequences located at the first, or second nucleotide of the codons. The entire first exon encodes the 5-untranslated sequence of the human c-met transcript (394 bp) (Figure 32.3) (Liu, 1998) and is separated by approximately 26 kb from the second exon. The second exon is the largest coding exon, containing a 14 bp of 5-untranslated sequence as well as a stretch of 400 amino acids that includes the signal peptide. The cleavage site that yields the a and b subunits from the single-chain polypeptide precursor is located within exon 2 and the entire extracellular portion of HGF receptor is encoded by 11 exons (from exon 2 to exon 12) distributed over a region of approximately 70 kb. Exon 13 codes for the complete hydrophobic transmembrane domain of MET. The cytoplasmic domain

IV

III

Cys (IgLD)n Cys

KI

PTK

EGF-R HER2/neu c-erbB-3

Insulin-R IGF-R

793

PDGF-R(A/B) MET CSF-1R (fms ) RON FGF-R SEA KGF-R(bek)

FIGURE 32.2 Schematic representation of the four receptor tyrosine kinase subclasses described by Comoglio (1993). For each subclass the prototype receptor is listed first with known related family members below. For example, MET is the prototype of the tyrosine receptor subclass IV that also includes RON and SEA. Cysteine-rich domains (Cys) are shown as unshaded rectangles. Immunoglobulin-like domains (IgLD) are depicted as circles. Protein kinase domains (PTK) are shown as darkened cylinders. KI, kinase insert; EGF, epidermal growth factor; IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; CSF, colony-stimulating factor; FGF, fibroblast growth factor; KGF, keratinocyte growth factor.

Transmembrane domain Extracellular domain 1

5-Noncoding region

2

3

4 5

cleavage site separating

and  chains

signal peptide

6

Cytoplasmic domain

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

tyrosine kinase domain alternative splicing (-18 aa)

3-Noncoding region

alternative exon (-47 aa)

FIGURE 32.3 Schematic representation of the human MET exons with their corresponding structural and functional domains. Exon numbers are shown above the boxes. Two alternative splice sites described by Rodrigues et al. (1991) and Lee and Yamada (1994), are indicated by arrows. Adapted from Liu (1998), with permission from Elsevier Science. THE CYTOKINES AND CHEMOKINES

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HEPATOCYTE GROWTH FACTOR AND ITS RECEPTOR , MET

of MET is encoded by exons 14 to 21 and spans approximately 30 kb of genomic DNA with the juxtamembrane region encoded by exon 14 and the protein tyrosine kinase domain encoded by exons 16 to 21. Exon 21 is the largest of the 21 exons and includes the coding region for the carboxy terminus of the protein as well as the large 3-untranslated region of the gene.

MET promoter structure and function The promoter region of the c-met gene has been isolated and structurally characterized (Liu, 1998; Seol and Zarnegar, 1998). Analysis of the full sequence reveals that the c-met promoter lacks either TATA or CAAT elements, has greater than 70% G–C content within a 600-bp region around the transcription initiation site (positions 400 to 200), and contains seven repeats of the consensus Sp1-binding sequence (Liu, 1998). A number of potential regulatory elements, including AP1, AP2, NFjB and IL-6RE binding sites, are found in the regulatory region of human c-met gene. The presence of these elements suggests that control of c-met expression involves interactions between multiple cis-acting elements and transacting factors (Liu, 1998; Seol et al., 2000). To delineate the sequences essential for c-met transcription, a series of human c-met promoter–reporter chimeric plasmids were constructed in which various lengths of the 5-flanking region of the human c-met were fused to the coding region of the chloramphenicol transferase (CAT ) gene (Liu, 1998) and used in transient transfection assays. The 0.1met–CAT containing the minimal promoter region (68 to 60) was expressed at a low level in renal epithelial mIMCD-3 cells. This basic promoter activity was modulated by multiple cis-acting regulatory elements in the 5-flanking region of the c-met gene. Two positive regulatory elements were found at nucleotide positions 2615 to 1621, and 223 to 68, respectively. In contrast, a negative element was identified at nucleotide position of 1621 to 1093 (Liu, 1998). Sequence analysis indicates that the strongly positive element found at nucleotide positions 233 to 68 is concomitant with three Sp1 sites that are contained within this region. Deletion, mutation and transfection studies indicate that multiple Sp1 sites in the proximal promoter region are responsible for consti-

tutive expression of c-met in renal epithelial cells (Liu, 1998; Seol and Zarnegar, 1998).

MET expression During development c-met is expressed in the mouse embryo at gastrulation and early organogenesis (Andermarcher et al., 1996). The expression of c-met overlaps with HGF expression during this period and is seen in the endoderm and the mesoderm along the rostro-intermediate part of the primitive streak, followed by expression in the node as well as the notochord. Neither c-met nor HGF are expressed in the ectoderm at any time during gastrulation. In early organogenesis, overlapping expression of HGF and c-met is found in the heart, condensing somites and neural crest cells. A second distinct expression pattern of c-met is seen in the ectodermal areas of bronchial arches and limb buds. At later stages of gestation (E13 p.c.), expression is observed in the differentiated somites and in several organs such as liver, lungs and gut. A similar distribution of c-met is also observed in the developing liver of rat (DeFrances et al., 1992). c-met transcripts are also observed in neural, endothelial and muscle cells during embryogenesis (Sonnenberg et al., 1993). Elevated expression of c-met and HGF during the early development of the pancreas has also been reported and appears to be age dependent with the highest expression occurring before weaning (Calvo et al., 1996). The expression of c-met has also been observed during the early, critical periods of human organogenesis at 6 to 13 weeks of gestation (Kolatsi-Joannou et al., 1997). Liver, kidney, intestine and lungs express both HGF and c-met and clearly indicate the role of this pathway in inductive interactions between mesenchyma and epithelia. Between 7 and 8 weeks of gestation, c-met is localized to the epithelia and especially the fundic parietal cells, pancreatic and gut endocrine cells and in muscular layers (Kermorgant et al., 1997). c-met has also been detected in the trophoblastic cells of the chorionic villi as early as 5 weeks of gestation. The generation and analysis of HGF and c-met knockout mice have definitively proven the essential role of HGF–MET interactions during embryogenesis. Ablation of the c-met gene results in the complete absence of myogenic precursor cells in the limb buds as well as the diaphragm (Bladt et al., 1995). In con-

THE CYTOKINES AND CHEMOKINES

THE HGF RECEPTOR , MET

trast, the development of the axial skeletal muscles is normal. In HGF null mice, defects also include a decrease in liver size and the failure of embryonic stem cells harboring the null mutation to contribute to the adult liver (Schmidt et al., 1995; Uehara et al., 1995). In the developing nervous system, the expression pattern of c-met is consistent with a role in axon guidance of the motor neurons that are abundant in the limb and trunk innervating segments (Ebens et al., 1996; Yamamoto et al., 1997). Apart from changes in motor neuron branching in the developing limbs, c-met-null mutant embryos also exhibit a small increase in cell death of the sensory and sympathetic neurons (Maina et al., 1996, 1997). Unfortunately, the ultimate death of the null embryos has precluded an analysis of the c-met gene at later gestational stages when development of the extensive peripheral and central nervous system occurs. The development of Xenopus liver also requires MET and HGF. Overexpression of a transdominant c-met (without a tyrosine kinase domain) has established a positive role of c-met in liver development as the hepatic compartment failed to develop (Aoki et al., 1997). Other notable defects in these animals include problems in the development of the gut epithelia and pronephros. Thus, fundamental similarities in the expression of HGF and c-met exist in the epithelial and mesenchymal cells of Xenopus. This is consistent with the review by Birchmeier and Gherardi (1998) that demonstrates the role of HGF as a mediator of epithelial–mesenchymal interactions is evolutionarily conserved across vertebrates.

MET in disease states In adult, c-met is expressed at relatively low levels in a variety of tissues such as breast, liver, lung, kidney, large and small intestine, placenta, skin, stomach and thyroid (Prat et al., 1991). The most abundant transcript observed is the 8-kb species, although other species with sizes of 9, 7, 5 and 3.4 kb have also been observed (Giordano et al., 1989). In addition to normal tissues, a number of cancer cell lines have also been found to express c-met. For example, carcinoma cell lines from gastric and colorectal cancers show elevated expression of c-met (Kuniyasu et al., 1992; Liu et al., 1992). A comprehensive list of c-met expression in human malignancies is provided in Table 32.2.

795

An oncogenic form of c-met (tpr-met) was first identified in the DNA from an N-methyl-N -nitro-Nnitrosoguanidine-treated human osteosarcoma cell line (Cooper et al., 1984). The tpr-met chimera is activated via leucine zipper dimerization of the tpr portion of the protein. Dimerization leads to transphosphorylation of the met kinase and hence, a constitutively active state (Landschulz et al., 1988; Rodrigues and Park, 1993). Another piece of strong evidence suggesting a distinctive role for MET in tumor formation comes from experiments with NIH 3T3 fibroblasts. Transfection of the fibroblasts with the normal c-met cDNA results in tumor formation when the altered cells are injected into immunologically compromised ‘nude’ mice (Rong et al., 1992, 1993). Presumably, transformation results from the formation of an autocrine loop between HGF and MET. Immunohistochemical studies have demonstrated elevated MET staining in several tumors such as hepatocellular carcinoma (Prat et al., 1991). Overexpression of c-met (enhanced tyrosine kinase activity) has also been noted in a variety of human tumors (Di Renzo et al., 1991; Comoglio, 1993) and the c-met gene itself is frequently amplified (Rong et al., 1993). In vivo studies have also demonstrated the oncogenic capabilities of c-met. Deregulated c-met expression of tpr-met has been shown to result in mammary tumorigenesis in a transgenic mouse model (Liang et al., 1996) and inherent missense mutations have been shown to be associated with papillary renal carcinoma (Schmidt et al., 1997). In animal models of chemically induced carcinogenesis using 2-acetylaminofluorene and diethylnitrosamine, there is a transient increase in c-met expression (Imai et al., 1996). Finally, autocrine activation of MET has also been reported in melanoma (Otsuka et al., 1998). The role of MET in assessing the prognosis and survival of cancer patients has also been investigated. In pancreas, it has been shown that there is no significant MET immunoreactivity in normal duct epithelium. On the other hand, a subset of patients with resectable ductal adenocarcinoma exhibits diffuse MET immunostaining. This has a prognostic significance as these patients have a significantly lower survival rate than patient with tumors showing negative or focal staining for MET (Furukawa et al., 1995). In another study of gastric cancer patients, c-met

THE CYTOKINES AND CHEMOKINES

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HEPATOCYTE GROWTH FACTOR AND ITS RECEPTOR , MET

TABLE 32.2 MET expression in cancers Tumor

MET expression

References

Bladder cancer

c-met overexpressed

Joseph et al., 1995

Breast Cancer

Overexpressed c-met and higher levels in undifferentiated form Allelic loss in primary but more prominent in metastasis

Beviglia et al., 1997; Wang et al., 1994 Jin et al., 1997; Tuck et al., 1996 Ng et al., 1997

Bone tumors c-met overexpressed Chordoma, chondrosarcoma Osteosarcoma, enchondroma Chondroblastoma, Osteochondroma

Naka et al., 1997; Scotlandi et al., 1996

Burkitt’s lymphoma

c-met overexpressed

Jucker et al., 1994; Weimar et al., 1997

Colorectal cancer

c-met overexpressed, increased in later stage and metastasis

Di Renzo et al., 1995; Hiscox et al., 1997

Esophageal cancer

c-met overexpressed

Iwazawa et al., 1996

Gastric cancer

Overexpressed c-met and related to disease stage Increased expression in invasive and diffuse disease

Kaji et al., 1996; Takeuchi et al., 1996 Wu et al., 1997; Kaji et al., 1996

Glioblastoma

Expressed c-met

Moriyama et al., 1996; Moriyama et al., 1995; Rosen et al., 1996; Shiota et al., 1996

Glioma

Overexpressed c-met

Moriyama et al., 1994; Moriyama et al., 1996; Yamamoto et al., 1997

Hepatoblastoma

Overexpressed c-met

Kuniyasu et al., 1996; Sunitha et al., 1994

Hepatocellular cancer

Overexpressed c-met Mixed expression of c-met

Rahimi et al., 1994; Ueki et al., 1997 Selden et al., 1994; Shiota et al., 1992 Ljubimova et al., 1997; Shiota et al., 1994 Kiss et al., 1997

Hodgkin’s disease

c-met overexpressed

Jucker et al., 1994

Kaposi’s sarcoma

Highly elevated expression

Maier et al., 1996; Naidu et al., 1994

Leukemia

c-met overexpressed

Hino et al., 1996; Jucker et al., 1994

Lung cancer

Adenocarcinoma and SCC Overexpressed c-met

Takanami et al., 1996; Ichimura et al., 1996

Melanoma

Overexpression, activation Livant et al., 1995; Saitoh et al., 1994 and abnormal distribution of c-met Halaban et al., 1992

Myeloma

c-met overexpressed in myeloma cells

Borset et al., 1996a; Borset et al., 1996b Seidel et al., 1998

Ovarian cancer

Overexpressed c-met

Corps et al., 1997; Wagatsuma et al., 1998

Pancreatic cancer

Overexpress c-met and related to survival

Tani et al., 1997; Vila et al., 1995 Furukawa et al., 1995

Prostate cancer

Overexpressed c-met, especially in metastatic disease

Humphrey et al., 1995; Pisters et al., 1995; Di Renzo et al., 1995; Ebert et al., 1994

Renal carcinoma

Overexpression c-met

Kobayashi et al., 1994

Rhabdomyosarcoma

Abnormal c-met expression

Ferracini et al., 1995

THE CYTOKINES AND CHEMOKINES

HGF/MET SIGNALING

expression was found in about 43% of the patients. These tumors had a greater tendency towards local and remote metastases with enhanced lymphatic and peritoneal dissemination and were associated with poorer prognosis.

HGF/MET SIGNALING Much research interest has focused upon the fact that a single ligand, HGF, can induce such pleiotrophic responses (i.e. mitosis, migration, tubulogenesis, cytotoxicity) via interaction with a single receptor, MET. The logical assumption is that the cellular environment determines what the final outcome will be; however, there are many aspects to what constitutes this ‘cellular environment’. For example, HGF is a heparin sulfate binding protein and it could be hypothesized that the glycosaminoglycan content on the cell surface determines which biologic outcome the HGF signal will induce. On the other hand, costimuli or repressors, simultaneously signaling via different cell surface receptors, could be what determines the final endpoint. Another possibility is that specific internal proteins (i.e. kinases, phosphatases, transcription factors) within the cells are responsible for the final result. In fact, evidence exists to suggest that all of these mechanisms, and possibly more, help to determine which pathways the cells eventually take in response to HGF.

The role of proteoglycans HGF is a heparin-binding protein. Mutation analyses have determined that a very few amino acids within the N terminus of the protein (Lys60, Lys62, Arg73, Arg76, Lys78) and a region from kringle 2 are primarily responsible for this characteristic (Mizuno et al., 1994; Zhou et al., 1999). It has long been assumed that binding of HGF to cell surface proteoglycans has a role in regulating HGF–MET interactions; however, the mechanism by which this occurred was unclear. Recently, Rubin et al. (2001) showed that MET is also able to bind heparin. In cells that lack proteoglycans, the K1 variant of HGF (lacking a heparin-binding domain) is only able to induce mitosis when heparin is present. Interestingly, motility is heparin independent, suggesting proteoglycans may specifically regulate

797

the mitotic response. In support of this hypothesis is the finding that HGF knockout mice with a deletion of exon 2 (the primary heparin-binding region) have impaired hepatic growth as their overriding defect (Schmidt et al., 1995). Mice with a deletion in exon 5 (encoding kringle 1) have a more severe phenotype involving placenta as well as liver (Uehara et al., 1995).

The role of co-receptors Multiple proteins, including CD44, the EGF receptor (EGF-R), RON, b-catenin and the u-PA receptor (u-PAR) have been shown to exhibit functional interactions with HGF and MET. A heparin-containing splice variant of CD44 has specifically been shown to facilitate HGF–MET interactions via HGF binding, resulting in the phosphorylation of MET and subsequent downstream activation of ERKs 1 and 2 (van der Voort et al., 1999). In transformed rat liver epithelial cells, EGF-R and MET are found to co-precipitate. In these cells constitutive activation of MET is inhibited by antibodies against EGF-R or one of its ligands, TGFa (Jo et al., 2000), suggesting that direct interaction between EGF-R and MET is responsible for the continuous phosphorylation of MET. In addition to EGF-R, direct cross-talk between MET and the related type IV receptor, RON, has been described (Follenzi et al., 2000). Although there is no direct evidence linking E-cadherin and MET, the two proteins coendocytose when either receptor is stimulated. Endocytosis involves Rho and Rab family members and, for the HGF ligand only, PI-3 kinase (Kamei et al., 1999). This phenomenon can be partially explained by the observation that MET and b-catenin are able to co-precipitate (Monga et al., 2001) as b-catenin and E-cadherin have long been known to associate (Wheelock and Knudsen, 1991). Finally, MET is able to interact with the u-PA receptor as well as u-PA (Naldini et al., 1995; Stolz et al., 1999). This finding indicates that complex formation occurs between MET and some of the proteins relevant for HGF activation (Mars et al., 1995, 1996). Although u-PAR does not span the membrane, u-PA is able to induce intracellular signaling through many of the same effectors as MET (Nguyen et al., 2000). Thus, MET–u-PAR interactions may be an important part of the paracrine/ endocrine-mediated signal transduction pathway.

THE CYTOKINES AND CHEMOKINES

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HEPATOCYTE GROWTH FACTOR AND ITS RECEPTOR , MET

Signal transduction The carboxy terminus of MET contains a multifunctional docking site. Following tyrosine phosphorylation, this site is able to bind a multitude of SH2-containing proteins including phosphatidylinositol 3-kinase (PI-3) kinase, phospholipase Cgamma (PLC-c), Src, the Grb2 and Shc adapters and Gab1 (Ponzetto et al., 1994; Schaeper et al., 2000). Given the variety of responses that MET is capable of inducing, much interest has centered upon discerning which interactions between the various docking molecules are required and/or involved in the diverse biologic pathways. Experiments using transgenic mice with specific mutations introduced into the docking site have done much to clarify which roles the various adapters play. In 1996, Maina et al. showed that tyrosines 1349 and 1356 are essential for normal development. In addition to producing a lethal phenotype similar to loss of HGF or MET (Maina et al., 1996), functional loss of these two tyrosines results in the loss of binding to Gab1, Grb2, Src and PI-3 kinase (Maina et al., 2001). Phosphorylation of Gab1 and other downstream molecules is reduced but not completely absent in these animals, suggesting a signaling threshold may be necessary for their effect. When the multifunctional docking sites were mutated to reproduce optimal binding motifs for PI-3 kinase, Src or Grb2, the data showed that both PI-3 kinase and Src are necessary for hepatocyte survival and myoblast migration. On the other hand, Src binding (without PI-3 kinase) was sufficient for both placental and myoblast proliferation and PI-3 kinase (without Src) was sufficient for axon outgrowth and branching (Maina et al., 2001). In addition to migration, mitosis and cell survival, an important downstream effect of HGF is branching tubulogenesis. In 1998, Boccacio et al. showed that tubule formation by epithelia is dependent upon the STAT pathway. The way in which this occurs is unclear although there is evidence suggesting that STAT can bind directly to MET via its SH2 domain. Recent studies by Gual et al. (2000) have shown that the length of induced signaling may also affect outcome. When downstream signaling using EGF (incapable of inducing tubules) was compared with HGF, the primary difference between the effects of the two growth factors was the length of time that Gab1

remained phosphorylated; sustained phosphorylation of Gab1 was associated with tubulogenesis. The prolonged phosphorylation of Gab1 is likely to involve PLC-c as overexpression of a Gab1 mutant unable to bind PLC-c allowed migration and mitosis, but not branching morphogenesis. Interestingly, serine/ threonine kinase-induced hyperphosphorylation of Gab1 leads to tyrosine hypophosphorylation, with a resultant down-regulation of HGF-induced signaling (Gual et al., 2001). These data are in agreement with the fact that the juxta-membrane domain of MET, thought to be the region that controls the negative regulation of MET (Vigna et al., 1999), binds the tyrosine phosphatase known as PTP-S (Villa-Moruzzi et al., 1998)

SUMMARY AND FUTURE DIRECTIONS The HGF/MET system is unusually complicated. As such, interactions between HGF and MET can lead to a variety of responses including mitosis, motility, tubulogenesis and cytotoxicity. Partly, this is due to a multifunctional docking site that is unique to the carboxy terminus of class IV tyrosine kinase receptors; however, specific intra- and extracellular effectors also contribute to biologic outcome. Although most HGF–MET interactions are believed to occur in a paracrine/endocrine manner, there is evidence to suggest that in some cases the same cells that produce HGF can also respond to it. Furthermore, HGF must be cleaved to induce signaling and there are multiple known molecules that are capable of performing this function, some of which (u-PA and u-PAR) that may be contributing to the cell signaling process via complex formation. Finally, multiple forms of HGF exist and there is evidence suggesting that two of these forms (NK1 and NK2) can confer either agonistic or antagonistic effects on HGF/MET signaling, depending upon the situation. Thus, when one describes HGF and MET, there are no simple discussions regarding how this system functions. Multiple areas of HGF/MET biology are likely to be expanded in the very near future. One of the main hindrances to understanding HGF–MET biology in vivo has been the fact that ablation of either

THE CYTOKINES AND CHEMOKINES

REFERENCES

HGF or MET results in an embryonic lethal phenotype. This has been partially overcome by introducing point mutations within the tyrosine kinase domain of MET that allows the embryos to complete development; however, these animals are still developmentally challenged. To get around this problem, the next avenue of exploration is likely to be conditional ablation of these genes using cre/lox technologies (Rohlmann et al., 1996). For example, by generating mice with ‘floxed’ HGF and/or MET genes and then breeding them with mice that express the cre recombinase under organ-specific promoters (i.e. albumin in the mouse to introduce a liver-specific phenotype), one can generate adult animals with regional ablation of the genes. The second area of exploration that is likely to expand is the understanding of HGF activation. Currently, there are at least six proteins that can facilitate the generation of mature HGF. With the exception of one (HGFa), all are known to have other functions and it remains an intriguing question to understand which factors control the utilization of one activator versus another or, why one activator chooses one substrate versus the other. The final area that will most surely expand is the understanding of how a single ligand/receptor pair induces such a variety of responses. This last area of research will probably continue to explore signal transducers; however, it is also likely to expand heavily into the area of co-receptors.

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hepatocyte growth factor, transforming growth factor beta1, and activin A: comparison of a cholangiocarcinoma cell line with primary cultures of non-neoplastic biliary epithelial cells. Hepatology 32, 26–35. Zarnegar, R. and DeFrances, M.C. (1993). Expression of HGFSF in normal and malignant human tissues. EXS 65, 181–199. Zarnegar, R. and Michalopoulos, G. (1989). Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res. 49, 3314–3320. Zarnegar, R. and Michalopoulos, G.K. (1995). The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J. Cell Biol. 129, 1177–1180. Zarnegar, R., Muga, S., Enghild, J. and Michalopoulos, G. (1989). NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes. Biochem. Biophys. Res. Commun. 163, 1370–1376. Zarnegar, R., DeFrances, M.C., Oliver, L. and Michalopoulos, G. (1990a). Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes. Biochem. Biophys. Res. Commun. 173, 1179–1185. Zarnegar, R., Muga, S., Rahija, R. and Michalopoulos, G. (1990b). Tissue distribution of hepatopoietin-A: a heparin-binding polypeptide growth factor for hepatocytes. Proc. Natl Acad. Sci. USA 87, 1252–1256. Zarnegar, R., Petersen, B., DeFrances, M.C. and Michalopoulos, G. (1992). Localization of hepatocyte growth factor (HGF) gene on human chromosome 7. Genomics 12, 147–150. Zhou, H., Mazzulla, M.J., Kaufman, J.D. et al. (1998). The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site. Structure. 6, 109–116. Zhou, H., Casas-Finet, J.R., Heath Coats, R. et al. (1999). Identification and dynamics of a heparin-binding site in hepatocyte growth factor. Biochemistry 38, 14793–14802.

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33 Toll-like receptors Bruce Beutler The Scripps Research Institute, La Jolla, CA, USA

Misbalance of the constitution is the illness, yet the cause is the practical point. There are causes we do not know. Jean-François Fernel

INTRODUCTION Inflammation denotes the ‘fire’ that attends infectious, traumatic or autoimmune reactions, and cytokines coordinate its every aspect. But what is the primary cellular event that leads to cytokine production? This question of causa causans must be ranked among the most profound that cytokine biologists have confronted – and among the most therapeutically enticing. The daunting complexity of cytokine networks, with their many instances of mutual induction, synergy, cross-talk, and inhibition, belies the fact that the initial events in inflammation are comparatively simple. Yet for many years, insight into these events remained elusive. As described in this chapter, the inflammatory cytokine cascade is chiefly triggered by members of a family of receptors that act as the primary sensors used by the innate immune system. Named for a Drosophila prototype, these are the Tolllike receptors (TLRs).

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

HISTORY Innate versus adaptive immunity In the late nineteenth century, when immunology gained acceptance as a legitimate science, a mechanistic dichotomy immediately became evident (Mörner, 1908). On the one hand, Ehrlich and his disciples determined that ‘antitoxins’ (later known as antibodies) could mediate protection of the host. This form of protection was absent in naive animals, but was stimulated by exposure to a toxin (antigen), and grew stronger over a period of days or weeks. The response had properties suggestive of ‘memory’ in that secondary responses to the antigen were more intense than primary responses. The system seemed ideally attuned to the elimination of toxins, though clearly, it could also eradicate pathogens per se, including both bacterial and viral agents. This humoral mode of host defense was strictly confined

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to vertebrates, and hence, seemed to have arisen rather recently in evolution. On the other hand, Metchnikoff and his followers recognized the existence of a far more primitive – yet utterly essential – type of host defense, rooted in the predatory behavior of wandering phagocytic cells that were represented in some form in practically all metazoans. These cells, exemplified in mammals by macrophages and granulocytes, were capable of engulfing and killing invasive microorganisms. No prior exposure to a pathogen was required for activation of this form of immunity. Rather, responses to infection were ‘innate.’ In the course of time, many connections between the two systems became apparent. The mammalian host had, it seemed, evolved so as to integrate the killing potential of what came to be known as ‘adaptive’ and ‘innate’ immunity, and in certain aspects, the two systems had become heavily dependent upon one another. Indeed, neither system is capable of providing adequate protection in the absence of the other. Many of the links between the two systems are now known to be dependent upon cytokines (Plate 33.1) (see Plate section). For example, antigen presentation by macrophages and dendritic cells occurs concomitantly with the expression of surface protein B7, which interacts with the T-cell receptor CD28, abetting the adaptive immune response. IL-12 produced by macrophages, is required to direct T cells to differentiate along the TH1 line, a process that involves T-cell production of the cytokine interferon c. Interferon c, in turn, augments the microbicidal activity of macrophages, and enhances their production of TNF. Macrophage-derived TNF can, through interaction with the p75 TNF receptor, stimulate T-cell proliferation that is required in the course of infection. Likewise, interleukin-1 derived from macrophages and other cells plays a co-stimulatory role, eliciting the secretion of IL-2, which, in an autocrine loop, evokes Tcell proliferation. Other bridges have formed between distinct populations of lymphoid cells themselves. For example, T-cell-derived CD40 ligand stimulates B-cell CD40, permitting IgG synthesis. The antibodies produced by B lymphocytes – both IgG and other forms of immunoglobulin – are known to opsonize invasive pathogens for destruction by phagocytic cells, including macrophages and granulocytes.

In both the adaptive and innate immunity fields, the question of ‘first cause’ quite naturally arose. What triggers an immune response? How does the body recognize that it is infected? The adaptive and innate immune systems were, for the most part, examined separately from one another. Ehrlich’s own view concerning the adaptive immune response revolved around the concept of ‘side chains’ that would bind antigen, and would, in turn, be synthesized in response to specific antigenic stimulus. Later, a concept of template-driven (‘instructional’) antibody formation held that the receptors of adaptive immunity formed by coupling with antigens and folding to accommodate the variable molecular surfaces of the latter. These models of adaptive immune recognition endured for several decades, but gave way to present concepts in the wake of discoveries concerning the structure of antibodies themselves. Abundant plasma proteins, accessible to measurement and isolation from the early days of the twentieth century, antibodies were found to be proteins with constant and variable regions, produced in an array of isoforms too numerous to explain by a model of germline transmission. Ultimately, antibodies – and the more elusive T-cell receptors – were shown to be produced by genomic rearrangements, and called into service as the result of clonal selection and amplification. Comparable understanding of the receptors that serve innate immune recognition lagged far behind. Although it was universally accepted that innate immunity was of paramount importance to host defense, the insoluble state of innate immune receptors (they are integral plasma membrane proteins), their low copy number, and uncertainty as to just what they target, all hampered inquiry into the receptive process utilized by cells of the innate immune system.

How are microbes perceived? The issue of innate immune recognition was informally divided into two fundamental questions. One of these was the question as to what was perceived by the host. Among microbial molecules, which were of primary importance? As specific molecules capable of strongly activating host innate immunity were found and chemically characterized, the question as to how these molecules were perceived came to the fore.

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The concept of receptors for molecules that elicit biological responses even at highly dilute concentrations gained credence during the early half of the last century, and it was widely assumed by immunologists that cells of the innate immune system must be endowed with specific receptors for the recognition of microbes and their products. In time, it was accepted that receptor diversity was far less prominent in the innate immune system than in the adaptive immune system, for there was no evidence of clonality, nor sufficient space in the genome to encode a different receptor for every microbe or molecular species with which the innate immune system might have to deal. The recombinase system that generates diversity among antibody and T-cell receptor molecules is, so far as we know, limited to those two systems. While it remained possible that a separate mechanism, such as alternative splicing, might render great diversity among the innate immune receptors, no evidence ever emerged in support of such a hypothesis. Rather, it seemed more and more likely that innate immune cells would detect microbial molecules that were shared across broadly defined taxa, and were essential for microbial survival. The question of how microbes are perceived is solidly linked to the question of how microbes create injury. The toxicity of microbial infections had been explored almost since the discovery of microbes themselves. One of the first molecules to be identified in this connection, because of its abundance and its potency, was that substance called ‘endotoxin’ (Pfeiffer, 1892), and later designated as lipopolysaccharide (LPS) to convey its chemical composition. A fairly constant structural feature of the outer membrane of Gram-negative organisms, LPS evokes shock, fever and metabolic disturbances in many mammalian genera, including humans. Other molecules of microbial origin yield a similar effect. These include the zymosan of yeast, the peptidoglycan of many bacterial species, bacterial lipopeptides, flagellin and the unmethylated DNA that characterizes all subvertebrate species. The double-stranded RNA of numerous viral genomes, and poly I:C, which mimics it, provide other examples. Such molecules are obvious landmarks for detection, and have long been considered to be some of the prominent features of microbes that are recognized by the host innate immune system.

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Despite fairly intensive inquiry, utilizing classical techniques of biochemistry, and ultimately, cDNA cloning approaches, the proteins responsible for detection of these ubiquitous microbial signals never emerged. Ultimately, the search for the LPS receptor took center stage in the story, since the signal evoked by LPS was quantitatively greater than that evoked by any other microbial inducer. Furthermore, LPS was, by the 1970s, chemically characterized in great detail (Luderitz et al., 1973), and ultimately, the toxic moiety of LPS (lipid A) was synthesized artificially (Imoto et al., 1984). Notwithstanding these advances, the LPS sensor remained elusive.

Genetic approach to the LPS sensor In 1990, CD14, a glycosylphosphoinositol-tethered protein on the surface of mononuclear cells, was identified as an essential component of the LPS receptor through expression studies involving the B-cell line 70Z3 (Wright et al., 1990). While these cells were normally incapable of responding to LPS, sensitivity to LPS was conferred when CD14 was expressed on the plasma membrane. There was, however, general consensus that CD14 could not be the sole avenue of signal transduction, since it lacked a cytoplasmic domain and had no obvious means by which to transduce the signal across the cell membrane. Identification of the LPS receptor ultimately depended upon studies of a genetic defect in mice that had been known since 1965. Animals of the C3H/HeJ substrain of C3H were, at that time, noted to be highly resistant to the lethal effect of LPS (Heppner and Weiss, 1965). This phenotype did not exist in closely related strains such as C3H/HeN or C3H/OuJ from which C3H/HeJ had recently diverged. The LPS-unresponsive state appeared to result from a spontaneous mutation that had become fixed in the C3H/HeJ population during the early 1960s (Vogel, 1992). In time, the C3H/HeJ defect was found to be global, in that truly pure preparations of LPS evoked no biological responses of any kind. In many laboratories, LPS came to be defined as ‘that which evokes a biological response in cells from C3H/HeN mice, but in cells from C3H/HeJ mice,’ and hence, the strain combination was used on many occasions to exclude the presence of contaminating LPS in assays of cell activation.

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C3H/HeJ were also used to demonstrate that the lethal effect of LPS is mediated by cells of hematopoietic origin. Hence, when hematopoietic progenitor cells derived from mice of the LPS-sensitive C3H/HeN strain were infused into C3H/HeJ mice in adoptive transfer studies, the animals were rendered LPS sensitive (Michalek et al., 1980). Furthermore, it became evident that TNF (and to a lesser extent, other cytokines) delivered the lethal effect of LPS (Beutler et al., 1985). Among cells of hematopoietic descent, the macrophage was shown to be of predominant importance in the LPS response (Freudenberg et al., 1986). In 1978, the LPS-resistant phenotype was traced to a single mutation located between the classical markers Mup and Ps on mouse chromosome 4 (Watson et al., 1977, 1978). Also, a second mutation affecting the same locus was identified in mice of the unrelated C57BL/10ScCr strain (Coutinho et al., 1977, 1978). The locus in question was named Lps. The fact that a single mutation could entirely abolish responses to LPS in C3H/HeJ mice revealed that a single receptor (or at any rate, a single protein) was absolutely essential for LPS signal transduction. While LPS might interact with many proteins outside the cell, and while the LPS signaling pathway might ramify extensively within the cell, a ‘bottleneck’ must exist at one point in the signaling pathway. This bottleneck most probably was embodied by the LPS receptor itself. Widespread interest in the LPS signaling pathway reflected the popular conviction that most, and perhaps all microbial inducers must utilize a similar pathway. Hence, the pathologic effects of many different microbial inducers were indistinguishable at the whole organism level. Gram-negative, Gram-positive and fungal infections all produced shock and tissue injury that were, at a clinical level, indistinguishable from one another. The identification of the LPS receptor would provide the key to the initial events in microbial pathogenesis. Between 1993 and 1998, the Lps mutation of C3H/ HeJ mice, previously mapped within broad limits to chromosome 4, was narrowed to a 2.6 megabase region flanked by newly identified markers, and identified through positional cloning (Poltorak et al., 1998a, 1998b). This solitary approach, entirely different from the biochemical, immunological and expression cDNA cloning methods that had preceded it,

revealed that the Toll-like receptor 4 was required for all responses to LPS. In C3H/HeJ mice, a point mutation (P712H) modified the cytoplasmic domain of this single spanning transmembrane protein. In C57BL/10ScCr animals, the TLR4 locus was deleted entirely. The following year, a phenocopy of the mutations in C3H/HeJ and C57BL/10ScCr mice was rendered by gene targeting (Hoshino et al., 1999).

The orphan receptors At the time that the Lps mutation was identified, TLR4 was one of a group of five orphan receptors, identified by virtue of their similarity to the Toll protein of Drosophila melanogaster. Toll was originally described as a receptor involved in dorsoventral polarization of the embryo (Anderson et al., 1985; Belvin et al., 1996). Subsequently, it had been determined that Toll played a vital part in the immune response of adult flies following inoculation with fungi (Lemaitre et al., 1996). Hence, the discovery that one of the mammalian paralogues of the Tolllike receptor family also had a well-defined immune function (Poltorak et al., 1998a) caused immediate excitement. By coincidence, two independent groups of investigators observed that cells of the human embryonic kidney line 293 would respond to rather crude preparations of LPS if transfected to express TLR2, another member of the mammalian family (Yang et al., 1998; Kirschning et al., 1998). Although it was widely believed for a period of months that two separate endotoxin-signaling pathways might exist, the notion was inconsistent with the fact that LPS responses were entirely abolished by mutations at the TLR4 locus. In the fullness of time, it has become clear that data concerning LPS signal transduction by TLR2 were artifactual, and resulted from massive overexpression of the TLR2 protein in conjunction with the use of impure preparations of LPS. TLR2 did, however, serve an important sensing function. It has been shown that TLR2 is the receptor for bacterial peptidoglycan, as well as certain lipopeptides, and probably certain glycolipids produced by microbes (Takeuchi et al., 1999a). TLR9 has become established as the receptor for unmethylated microbial DNA (Hemmi et al., 2000). TLR5 appears to be the receptor for flagellin (Hayashi et al., 2001), a protein

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produced by both Gram-positive and Gram-negative bacteria. Recent claims suggest that TLR3 may participate in the sensing of double-stranded RNA (Alexopoulou et al., 2001), although the defect in sensing observed in mice that lack TLR3 is only partial, and very large concentrations of poly I:C were used as a stimulus. A sixth Toll-like receptor was reported in 1999 (Takeuchi et al., 1999b), and three additional receptors were cloned on the basis of genomic sequence data in 2000 (Du et al., 2000; Chuang et al., 2000). A tenth (and apparently final) Toll-like receptor was reported in 2001 (Chuang et al., 2001). Collectively, these receptors are believed to represent the most important avenue for microbial sensing by the innate immune system. Where the immune response is concerned, they represent the point of first contact between pathogen and host.

The full genomic complement The ten human Toll-like receptors (Plate 33.2) are scattered in the genome, although half of them are resident on human chromosome 4. To some extent, the physical separation of genes within the genome is reflective of the time that has elapsed since their duplication. The relatively recent descent of the IL-1 and IL-18 receptor group is reflected in the fact that five of the seven chains of receptors that have TIRs linked to Ig-repeat motif ectodomains are clustered on chromosome 2. Likewise, the recent descent of TLRs 1, 6 and 10 – as well as TLRs 7 and 8 – is reflected in the fact that these genes are very close to one another. These observations are consonant with measurements of the time elapsed since duplication based on sequence homology. Indeed, TLRs 1 and 6 are extremely similar to one another in both species, and may not be true orthologs, but rather ‘pseudoorthologues,’ and TLR10 is not represented in the mouse at all, though it is represented in humans. All of the TLR genes have three exons, although one, two or all three of these exons may have coding function, depending upon the gene concerned. There are also TLR pseudogenes (Du et al., 2000), which reflect the occurrence of recent gene duplication, followed by mutational destruction. None of the mammalian TLRs is known to be required for survival, and the preponderance of evidence suggests that all are

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involved in microbial sensing. Two of the TLR genes (TLR7 and TLR8) are X-linked, and reside adjacent to (but not within) the pseudo-autosomal region of the chromosome. TLR4 is now one of the most heavily sequenced loci in the human genome. It would appear, based on studies of the pattern of mutations observed in the gene, that the locus has been subject to weak purifying selection (Smirnova et al., 2001). That is, a relative excess of rare coding variants can be found in the human population, and all of these variants probably confer diminished fitness. The story is likely to be similar at all of the TLR loci, and collectively; variation at the TLR loci may foretell susceptibility to a number of human infectious diseases. Best studied in this regard are the TLR2 and the TLR4 loci, variants of which appear to influence susceptibility to meningococcal sepsis (Smirnova et al., submitted). It is suspected, although unproved, that mutations affecting the two X-linked TLRs might be important susceptibility loci in those diseases in which male predominance is observed (e.g. leptospirosis).

The Drosophila story: Toll As already alluded to, a parallel story of innate immunity developed in Drosophila, and indeed, anticipated discoveries in the mammalian field. In Drosophila, antimicrobial peptides are of paramount importance in the response to infection. A total of eight antimicrobial peptides are known in flies, and in each case, the genes encoding these peptides bear NFjB-like elements in their promoters. Seizing upon this discovery, and taking account of the fact that in Drosophila there are only three rel family transcription factors capable of engaging NFjB-like motifs, Hoffmann and colleagues performed a retrograde analysis which led to the discovery that Toll, a plasma membrane protein previously known to participate in dorsoventral polarization of the fly (Plate 33.3) occupies a central position in signaling pathways that permit adult animals to respond to fungal infection. Toll has no direct contact with molecules of microbial origin. Rather, it engages a protein ligand, spätzle, which is generated as a proteolytic cleavage product through interaction between components of the hemolymph and microbial inducers. Toll signals by activating a serine kinase (pelle) in conjunction with tube, a protein of unknown

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function. These proteins, in turn, cause the disassociation of cactus (an IjB homolog) from dif (one of the three NFjB family members in Drosophila). The terminal event in the defensive pathways is the activation of drosomycin synthesis. Drysomosin, an antifungal peptide, protects the adult fly against infection. In Drosophila there are nine other ‘Tolls’ (so called to distinguish them from the mammalian Toll-like receptors). None of the other Tolls (2–8) is known to have a role in Drosophila immunity. Rather, in Drosophila, the Tolls seem to have been ‘co-opted’ for developmental tasks. In 1991, with the cloning of the IL-1 receptor, it was noticed that there was substantial homology between this novel protein and Drosophila Toll (Gay and Keith, 1991; Heguy et al., 1992). Moreover, it emerged that the IL-1 receptor could signal by way of the serine kinase IRAK (homologous to pelle) (Cao et al., 1996). This in turn would lead to NFjB activation, and the synthesis of numerous protective cytokine genes. It might have been thought that the homologs pathway for Toll signaling had indeed been discovered. However, the identification of even better homologs of Toll (the TLRs) was yet to come. In 1994, the first Toll-like receptor (later known as TLR1) was cloned by Nomura et al. (1994). By 1996, this TLR gene had been mapped to human chromosome 4 (Taguchi et al., 1996). Its immune function remained obscure, however, and was not formerly considered until after the discovery that Drosophila Toll was an essential participant in insect immunity (Lemaitre et al., 1996). By 1997, it was shown that another member of the TLR family (four more of which were identified within the space of a year) – hToll or TLR4 – could activate NFjB in mammalian cells when crosslinked by chimerization (Medzhitov et al., 1997). In a sense, discoveries in insects and mammalians were mutually reinforcing, each leading to discoveries in the opposite species. The longstanding awareness of the importance of NFjB in mammalian immune responses encouraged the search for NFjB motifs in the promoter region of anti-microbial peptides in flies (Reichhart et al., 1992; Georgel et al., 1993; Engstrom et al., 1993; Hultmark, 1993). The discovery of these motifs, and the fact of homology between the IL-1 receptor and Toll, encouraged speculation that the latter might have an important immune function as

well. The finding that Toll did have an immune function aroused suspicion that members of the mammalian TLR family might also have immune importance (although there was a tendency to believe that the TLR in question acted to serve the adaptive immune system) (Lemaitre et al., 1996). The positional cloning of Lps (Poltorak et al., 1998a), which provided a formal demonstration that LPS sensing is dependent upon TLR4, cemented the conviction that Toll-like receptors are of central importance in mammalian innate immune recognition.

STRUCTURE AND EVOLUTION The TIR domain The Tolls and Toll-like receptors are characterized by a modular domain structure involving leucine-rich repeats (LRRs) that reside outside the plasma membrane, and a so-called Toll/IL-1 receptor (TIR) motif that comprises the bulk of the cytoplasmic domain (Plate 33.4). As previously mentioned, the TIR motif was first recognized in 1991, subsequent to the cloning of one chain of the IL-1 receptor. It is the most conserved part of all TLR molecules, and as such, provides a slow ‘clock’ for analysis of TLR ancestry. The fold of the TIR motif has been solved crystallographically by Xu and colleagues (2000). This portion of the molecule is believed to represent a bimolecular interaction motif. A TIR domain is found at the C-terminal end of the principal cytoplasmic transducer for all of the TLR proteins, MyD88. Arguably, a similar motif is observed in MyD88’s closest homolog, a protein now known as MAL (Fitzgerald et al., 2001) or TIRAP (Horng et al., 2001). Although no inkling of the nature of the interaction between TIR domains was provided by crystallographic studies, it is likely that TLRs exist in a multimeric form. Certainly the IL-1 and IL-18 receptors, which also bear a TIR motif, exist as heterodimers. It has been suggested that TLR1 and TLR6 associate with TLR2 in a heterodimeric arrangement (Ozinsky et al., 2000). Possibly, receptor activation is associated with recruitment of MAL/ TIRAP and or MyD88 to the receptor complex, and the exchange of one heterodimeric interaction for another.

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TLR4 is widely believed to exist as a homodimer, based on the observation that antibodies against TLR4 have an LPS-mimetic effect (Nattermann et al., 2000), and on the finding that chimeric versions of TLR4 receptor are capable of constitutively activating NFjB (Medzhitov et al., 1997). In this case, then, the exchange of the homodimeric interaction for a heterodimeric interaction with the transducing proteins is envisioned. The TIR is an ancient motif, observed not only in vertebrates and invertebrates, but in plants, where, as in animals, it fulfills a defensive function (Meyers et al., 1999; Pan et al., 2000; Young, 2000). The TIR of plants is associated with cytoplasmic proteins. It is not directly connected to any plasma membrane receptor per se, but may transduce signals that emanate from such receptors in response to infection.

LRRs Leucine-rich repeats occur in many proteins, and in the TLR ectodomains, where they play an uncertain role. While there is a tendency to believe that they are involved in hydrophobic interactions, this has not been proved in a formal sense. They may, in fact, simply maintain the overall shape of the ectodomain through interactions with one another that are reminiscent of those observed in cytoplasmic ribonuclease inhibitor, a protein almost entirely composed of leucine-rich repeats (Kobe and Deisenhofer, 1993). There is great curiosity about the overall structure of the TLR ectodomains. In all probability, they bear little resemblance to one another, except in the case of close homologs like TLR1 and TLR6. The arrangement of the LRRs is entirely dissimilar in different family members, and there is little or no homology when sequence comparisons of intervening parts of the protein backbone are performed. At present, a major obstacle to structural studies of the TLR ectodomains has been a technical one: it is extremely difficult to produce large quantities of these proteins in a soluble form. It has recently been reported that GP96, a heat-shock protein, permits much higher expression of the TLRs when co-expressed with them in a mammalian transfection system (Randow and Seed, 2001). It will be of particular interest to determine precisely how the TLRs interact with their spe-

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cific ligands, beginning with the interaction between LPS and TLR4. A small secreted protein, MD-2, appears to interact with the ectodomain of TLR4, and may participate with CD14 in transduction of the LPS signal. While no ‘hard’ data (i.e. gene knockout mutations) yet support the thesis that MD-2 is required for LPS signaling, transfection studies suggest that it may be (Shimazu et al., 1999; Akashi et al., 2000). Still other components of the LPS receptor may yet elude detection. It has been reported that co-resistance to LPS and IL-1 are conferred by what may be a single form fruste mutation in humans (Kuhns et al., 1997). Although one might immediately suspect that a mutation affecting signal transduction from the TIR might be at fault, no such mutation has been identified to date. For this reason, it is possible that another protein may be required for membrane targeting or transduction from these two receptors.

Ig repeats As already mentioned, some TIR-bearing receptors do not have leucine-rich ectodomains. These include the IL-1 and IL-18 receptor chains, as well as the ST2 and SIGIRR receptors, which remain orphans. All of these protein have ectodomains based on repeats of the primordial immunoglobulin (Ig) motif. It would appear that, in the course of evolution, vertebrates fashioned a means of eliciting TLR-like signals through the reception of host proteins, such as IL-1 and IL-18. These receptors form a bridge between innate immunity and adaptive immunity, residing upon lymphocytes, and responding to signals that are initiated by cells of the innate immune system. While insects and other invertebrates possess the Ig fold, there is not, as yet, any known instance in which they have employed it in conjunction with the TIR domain. Hence, this ‘swapping’ of ectodomains occurred in the course of vertebrate evolution, and as such, is a relatively recent development – as are most cytokines themselves, including IL-1 and IL-18.

Cytoplasmic transducers MyD88 appears to be a universal transducer of TIR signals, interacting with TIRs as diverse as those observed in the IL-1 receptor and the TLRs. The

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N-terminal half of MyD88 contains two death domains, which are known to interact with similar motifs in the serine kinase IRAK. It is possible that the death domains also interact with components of cell death pathways, and such interactions might account for the proapoptotic effect of LPS that has been observed in some systems, as in cultured human umbilical vein endothelial cells, where FADD is believed to be a conduit to LPS-induced cell death (Choi et al., 1998). MAL/TIRAP does not contain death domains. In a manner as yet unknown, it seems to trigger the MAP kinase cascade (Fitzgerald et al., 2001; Horng et al., 2001), which is activated even in cells that lack MyD88 (Kawai et al., 1999). As illustrated in Figure 33.5, MyD88 and MAL, together with the Ig motif-bearing representatives of the TIR superfamily, form an evolutionarily conserved cluster. A second branch of the tree encompasses Stem Metazoan

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FIGURE 33.5 Protein evolution of the TIR domain over greater evolutionary distance. Since the TIR changes more slowly than the ectodomain and is represented in proteins that lack an LRR-filled ectodomain, it is the most useful part of the TLR to analyze in evolutionary studies. In this illustration, Drosophila proteins are denoted with a ‘d’ while proteins from other species are not. The fact that sea urchin TIR domains are more distant from human TIRs than insect TIRs suggests that they are a remnant of a very ancient form of TIR, present in early metazoan life forms. One of the Drosophila TIR domains (from Toll-9) is very similar to the TIR domains in the human TLR cluster containing TLRs 4, 2, 1, 6 and 10. Hence, the human TLRs and Drosophila Toll-9 descend from a second ancient precursor. A third ancient precursor gave rise to the remaining Drosophila Tolls, and a fourth gave rise to MyD88 of both insects and humans, and to all of the Ig motifassociated TIRs. Note that the divergence of the IL-1, IL-18 and ST2 TIR motifs was a very recent event.

Drosophila Tolls 1–8. A third branch of the tree includes all of the mammalian TLRs, as well as Drosophila TLR9. Hence, in the last common ancestor of humans and flies, at least three TIR genes were clearly in place. In fact, at least four existed, insofar as the TIR-bearing proteins of the purple sea urchin seemed to form a distinct branch, and the sea urchin diverged from vertebrates long after the final common ancestor of vertebrates and insects lived. It is impossible to know how many TIRs exactly existed in our remote metazoan ancestors, but in all likelihood, there were several, just as there seems to be in most organisms today.

SPECIFICITY AND FUNCTION Knockouts as the most reliable guide A common error, often repeated in the TLR field, has been rooted in the assumption that all cells would be capable of responding to TLRs, if only they expressed them. Hence, the strategy of transfecting HEK 293 cells with different members of the TLR family and then stimulating these cells with various microbial or mammalian ligands, has led to a good deal of misunderstanding. It has been inferred that a large number of microbial inducers trigger TLR2 (Lien et al., 1999; Golenbock and Fenton, 2001), and that a large number of mammalian host proteins, as well as viral proteins, trigger TLR4 (as discussed below). The further analysis of ligand specificity should be carried out with two guiding principles in mind. First, mutational data are far more robust than those acquired by means of transfection alone. As all of the TLR genes have now been knocked out, the conclusion that a given ligand acts through a given TLR ought to be minimally dependent upon the absence of a signal in animals that lack that TLR. Second, one must be mindful of the purity of the reagents used. As endotoxin is practically ubiquitous in the environment, it is difficult to purify any protein without introducing it, and no amount of reassurance provided by the addition of polymyxin B nor by Limulus testing should be sufficient to assuage doubts that LPS, rather than a vertebrate protein, is responsible for cell activation via TLR4. The same general caution applies in the case of other TLRs and their specific lig-

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ands. Hence, where microbial inducers are considered as candidates for induction via one TLR or another, these inducers ought to be produced by chemical synthesis. Where proteins are considered as the inducers, they ought to be produced in a wellcontrolled transfection system (i.e. as the secretion products of stably transfected cells), rather than introduced to the culture as purified materials.

Multimeric structure There is evidence that, where TLR2 is concerned, heterodimer formation with TLR1 provides different specificity than heterodimer formation with TLR6. By inference, Tlr10 might further alter the spectrum of response. Hence, the formation of heteromers between TLRs may be expected to broaden the spectrum of microbial inducers to which the TLRs collectively can respond. There is, of course, a limit to this ‘broadening’ effect in that even if promiscuous interaction between all of the TLRs were observable (and it seems that it probably is not), only 100 dimeric interactions are possible. On no account can the complexity of the microbial repertoire be met through the multimeric interaction of TLR receptors, save for the consideration that the determinants recognized are broadly shared by many microbial taxa.

Endogenous ligands It has been reported that HSP60 (Ohashi et al., 2000; Vabulas et al., 2001), HSP70 (Asea et al., 2000; Basu et al., 2000), fibrinogen (Smiley et al., 2001), and fibronectin (Okamura et al., 2001) and other proteins act as endogenous ligands for TLR4. The F protein of respiratory syncitial virus (RSV) has also been proposed as a ligand for TLR4 (Kurt-Jones et al., 2000; Haynes et al., 2001). There has also been wide, unpublished speculation that an endogenous mammalian version of the Drosophila protein spätzle might be interposed between endotoxin and the TLR4 protein. Without exception, however, these reports fail to meet the standard of evidence set forth in the section ‘Knockouts as the most reliable guide’ and accordingly, the evidence is less than compelling. In the specific case of the F protein of RSV, one critical piece of evidence is insupportable. While it was noted that C57BL/10ScCr mice show enhanced susceptibility to

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the virus, which multiples more rapidly in these animals than in C57BL/ScSn controls (Kurt-Jones et al., 2000; Haynes et al., 2001), it has recently been shown that the Cr substrain has, in addition to the earlier established Tlr4 deletion, a point mutation that leads to premature termination of the Il-12 receptor b2 chain (Poltorak et al., 2001). This, rather than the Tlr4 defect, is probably responsible for augmented viral replication in vivo. It is, none the less, a fact that tissue necrosis of any cause can trigger an inflammatory response. Might the TLRs be the first cause of inflammation in this case also? It is possible that they are, but the mechanism involved remains unclear. Perhaps the systemic release of mitochondrial DNA (which is unmethylated), or cellular proteins yet unknown, might trigger a response from some TLRs. Very rigorous experimental tools need to be applied to answer this question.

Signal transduction The similarities of signal transduction from different TLRs are, for the most part, quantitative rather than qualitative (although it cannot be doubted that some specific endpoints of response are uniquely triggered by individuals TLRs). It is chiefly for this reason that a Gram-positive infection is clinically indistinguishable from a Gram-negative infection, although no TLR4 signaling takes place during the latter, other TLRs are fully capable of triggering the release of cytokines that cause fever, shock and tissue damage. Although the discovery of the TLRs offers an opportunity to access the most crucial signaling events that evoke inflammation ab origio, the picture remains very complex, and is fraught with phenomena that defy explanation. To begin with, the full complement of proteins that engage the TLRs has not been established. MyD88 has already been mentioned. Less well substantiated is MAL/TIRAP, which has not been mutationally inactivated, and hence, is less certain to play an essential role in signaling. Tollip, a protein that appears to interact with the IL-1 receptor based on immunoprecipitation studies (Burns et al., 2000), may also contribute to signaling; specifically to the recruitment of IRAK. However, as with MAL/TIRAP, it has not yet been studied by definitive methods. The signals elicited by TLRs are similar to those elicited by IL-1, and the present concept of IL-1 signal

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transduction is largely attributable to Muzio et al. (1997) and Adachi et al. (1998), who showed that MyD88 is essential for such signaling to occur. As previously mentioned, since 1997 it has been believed that a single mutation can cause co-resistance to IL-1 and LPS signaling (Kuhns et al., 1997). The present interpretation of this observation holds that TLR4 and the IL-1 receptor are dependent upon a shared constituent of each receptor complex, required for signaling. However, the identity of this protein remains unknown. Although the signaling pathway utilized by each of the TLRs remains incompletely understood, a basic outline of what occurs when TLR4 is activated by LPS is presented in Figure 33.6, wherein it is shown that both MAL/TIRAP and MyD88 participate as receptor interacting proteins, with each signaling in a different direction. Hence, the activation of several protein kinase systems within the cell occurs independently of NFjB activation. It is known from previous studies that where TNF synthesis is concerned, both transcriptional and translational activation steps are

LPS

? CD14

p38

?

MD-2

Tlr4

MAL/TIRAP

MyD88, IRAK

PI3K JunK MAPK

TRAF-6 MEKK1

TAK1

Iκ B:NF-κB Iκ B

NF-κB TNF, IL-1, etc.

PLATE 33.6 Principal signaling events in the LPS response pathway. MD-2, Tollip (not shown) and MAL/TIRAP may or may not be required for signal transduction, and a final assessment of their role will depend upon the production of animals with targeted deletion of the respective genes.

required (Han et al., 1990a, 1991; Kruys et al., 1992), and it now appears that the dissociation of signals may occur at the level of the TLR4 protein itself. Because LPS signaling has been studied in great detail over the past few decades, a number of enduring questions have been re-examined in light of the newfound identity of the receptor. The phenomenon of endotoxin tolerance is still not understood, although it may be partially attributable to downregulation of the receptor, which occurs after LPS stimulation. Glucocorticoid hormones (Beutler et al., 1986; Han et al., 1991), tyrosine kinase inhibitors (Novogrodsky et al., 1994), and phosphodiesterase inhibitors (Strieter et al., 1988; Giroir and Beutler, 1992; Han et al., 1990b) are each well-known antagonists of LPS signaling. However, their mechanism of action remains unresolved. A likely ‘checkpoint’ at which glucocorticoids might exercise their impressive broad-spectrum anti-inflammatory effects might be MyD88, since it is believed to be required for signal transduction initiated by all TLRs, as well as IL-1 and IL-18.

PROMOTER FUNCTION AND TISSUE DISTRIBUTION Many cells that have no overt defensive function none the less express some of the TLRs. For example, TLR4 is expressed on cardiac myocytes (Frantz et al., 1999), which are not generally regarded as defensive cells that would need to respond directly to LPS. Moreover, tissue distribution of the TLRs is quite variable among species. These facts may be taken to indicate that the expression of some TLRs is superfluous in some cells, which may or may not have any means of responding to signals that these receptors transduce. The level of expression of some TLRs is probably subject to regulation by mediators that enhance or diminish sensitivity to specific microbial inducers. For example, interferon-c is known to regulate the expression of TLR4 in human mononuclear phagocytes (Bosisio et al., 2001). The proximal promoter of TLR4 is sufficient for expression in human myeloid cells. The sequence of this region is similar in human and mouse TLR4 genes, both of which lack a TATA box, typical Sp1 sites or CCAAT box sequences. Rather, the proximal pro-

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moter contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif. The activity of the promoter in macrophages is dependent upon the integrity of both half-sites of the composite interferon response factor/Ets motif. Hence, two tissue-restricted transcription factors – PU.1 and interferon consensus sequence-binding protein – participate in the basal regulation TLR4 (Rehli et al., 2000). Less is known about the sequences that ensure accessibility of the gene. However, C57BL/10ScCr mice, which lack an endogenous TLR4 locus, can be rendered transgenic using BAC clones that contain either the human or mouse genes. Such mice do express TLR4, and are LPS-sensitive (M.A. Freudenberg, et al., in preparation).

POLYMORPHISM AT THE TLR LOCI Vigorous efforts to characterize differences in TLR4 coding sequence within and among ethnically diverse human populations have led to the conclusion that TLR4 – and by implication, other TLRs – are quite stringently conserved among Homo sapiens. About 90% of human chromosomes encode the most common isoform of TLR4 (Smirnova et al., 2001), and the locus has, overall, been subject to weak purifying selection, rather than the diversifying selection that is observed at some other loci of immunological importance. A common variant isoform, called TLR4B, is observed in Caucasian populations and is defined by two amino-acid replacements in the mid-ectodomain of the receptor. Each of the component substitutions is observed at far lower frequency in isolation, although they are in exceedingly strong linkage disequilibrium with one another. The component mutations are more commonly observed by themselves in African populations, while the compound variant is not. This suggests that the two mutations might have been united by a crossover event in an early progenitor of modern-day Caucasians. The TLR4B isoform has been associated with relatively mild (and so far as is known, clinically insignificant) hyporesponsiveness to LPS (Arbour et al., 2000). On the other hand, rare mutations of TLR4 are

observed at exaggerated frequency among individuals with meningococcal sepsis (Smirnova et al., submitted). Just as mice lacking a functional TLR4 gene are more susceptible to Gram-negative infection, humans with uncharacterized (but likely hypomorphic) TLR4 mutations seem more susceptible to at least one Gram-negative infection. Although fewer data are available concerning mutations at other TLR loci, the TLR2 locus (Smirnova et al., submitted) as well as the TLR7 and TLR9 loci (Beutler et al., unpublished data) seem nearly monomorphic as well.

CONCLUSIONS The Toll-like receptors represent the first point of meaningful physical contact between pathogens and host. They are the ‘eyes’ of the innate immune system, and they supply the information upon which the host must rely in forming an immune response, with all of the benefits and potential problems that such a response entails. While other innate immune mechanisms work in parallel with the TLRs and support their function, the overall pattern of the response to a pathogen is cast in stone from the moment that the TLRs are activated. Where inflammation at large is concerned, there are many questions worth asking. Do the TLRs participate in inflammation that has a non-infectious etiology? Is it possible to speak of autoimmunity rooted in the innate immune system rather than the adaptive immune system? More specifically, we are far from deciphering the molecular events that transpire in TLR signal transduction. While all of the human TLRs have been identified, other proteins may participate in forming the macromolecular complex that detects pathogens at the surface of innate immune cells. Some of these proteins may be detected through direct biochemical techniques; others may be identified only through mutagenesis.

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34 Lymphotoxins Steve W. Granger and Carl F. Ware La Jolla Institute for Allergy and Immunology, San Diego, CA, USA

As to diseases, make a habit of two things – to help, or at least, to do no harm. Hippocrates, Epidemics

INTRODUCTION Lymphotoxin (LT) was originally identified as a soluble factor produced by lymphocytes that was cytotoxic to tumor cells (Granger and Williams, 1968; Ruddle and Waksman, 1968) and later recognized as a cell surface protein on activated T cells (Ware et al., 1981). Two distinct forms of LT are now recognized, the original secreted form that is homologous to tumor necrosis factor (TNF) (Gray et al., 1984), known as LTa (previously referred to as TNFb), and the membrane form, comprised of a heterocomplex between LTa and a second related protein LTb (p33) (Androlewicz et al., 1992; Browning et al., 1993). LTa and TNF both bind the cell surface receptors TNFR1 (p60, CD120a) and TNFR2 (p80, CD120b), whereas the LTab complex binds a distinct receptor with specificity determined by the LTb subunit (LTbR) (Crowe et al., 1994; Williams-Abbott et al., 1997) (Plate 34.1) (see Plate section). The receptors for LT and TNF are members of a corresponding family of cell surface The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

and secreted proteins each with distinct roles in physiology. Gene disruption studies in mice have linked LTab-LTbR signaling to lymphoid organogenesis, splenic architecture and the differentiation of NK and NK-T cells, whereas LTa-TNFR1 signaling has been linked to the formation of tertiary lymphoid tissues and inflammation. The shared ligand specificity of these receptors is further revealed by the interaction of LTbR with the LT-related ligand LIGHT (TNFSF14) (Mauri et al., 1998). LIGHT also binds the TNFR superfamily member, HVEM (herpes virus entry mediator), which can engage LTa. This shared receptor usage indicates these ligands form an integrated network of signaling systems that orchestrate immune function, underscored by their conserved genetic organization and linkage to the major histocompatibility gene complex (MHC). This chapter will focus on recent aspects of LTab and LTa as ligands for LTbR and TNFR, respectively. Additional reviews are available on the general features of the TNF superfamily (Ware et al., 1998; Wallach et al., 1999; Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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Lockesley et al., 2001) and LT (Ware et al., 1995; Ware and Ruddle, 1999).

STRUCTURE OF LTa AND LTb The genes for LTa, LTb and TNF reside on human chromosome 6 (17 in the mouse) (Spies et al., 1986; Lawton et al., 1995) in a compact locus in the middle of the MHC (Figure 34.2). The TNF/LTab locus is recognized as one of four MHC paralogous regions found on chr 1, 9 and 19 (Granger et al., 2001), each containing genes for several other TNF related ligands. The genes encoding the receptors for LTab and TNF are

FIGURE 34.2 Organization of the MHC/TNF region in the human genome. Top, the ideogram of human chromosomes 6 is shown including the chromosomal location of the MHC locus at 6p21.3. Middle, the gene distribution of the MHC locus including genes that border each region and the complement genes C4B and C2 are shown. Bottom, the TNF gene cluster is represented. Solid blocks represent exons and arrows indicate transcriptional orientation. TNF is 2.9 kb from LTb and 1.3 kb from LTa.

also linked and appear to be generated in part by chromosomal duplication. Members of the TNF ligand family are type II (amino terminus inside the cell) transmembrane proteins. Lymphotoxin-a is an exception because it contains an efficiently processed signal peptide at its amino-terminus and is exclusively secreted. By contrast, the secreted form of TNF is derived by cleavage of the membrane protein at the extracellular surface by a metalloprotease. The crystal structure of LTa (Eck et al., 1992) reveals a compact trimer assembled from subunits folded into a b-sheet sandwich (Plate 34.3). This trimeric structure is common to all TNF-family members despite relatively low sequence homology (35% amino acid identity) (Eck and Sprang, 1989; Jones et al., 1989; Karpusas et al., 1995; Hymowitz et al., 1999; Cha et al., 1999; Mongkolsapaya et al., 1999). The ~150 amino acid region of structural homology referred to as the TNF homology domain (THD) (Bodmer et al., 2002), encoded primarily by the fourth exon, is composed of eight anti-parallel beta strands forming two beta-pleated sheets creating an overall ‘jellyroll’ structure (Eck and Sprang, 1989; Bodmer et al., 2002). The similarity among members is largely restricted to the amino acids that form the b-strand scaffold, important for trimer assembly and to the residues that form the loops involved in receptor binding. The LTab complex can assemble as two isomers differing in their subunit ratio, LTab2 and LTa2b (Androlewicz et al., 1992; Browning et al., 1997). The LTab2 isoform predominates as the most abundant form of LT on the surface of T cells. LTb is not cleaved and thus initiation of signaling depends on cell-tocell contact. The LTb subunit provides specificity for the LTbR to bind the LTab2 isoform, while the LTa subunit is required for assembly of stable heterotrimers (Williams-Abbott et al., 1997). The LTa2b isoform binds TNFR1 and LTbR, but does not activate cell death, and no additional evidence is available that this isoform exists in a physiologic setting. The LTbR, like TNFR1 and TNFR2, has four repeats of the cysteine-rich motif that defines the ligandbinding domain of these receptors (Plate 34.3). The six cysteines in each motif create a ladder of disulfide bonds that together form an elongated molecule. The crystal structure of TNFR1 bound to LTa reveals that each of three receptor molecules lies in a cleft formed

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between adjacent ligand subunits (Banner et al., 1993). In order to form a signaling complex the receptor and ligand must reside on opposing membranes. Mutational analysis of LTa has demonstrated that residues in the A-A and D-E loops (e.g. Asp-50 and Tyr-108) make the major contacts with TNFR1. Each trimeric LTa ligand has three equivalent receptor binding sites indicating that aggregation or clustering of two or three receptors by the ligand appears to be the initiating event in signal transduction. Consistent with this concept is the finding that multivalent antibodies often mimic the ligand (Engelmann et al., 1990).

SIGNAL TRANSDUCTION The cytosolic portion of the LTbR is relatively large and predicted to fold into several domains, however a collagen-like stretch of ~50 amino acids rich in proline accounts for all of the recognized signaling activity of this receptor (Force et al., 2000). Deletion analysis has revealed discrete regions within this proline-rich sequence that control adaptor binding, activation of transcription factors, receptor processing and compartmentalization. The LTbR, like HVEM and TNFR2, binds to the TNFR associated factors (TRAF) family of zinc RING finger proteins. Deletion of the amino acid sequence PEEGDPG in the LTbR abolishes binding of TRAF2, 3 and 5 and disrupts activation of NFjB1(p65/p50). LTbR signaling bifurcates at the level of TRAF recruitment, both TRAF2 or 5 can activate NFjB, however dominant negative mutants of TRAF3 inhibit the slow apoptotic anti-tumor action of LTbR (VanArsdale et al., 1997; Force et al., 1997). TNFR1 is a member of the death domain (DD) containing subgroup of receptors characterized by a region of approximately 80 amino acids in length at its C-terminus (Tartaglia et al., 1993). The death domain binds other death domain containing signaling adapters that are mainly involved in the initiation of apoptosis. One such molecule is the TNF receptor associated death domain protein (TRADD), which associates with the DD on TNFR1 and recruits the Fas-associated death domain protein (FADD). FADD in turn activates caspase 8 leading to the activation of executioner caspases that induce cell death (MacEwan, 2002). TRADD also has the ability to

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recruit TRAF2 and activate the transcription factor AP1. LTa signaling via TNFR1 is comparable to TNF in inducing the death of murine L929 cells, but in all other assays, including human tumor cell death and inflammation, LTa is far less efficacious and behaves as a partial agonist (Browning and Ribolini, 1989; Andrews et al., 1990). The basis for this effect is not well understood, but is independent of binding affinity, suggesting that subtle differences in signaling complexes may contribute to biological actions. Activation of the NFjB transcription factor family is an important aspect of LT- TNF family receptor signaling. The activation of NFjB by the LTbR is mediated via TRAFs through the NFjB inducing kinase (NIK). NIK in turn activates IKKs to induce the degradation of IjB leading to the formation and translocation of NFjB1 to the nucleus (Yin et al., 2001). LTbR also induces the formation of NFjB2 complexes. In contrast, TNFR1 does not use NIK and is limited to activation of NFjB1, which apparently favors induction of genes involved in proinflammatory processes. Genes inducible by NFjB2 are often involved in cell survival or differentiation. Together these signaling attributes may partially account for the unique biological responses initiated by the LTbR, which differ dramatically from TNFR (Plate 34.4) (Dejardin et al., 2002).

REGULATION OF EXPRESSION AND TISSUE DISTRIBUTION As with many other TNF superfamily ligands, LTab is inducibly, but transiently expressed in T cells, remaining at the cell surface for only a few hours in tissue culture models (Ware et al., 1992).WhenT cells are activated, LT-a and LT-b are coinduced to form the LTab heterotrimer. In a similar manner toTNF mRNA, degradation of LTa mRNA is partially controlled by AU-rich sequences in the 3 untranslated region of the transcript that impart message instability (Kontoyiannis et al., 1999). In contrast, LTb mRNA is not regulated by this mechanism. The cellular distribution of LTab expression includes activated human T- and B-lymphocytes and natural killer (NK) cells (Ware et al., 1992). Stimuli such as TCR activation or IL2 effectively induce surface LTab on NK and T cells. In

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the mouse, LTab is induced on naive T cells responding to specific antigen and in the presence of IFNc, expressed transiently on CD4 type 1 T helper (TH1) effector cells, but not by IL4 influenced TH2 cells (Gramaglia et al., 1998). In this case, TH1 expression may be responsible for some inflammatory reactions mediated by LTab. Expression of LTab by B cells is responsible for splenic follicle formation and follicular dendritic cell networks. Naive B cells have been found to express LTab in response to specific chemokines in the mouse (Ansel et al., 2000). B lymphocyte chemoattractant (BLC) efficiently induces LTab expression, an activity demonstrated to be critical for the proper development of primary lymphoid follicles in the spleen and lymph nodes (Ansel et al., 2000). The cellular expression and tissue distribution of LT-specific receptors vary and may be a significant control mechanism. LTbR is constitutively expressed on stromal cells, but conspicuously absent on lymphocytes (Murphy et al., 1998). LTbR expression is elevated in specific embryonic mucosal epithelial layers during fetal development in the mouse (Browning and French, 2002), which may account for the role this receptor plays in the development of lymphoid tissues. TNFR1 is expressed constitutively at extremely low levels, typically less than a few thousand molecules per cell, by most cells including fibroblasts, lymphoid and epithelial cells. In contrast TNFR2 is expressed primarily in the hematopoietic compartment and prominently on T and B lymphocytes, but only after activation (Gehr et al., 1992; Crowe et al., 1993; Ware et al., 1991). When restimulated, activated T cells rapidly (within minutes) downregulate TNFR2 by two distinct mechanisms, one that involves shedding of the receptor by a metalloproteinase to release a soluble extracellular domain with ligand-binding activity, and a second mechanism involving internalization, resulting in a loss of surface expression independent of proteolysis (Crowe et al., 1995). Significant levels of TNFR2 accumulate in the plasma during inflammatory reactions. Since TNF is thought to function as an apoptosis signal for CD8 T cells, the down-regulation of TNFR2 during T cell activation may exist to protect these cells from apoptosis.

FUNCTIONS OF LYMPHOTOXINS Peripheral lymphoid tissue biogenesis LT-related cytokines have many functions in development, homeostasis and architecture of lymphoid tissue and the differentiation of cells in the innate and adaptive immune systems (Table 34.1). When LTa was disrupted in mice, they had no lymph nodes (De Togni et al., 1994; Banks et al., 1995). Since mice deficient in TNF have normal lymph nodes (Pasparakis et al., 1996), these studies firmly establish that LTa and TNF are not functionally redundant. Interestingly, there were no defects observed in T lymphocyte number or function in the LTa-deficient mice, T cell proliferation, activation and cytotoxic activity were comparable to those of wild-type littermates. The LTa-deficient mice did, however, display an increase in circulating B cells (De Togni et al., 1994). In addition, LTa-deficient bone marrow cells were able to home to both spleen and lymph nodes when adoptively transferred into mice with severe combined immune deficiency or lethally irradiated wild-type mice, indicating no intrinsic defect in the hematopoietic compartment (Banks et al., 1995; Mariathasan et al., 1995). In contrast, normal bone marrow was unable to reconstitute lymph nodes in irradiated LTdeficient mice, suggesting that lymph node organogenesis is developmentally fixed (Mariathasan et al., 1995). Mice deficient for the TNF receptors or TNF had no defects in lymph node organogenesis, demonstrating that an interaction between membrane-bound LTab and LTbR play the major role in the phenotype of the LTa-deficient mice. This was established by several different approaches including pharmacological TABLE 34.1 Some phenotypic differences of mice deficient in LT related cytokines Gene

Lymph Peyer’s Marginal Germinal NK-T NK nodes patches zone centers

LTa LTb TNF LIGHT LTbR TNFR1 TNFR2

      

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inactivation of the ligands for LTbR and receptor activation in the absence of ligands. LTbR-Fc fusion protein either expressed as a transgene (Ettinger et al., 1996) or injected into pregnant mice to attain embryonic circulation (Rennert et al., 1996) blocked lymph node formation. When LTbR-Fc was expressed as a transgene, neutralizing levels were not achieved until day 3 after birth and these mice had normal lymph node development, but Peyer’s patches were reduced or absent (Ettinger et al., 1996). By contrast, administration of LTbR-Fc fusion protein at different days during gestation showed a developmentally staged formation of lymph nodes (dorsal to ventral), with Peyer’s patches the last to develop. In LTa-deficient mice, injection of anti-LTbR monoclonal antibody restored lymph node formation (Rennert et al., 1998). Interruption of the LTb locus (Alimzhanov et al., 1997; Koni et al., 1997) gave similar lymphoid organ deficiencies as LTa gene deletion, but not an identical phenotype. LTb-deficient mice lack most inguinal and popliteal lymph nodes and Peyer’s patches, but retain mesenteric nodes. However, the phenotypes of LTb receptor / mice recapitulated the LTa/, indicating that the lack of LTab-LTbR signaling is in fact responsible for the striking lymphoid developmental phenotype (Futterer et al., 1998). This result suggested that an alternate ligand for LTbR or receptor for LTa may exist. LIGHT and HVEM indeed fit this profile and recent results with LIGHT/ deficient mice crossed to LTb-deleted mice demonstrate a redundant role for LIGHT in mesenteric lymph node formation, but not entirely. Hence a piece of this biological puzzle remains to be ascertained (Scheu et al., 2002). Upon closer inspection, TNFR1-deficient mice were found to have smaller and fewer Peyer’s patches (Neumann et al., 1996), suggesting that the LTa homotrimer may play a role in the development of some peripheral lymphoid structures (Kratz et al., 1996). LT is not required for all peripheral lymphoid organs as nasal lymphoid tissue forms in LT-deficient mice, yet the organization of this tissue is dependent on LT (Harmsen et al., 2002).

Microarchitecture of the spleen and lymph nodes LTa, LTb, LTbR-deficient mice and mice rendered LTab-deficient by employing Fc-decoy receptors dis-

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played abnormalities in cellular organization in the spleen and lymph nodes (De Togni et al., 1994; Banks et al., 1995; Matsumoto et al., 1996a; Le Hir et al., 1996; Ettinger et al., 1996; Rennert et al., 1996; Koni et al., 1997; Futterer et al., 1998). This phenotype is characterized by an absence of normal segregation between T and B lymphocytes in the white pulp and loss of well-defined marginal zones in the spleen. Furthermore, following exposure to antigen, both LT-deficient and the TNFR1-deficient mice fail to generate germinal centers. LTa, LTb and LTbR-deficient mice, in contrast to TNFR1-deficient mice, are negative for staining with MOMA-1, a marker that detects metallophilic macrophages of the marginal zone (Matsumoto et al., 1996b). By contrast, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), another marker for the marginal zone, is absent in TNFR1deficient mice, LTbR-deficient mice and those treated with the LTbR-Fc fusion protein (Neumann et al., 1996; Rennert et al., 1996; Futterer et al., 1998). Finally, follicular dendritic cell (FDC) networks are missing in LTa, LTbR and TNFR1-deficient mice (Le Hir et al., 1996; Matsumoto et al., 1996b; Pasparakis et al., 1996; Fu et al., 1997; Futterer et al., 1998). In this case signaling through both the LTbR and TNFR1 are required for FDC network development (Matsumoto et al., 1996b; Le Hir et al., 1996). Collectively these defects may explain the dysregulation of antibody responses to T dependent antigens seen in LTa, LTb, LTbR and TNFR1-deficient mice (Banks et al., 1995; Le Hir et al., 1996; Pasparakis et al., 1996), as well as the T cell independent type 2 antigens observed in TNF-LTadouble deficient mice (Ryffel et al., 1997). These results strongly support the idea that the LTbR and TNFR signaling pathways cooperate and are integrated to orchestrate the tissue architecture essential for efficient immune responses. LT signaling is important for mucosal immune responses. The phenotypes of mice rendered deficient in LT signaling include a dramatic reduction of IgA production in response to antigen and significantly lower baseline IgA levels (Banks et al., 1995). A likely cause of this deficiency is the lack of Peyer’s patches and mesenteric lymph nodes that house many IgA producing B cells. In a recent study, the LT-dependent IgA deficiency was rescued by adoptive transfer of LTa-expressing wild-type bone marrow into LTadeficient mice, despite the absence of Peyer’s patches

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and/or mesenteric lymph nodes or by transfer of normal intestinal tissue from immune-deficient mice lacking LTbR (Kang et al., 2002). LTa-deficient mice display reduced expression of chemokines known to be important for organization of secondary lymphoid tissues in the gut (Cyster, 1999a; Kang et al., 2002). Thus, LT signaling in the lamina propria induces chemokine and adhesion molecule expression critical for IgA production. Interestingly, constitutive expression of LIGHT in the T cell compartment induces a selective elevation of IgA suggesting this molecule may function as a ligand for LTbR in mucosal tissue (Shaikh et al., 2001). LTbR-Fc reduces intestinal inflammation induced by dextran sulfate or adoptive transfer of effector CD4 T cells (Mackay et al., 1998) further suggesting a significant role for LT or LIGHT in mucosal inflammation.

Reciprocal regulation of LT and chemokines in formation of lymphoid microarchitecture Membrane bound LTab signaling through the LTbR is critical for the maintenance of structures of splenic micro-architecture, such as follicular dendritic cells, marginal zones and the segregation of B and T cells. More recent studies employing various chemokinedeficient mouse models in conjunction with mice rendered deficient in LT signaling have further defined mechanisms in the genesis and maintenance of secondary lymphoid tissues (Okada et al., 2002; Reif et al., 2002; Luther et al., 2002; Ueno et al., 2002). Lymphotoxin-dependent expression of three chemokines, BLC/CXCL13, secondary lymphoid tissue chemokine (SLC/CCL21) and Epstein–Bar virusinduced molecule 1 ligand chemokine (ELC/CCL19), have been found to play central roles in the establishment and maintenance of secondary lymphoid structures (Cyster, 1999a, 1999b; Fu and Chaplin, 1999). In general, lymphocyte-derived surface LTab bind LTbR on stromal cells of secondary lymphoid organs to trigger the production of these chemokines. Chemokines expressed in specific regions of these organs are responsible for chemoattraction of lymphocytes and DCs to establish and maintain secondary lymphoid cell structure necessary for efficient immune function. For example, the chemokine BLC produced by stromal cells of the lymphoid follicles effectively

recruits B cells (Ansel et al., 2000; Okada et al., 2002; Reif et al., 2002), whereas stromal cells of the T cell areas produce ELC and SLC to attract T cells and DCs to these regions (Cyster, 1999a). When B lymphocytes engage antigen and become activated, they upregulate CCR7, the receptor for ELC and SLC, and migrate towards the T cell areas (Reif et al., 2002), facilitating T cell dependent help crucial for effective humoral immune responses. This example illustrates a portion of the well-choreographed interplay between lymphocyte chemokine receptor expression and hence differential responsiveness to chemokines that enables T and B cells to migrate to their proper areas in secondary lymphoid organs. These chemokines are responsible for adhesion and migration of lymphocytes across high endothelial venules and into secondary lymphoid organs (Cyster, 1999a; Yu et al., 2002; Okada et al., 2002). Several chemokines can induce LTab expression in B lymphocytes, including ELC, SLC and SDF, but BLC is the most effective at increasing LTab levels. BLC is the primary chemokine required for LTab expression on naive B cells in the mouse (Ansel et al., 2000). In fact, a positive feedback loop exists between BLC and LTab expression in lymphoid follicles (Ansel et al., 2000). Initial BLC levels increase the expression of LTab on B cells migrating to lymphoid follicles. Follicular stromal cells constitutively expressing LTbR produce more BLC upon engagement with B cell-derived LTab. The increased BLC levels in turn recruit more B cells to the follicle to engage B cell antigens on the resident APCs (Cyster et al., 2000; Ngo et al., 2001). Hence, LTab expression is critical to primary follicle development by the induction of BLC expression by follicular stromal cells. It is interesting to note that BLC-deficient mice share many phenotypes with the LT-deficient mice in that they fail to develop Peyer’s patches, FDC networks, organized follicles and most lymph nodes (Ansel et al., 2000). BLC has also been shown to be critical for B cell migration to body cavities (Ansel et al., 2002), however it is not required to retain B cells in the marginal zone (Lu and Cyster, 2002). In addition, recent studies have provided new insights into the requirement for LT signaling in the formation of the marginal zone (MZ) (Lu and Cyster, 2002). Membrane-associated LT induces adhesion molecule expression on stromal cells and macro-

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phages of the MZ. These molecules interact with integrins expressed by MZ B cells providing the mechanism that maintains MZ B cells as residents in this region. When these interactions are disrupted, MZ B cells enter the circulating B cell pool of the lymphoid follicles (Lu and Cyster, 2002). Hence, LTbR signaling is critical to the formation of the marginal zone and the retention of resident B cells in this region. Since the majority of B cells are in the marginal zones, loss of this structure decreases the efficiency with which B cells encounter antigens in the blood. Not surprisingly, mouse strains deficient in several other genes potentially linked to the LT-dependent signaling of lymphoid organogenesis have been revealed. For example, gene disruption of the NFjBinducing kinase (NIK), a critical molecule in LTbR signal transduction, results in a defect in LN formation (Shinkura et al., 1999). LTbR induces the formation of the transcription factor NFjB2 (p52/relb) via NIK and IKKa, which are responsible for inducing the expression of chemokines, such as SLC, involved in lymphoid tissue development and organization. In the development of Peyer’s patches, lymphoid ‘inducer’ cells express surface lymphotoxin in response to IL-7 signaling (Honda et al., 2001). The LT expressing progenitors bind and signal through the LTbR on mesenchymal ‘organizer’ cells inducing the expression of chemokines and the adhesion molecule ICAM. Interactions between these two cell types and the responses that occur thereafter appear to play a major role in the formation of the Peyer’s patch anlagen (Yoshida et al., 2001). Interestingly, a deficiency in RANK leads to loss of lymph nodes, but retention of Peyer’s patches, whereas IL7-deficient mice have a selective loss of Peyer’s patches. It is likely that RANK signaling may promote the expression of LTab in progenitors for lymph nodes akin to IL7. Other genes including CXCR5, CCL13, VLA4, ikaros, Id2 and RORc when deleted in mice result in a failure to form Peyer’s patches and/or lymph nodes (Kim et al., 2000; Ansel et al., 2000). Whether these genes are involved directly or indirectly in LTbR signaling remains to be established.

Tertiary lymphoid organ neogenesis Lymphotoxin may be a key cytokine responsible for lymphoid follicles that form outside lymphoid tissue

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in organs burdened with chronic immune and inflammatory reactions (also referred to as tertiary lymphoid tissue). Tertiary lymphoid structures regress with the tidal ebb of inflammation. When LTa is expressed as a transgene under the transcriptional control of the rat insulin promoter, lymph nodelike structures form in the pancreas and kidney, but without tissue destruction (Picarella et al., 1992; Kratz et al., 1996). The chemokines BLC and SLC are induced at the site of the organized lymphoid tissue, emphasizing their roles in lymphoid neogenesis associated with chronic inflammation and the dependence of LT signaling in their induction (Hjelmstrom et al., 2000, 2001). In another study, when BLC, SLC or SDF1 were expressed as transgenes in cells of the pancreatic islets, lymphoid-like structures developed and this process was likewise LTdependent (Luther et al., 2000). Thus, LT-induced chemokine expression contributes to the ectopic formation of organized lymphoid tissues associated with chronic inflammation that may facilitate efficient host defenses against pathogens causing persistent infections.

Innate defense systems and antiviral immunity mediated by LT Another surprising developmental phenotype common to both LTa, LTb and LTbR-deficient mice was the severe reduction of NK and NK-T cell numbers (Iizuka et al., 1999; Elewaut et al., 2000). This LT deficiency appears to effect the developmental differentiation of NK T cells and not their localization, long-term survival or ability to expand in response to antigen (Elewaut et al., 2000) since LTbR-Fc transgenic mice have a normal complement of NK and NKT cells. More recent studies have demonstrated that LTab-mediated LTbR signaling via the bone marrow stromal cells is an important event for early NK cell development (Wu et al., 2001). The consequence of lacking NK cells renders the LT-deficient mice susceptible to some tumors (Ito et al., 1999; Smyth et al., 1999). Lymphotoxin signaling pathways appear to be important for host defenses against intracellular pathogens, particularly viruses. LT signaling via either TNFR1 or LTbR, but not the death domain inducing Fas or TRAIL receptors, can limit the cytopathicity

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and spread of human cytomegalovirus (CMV) in cultures of infected fibroblasts (Benedict et al., 2001). The antiviral action does not induce apoptosis, but rather the induction of interferon-b by virus-infected cells. LT signaling activates NFjB to help override a block in IFNb gene expression placed by CMV. The induction of IFNb is cooperative between the virus and cytokine, leading to virus arrest without cellular elimination creating a state of coexistence between pathogen and host in this artificial environment. However, in a more physiological setting, LTadeficient mice are profoundly and selectively susceptible to murine cytomegalovirus, which is controlled by NK and T cell defenses (Benedict et al., 2001). The failure to resist this normally benign virus appears to be due to a failure in LTbR signaling during infection, and not to developmental defects known in the LTa/ mice. This is suggested by the susceptibility of mice expressing the LTbR-Fc decoy as a transgene, which have lymph nodes, NK and NK-T effector cells (Ettinger et al., 1998). LTa/ mice also have defects in response to herpes simplex virus apparently due to a block in differentiation of activated CD8 T cells (Kumaraguru et al., 2001), but have minimal phenotype in response to murine c-herpesvirus (Lee et al., 2000). Interestingly, LTbR-Fc decoy can inhibit lung inflammation in mice infected with lymphocytic choriomengenitis virus (LCMV) (Puglielli et al., 1999). Efficient immunity to this virus depends on LTab and is illustrated by the architectural defects associated with the absence of the LT signaling pathway during development (Berger et al., 1999). We now appreciate the wide variety of viruses, including DNA and RNA viruses that utilize specific genetic mechanisms to interfere with members of the TNF superfamily (Benedict and Ware, 2001). These mechanisms target divergent points in signaling pathways, from ligand-receptor binding to signal transduction. Hence each viral strategy illustrates the importance of these cytokines in regulating the immune responses necessary for efficient host defenses, particularly those in the immediate LT and TNF family. Highly efficient immune responses mediated by LT-related cytokines may be necessary to control pathogens that have sufficiently evolved to establish persistent infections, for example herpesviruses. Emerging evidence indicates that various LT and TNF systems are needed for the control of other

nonviral pathogens that establish persistent infections (Lucas et al., 1997, 1999). We expect that the LT-dependent functions, necessary to control a given pathogen, will vary depending on the specific agents and the niches established by them in modulating immune defenses.

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35 Tumor necrosis factor Haichao Wang 1,2, Christopher J. Czura1 and Kevin J. Tracey 1 1

North Shore-LIJ Research Institute, Manhasset, New York, USA 2 North Shore University Hospital, Manhasset, New York, USA

Science is wonderfully equipped to answer the question ‘How?’ but it gets terribly confused when you ask the question ‘Why?’ Erwin Chargaff

INTRODUCTION

STRUCTURE

Tumor necrosis factor (TNF, TNF-a, cachectin) is a pleiotropic pro-inflammatory cytokine that exerts multiple biologic effects. TNF was originally identified as an anti-tumor agent that induced necrotic cell death in sarcomas and other tumor types. Localized, low-level expression of TNF participates in beneficial tissue remodeling and host defense responses. The expression of TNF is tightly controlled, because systemic overproduction of TNF activates inflammatory responses to infection and injury, and mediates hypotension, diffuse coagulation, and widespread tissue damage. The diverse activities of TNF led to the simultaneous and apparently paradoxical pursuit of TNF as an anti-tumor strategy, and TNF inhibitors to attenuate lethal systemic inflammation. This chapter has been written as an introduction to the broad topic of TNF, its molecular biology, biochemistry, and physiology; by necessity, only a portion of the primary literature for each topic is reviewed.

Tumor necrosis factor is synthesized as a 26-kDa type II transmembrane precursor that is displayed on the plasma membrane, with the N-terminus in the cytoplasm and the C-terminus exposed to the extracellular space. The TNF precursor is proteolytically cleaved between alanine (1) and valine (1) to yield a biologically active 17-kDa mature TNF (Kriegler et al., 1988) that forms a trimer in solution (Smith and Baglioni, 1987). In solution, 17-kDa TNF is primarily comprised of two anti-parallel b sheets; these TNF monomers self-associate in a head-to-tail fashion into non-covalently linked, symmetrical bell-shaped homotrimers (Smith and Baglioni, 1987; Jones et al., 1989). The C-terminus of each subunit is embedded within the base of the trimer, while the N-terminus is relatively free from the structure and is not critical for TNF biologic activity (Creasey et al., 1987). Mutational analyses of mature TNF indicate that each trimer has three receptor interaction sites located in the

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intersubunit grooves near the base of the trimer (Zhang et al., 1992). The trimeric structure of TNF is important for its biologic activity, because mutations that destabilize the trimer or attenuate monomer association result in the loss of TNF biologic activity (Lin, 1992). Recently identified TNF muteins have been shown to possess decreased toxicity in mice without loss of its antitumor effects (Van Ostade et al., 1993; Barbara et al., 1994). For example, residues within the inter-subunit grooves at the base of the trimer dictate receptor specificity: the double mutant R32W/S86T exhibited wild-type binding to the 55kDa receptor (TNFR1) with no measurable binding to the 75-kDa TNF receptor (TNFR2). In contrast, the D143N/A145R double mutant fails to bind TNFR1, but does bind TNFR2 with five- to 10-fold lower affinity than wild-type TNF (Loetscher et al., 1993). There may be differences between species, because the R32W/S86T double mutant, which was non-toxic in mice, was significantly toxic to healthy baboons (Van Zee et al., 1994). A recently identified triple mutant (S52I, Y56F, plus deletion of the seven N-terminal residues) has 11- and 71-fold lower affinity for the 55kDa and 75-kDa receptors, respectively, and is 10-fold less toxic than wild-type TNF (Cha et al., 1998). These findings have raised hope for an engineered TNF protein that possesses tumor cytotoxicity activity, but with reduced systemic toxicity as compared with the native cytokine. Transmembrane pro-TNF is also biologically active, but less is known about its structure–function relationship. Prior to transport to the cell surface, proTNF is assembled into a bell-shaped trimer that is similar to the structure of mature TNF (Jones et al., 1989). A 20-residue linker sequence connects the transmembrane domain with the N-terminus, which facilitates but is not necessary for trimer formation. During the interaction of effector and target cells, membrane-bound pro-TNF in the effector cells binds TNF receptors on the target cells, and induces TNF responses via receptor aggregation (Kriegler et al., 1988). Pro-TNF is a more potent activator of TNF receptor 2 (TNFR2) than mature TNF (Grell et al., 1994). Highly acidic environments alter the tertiary structure of TNF such that the b-sheet structure is abandoned in favor of a helices (Narhi et al., 1996). TNF is unusual in that it has no a helix in its native structure

but forms an a-helical structure in the molten globule (an intermediate structure produced by acid-induced unfolding of proteins). With the resulting increase in hydrophobicity at low pH, TNF becomes more cytolytic, perhaps by insertion into membrane to form sodium ion channels (Tracey et al., 1986; Yoshimura and Sone, 1987; Baldwin et al., 1996). The conformational change at low pH may be physiologically relevant, because a pH of about 3.6 has been reported in the space between macrophages and their substrates (Silver et al., 1988). Recent membrane conductance studies have shown that the capacity of TNF to increase membrane conductance does not correlate with its ability to interact with membranes. These results suggest that TNF itself does not form a channel, but may increase sodium flux via interaction with endogenous ion channels or with plasma membrane proteins that are coupled to ion channels (van der Goot et al., 1999).

BIOSYNTHESIS AND REGULATION TNF is produced by numerous immune cells in response to an assortment of activating stimuli (Table 35.1). Among these stimuli, lipopolysaccharide (LPS) (in monocytes), T-cell receptor activation (in T lymphocytes), crosslinking of surface immunoglobulin (sIg) (in B lymphocytes), ultraviolet light (in epithelial cells), and viral infections have been widely studied. Suppressors of TNF expression in macrophages are also highly diverse and include prostaglandin E2 (PGE2), transforming growth factor b (TGFb), IFNa, IFNc, IL-4, IL-6, IL-10, G-CSF, certain viral products such as adenoviral proteins, dexamethasone, glucocorticoids, cyclosporin A, spermine, pentoxifylline cAMP, and cholinergic agonists including acetylcholine and nicotine. The biosynthesis of TNF is tightly controlled at many different levels to ensure the production of vanishingly small quantities in quiescent cells, yet TNF is capable of rapid and significant up-regulation in activated cells (Beutler et al., 1985). TNF is expressed as an immediate early gene, and a variety of stimuli induce high levels of TNF mRNA within 15–30 minutes with no requirement for de novo protein synthesis, suggesting that the factors necessary for the

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Table 35.1 Cell source and stimuli of TNF production Cell sources

Stimuli

Immune cells: Macrophages/monocytes, natural killer cells, Kupffer cells, B cells, T cells, basophils, eosinophils, glial cells, mast cells Non-immune cells: Astrocytes, granulosa cells, osteoblasts, cardiac myocytes, fibroblasts, keratinocyte, neurons, neutrophils, T cells, retinal pigment epithelial cells, smooth muscle cells, spermatogenic cells, tumor cells Bacterial endotoxin (LPS), antibodies to LFA-3, calcium ionophores, C5a, CD44, CD45, enterotoxin, GM-CSF, hypoxia, IL-1, leukotrienes, mellitin, MIP-1a, mycobacterial lipoarabinomannan, nitric oxide, oxygen radicals, parasites, phorbol esters, synthetic lipid A, TNF, toxic shock toxin-1, viruses Irradiation

induction of TNF expression pre-exist in unstimulated cells. The cell-type-specific regulation of TNF synthesis is reflected by the differential use of regulatory elements on TNF biosynthesis in different cells. Each one of the synthetic steps from sensing the presence of a stimulus to TNF secretion is controlled by different molecular events regulating the TNF gene, mRNA and protein.

Transcriptional regulation TNF is regulated at the transcriptional level by both activators and repressors. Besides a TATA box promoter located 20 base pairs (bp) upstream from the transcription start site, a number of regulatory sequences are also found upstream of the TNF gene (Figure 35.1), including three nuclear factor (NF)jB sites designated jl, j2 and j3 (Goldfeld et al., 1993); three nuclear factor of activated T cells (NFAT) binding sites for NFATp, NFAT-149, NFAT-117 and NFAT-76 (Tsai et al., 1996); one ‘Y’ box; one cAMP-responsive element (CRE) for activation transcription factor-2 (ATF-2)/Jun (Tsai et al., 1996); two SV40 promoter-1 (SP-1) sites (Kramer et al., 1994); one activating protein-1 (AP-1) and one AP-2 binding site for Fos/Jun (Leitman et al., 1992); one E26 transformation-

Y box -232

SP1 NFAT NFAT -164 -149 -117

j3 -89

AP1 -58

AP2 -27

TNF GENE j1 -577

j2 Egr-1 -201 -163

Ets-1 CRE NFAT -118 -100 -76

SP1 TATA -20 -45

FIGURE 35.1 Model of the TNF promoter. Putative cis-acting consensus sequences are denoted by boxes and numbers indicating the position in relation to the transcription start site. specific (Ets) binding site (Kramer et al., 1995); and an early growth responsive-1 (Egr-1) binding site (Kramer et al., 1994). Although j3 matches the binding consensus sequence for the transcription factor NFjB, j3 appears to bind to NFATp instead of NFjB. The CRE element in TNF promoter binds mainly to ATF-2/Jun. Genetic analyses suggest that additional regulatory elements may be important in regulating TNF transcription (Wong and Goeddel, 1988). TNF mRNA expression is negatively regulated by factors that are less well characterized. Several regions within the TNF promoter, including bases –280 to 172, 254 to 230, 125 to 82, and 95 to 36, relative to the transcription start site, seem to be critical for inhibition of TNF mRNA expression (Fong et al., 1995; An et al., 1999). The transcriptional regulators that bind these regions remain enigmatic. While AP-1 and AP-2 consensus binding sequences have been noted within these regions (An et al., 1999), evidence for binding of these repressors to the TNF promoter is lacking (Fong et al., 1995). More recently, Singh et al. (2001) have found that heat-shock factor 1 (HSF1) binds to regions 1080 to 845, 533 to 196 and 326 to 39 of the TNF promoter relative to the transcription start site, and represses TNF gene transcription. The regulation of TNF transcription is cell-type specific, partially because of differential use of these regulatory elements on the TNF promoter. In calcium- or T-cell ligand-activated Ar-5 T lymphocytes, a functional interaction between NFATp on the j3 site and ATF-2/Jun on the CRE element, but not AP-1 or AP-2, plays a crucial role in the induction of TNF expression (Tsai et al., 1996). In calcium-activated A20 B lymphocytes, NFATp on the j3 element is not required, but NFATp on NFAT-76 participates in the induction of TNF synthesis (Tsai et al., 1996). In phorbol myristate acetate (PMA)-activated T cells, the interaction between Ets and ATF/Jun is essential for both basal

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and induced TNF expression. Post-translational modification of the transcription factors that regulate TNF transcription, such as NFATp, NFjB and AP-1, has been implicated in the regulation of TNF induction. Protein kinases and phosphatases are also involved in the induction of TNF (Lee et al., 1994), as are proteases and phospholipase D (Balboa et al., 1992). Some of these enzymes modify the activity of TNF induction-related transcription factors such as NFjB and AP-1 (see below).

Translational regulation TNF synthesis is highly inducible upon cell stimulation, and is tightly regulated in quiescent cells; this enforced inhibition of TNF expression protects against the harmful effects of TNF overexpression. In response to LPS stimulation, macrophages and monocytic cells produce high levels of TNF protein, an effect that is blocked by pretreatment with dexamethasone, spermine or CNI-1493 (a tetravalent guanylhydrazone) (Bianchi et al., 1996; Beutler et al., 1986; Cohen et al., 1996; Zhang et al., 1997). Recent studies have shown that cholinergic agonists such as acetylcholine and nicotine inhibit TNF synthesis via post-transcriptional mechanisms (Borovikova et al., 2000). A key element for both negative and positive translational regulation of TNF has been identified in the 3 untranslated region (UTR) of TNF mRNA and many other cytokines, growth factors and oncoproteins (Caput et al., 1986; Han et al., 1990). The minimal motif UUAUUUAUU renders the TNF mRNA unstable (Shaw and Kamen, 1986; Zubiaga et al., 1995), and the presence of a single octanucleotide (UUAUUUAU) in the 3 UTR is sufficient to significantly inhibit translation (Kruys et al., 1989). This sequence physically interacts with the poly(A) tail of the TNF mRNA, preventing the mRNA from forming large polysomes (Crawford et al., 1997). Regulation of mRNA turnover is mediated through this region by trans-acting proteins such as tristetraprolin (TTP) (Lai et al., 1999), HuR (Dean et al., 2001), TIA-1 (Piecyk et al., 2000), and TIAR (Gueydan et al., 1999), which bind AU-rich elements and stabilize or destabilize the transcript. Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc-finger proteins, develop an inflammatory syndrome mediated by excess TNF (Lai et al., 1999). Other AU-rich-binding

proteins, such as TIA-1, normally function as translational silencers (Piecyk et al., 2000), because macrophages derived from TIA-1 knockout mice have a significantly higher proportion of TNF transcript associated with polysomes. TIA-1 knockout mice fail to inhibit TNF production and are exquisitely sensitive to endotoxemia (Piecyk et al., 2000). The 3 UTR of TNF mRNA is equally important for stimulating TNF synthesis, because a polymorphism (a GAU trinucleotide insertional mutation) present in the regulatory 3 UTR of TNF mRNA of NZW mice prevents the interaction of RNA-binding proteins with target sequences, and significantly reduces TNF production (Di Marco et al., 2001).

Signal transduction cascades regulating TNF production Mitogen-activated protein (MAP) kinases such as the extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 and Big MAP kinase (BMK)/ERK5 have a critical role in the regulation of TNF production (Zhu et al., 2000). The roles of ERK1/2- and p38 MAPK-dependent pathways in regulating cytokine production have been demonstrated primarily through the use of specific inhibitors (such as PD098059 for ERK1/2 activation, and SB203580 for p38 MAP kinase) (Cuenda et al., 1995; Alessi et al., 1995), which prevent cytokine production in LPSstimulated macrophage/monocytes (Feng et al., 1999; Guha et al., 2001). p38 MAP kinase may be critical in the translational control of TNF synthesis, because activation of the MAP kinase cascade enhances the translational efficiency of TNF mRNA (Lee et al., 1994). Mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) activates TNF induction-associated transcription factors such NFjB, c-Jun and p38 (Cohen et al., 1996; Lee S.Y. et al., 1996). An abridged listing of transcriptional and translational control mechanisms in TNF synthesis is shown in Figure 35.2. Multiple signaling pathways converge on NFjB, a ubiquitous transcription factor involved in the transduction of activation signals from the cytoplasm to the nucleus. NFjB resides in the cytosol as a trimer consisting of p50, p65 and IjB subunits. Upon stimulation, IjB is released, and the p50/p65 heterodimer migrates to the nucleus where it interacts with target DNA sequences

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modulate MMP levels (Panagakos et al., 1996), and plasminogen and plasmin increase secretion of MMPs and induce cleavage to their active forms (Lee et al., 1996).

Inflammatory Stimuli

Rac / Cdc42 MEKK1, 2

ASK 1

MKK4, 7

MKK3, 6

JNK1, 2, 3

p38 - a, b, c, d

Application

MAPKAP-K2 ATF-2 Elk-1, c-Jun NF-jB ?

TNF DNA

841

CREB ATF-1, Elk-1 NF-jB ?

mRNA

Protein

FIGURE 35.2 Schematic illustration of signal transduction pathways regulating TNF transcription and translation in macrophages. resulting in the transcription of TNF and other proinflammatory cytokines.

Post-translational regulation TNF is synthesized as a 233-amino-acid membraneanchored prohormone that is subsequently processed proteolytically to yield the mature 157 residue cytokine. Analysis of the cleavage site sequences in human TNF revealed significant homology with sites in collagen, a1-inhibitory proteins, and a1macroglobulin cleaved by matrix metalloproteinases (MMPs). The TNFa-converting enzyme (TACE) is an MMP-like enzyme (Black et al., 1997; Moss et al., 1997), and metalloproteinase inhibitors block TNF processing (Gearing et al., 1994). Gearing and colleagues (1995) have found that TACE activity is expressed ubiquitously, even in HeLa cells that do not produce TNF, and in insect cells where pro-TNF is expressed by means of a baculovirus transfectant. A serine protease, proteinase 3 (PR-3), may also process TNF, and serve as an extracellular alternate processing enzyme for TNF and IL-1b (Coeshott et al., 1999). Endotoxin, as well as TNF and other cytokines, can

Uncontrolled synthesis of TNF has been implicated in many human diseases of systemic inflammation, including sepsis, arthritis and Crohn disease. Consequently, there has been a great deal of interest in developing therapeutic strategies to inhibit TNF activity, and several inhibitors have been developed to attenuate TNF activity, release, translation or transcription. Corticosteroids, the most studied class of compounds that suppress the production of TNF, inhibit the transcription of TNF and other cytokines. One immunosuppressive mechanism of these drugs is through the stimulation of IjBa production (Auphan et al., 1995; Scheinman et al., 1995), which in turn results in the inactivation of NFjB. Corticosteroids also inhibit gene transcription by preventing the transcription factor, AP-1, from binding to its target promoter (Marx, 1995). Pentoxifylline and other protein kinase C (PKC) inhibitors regulate the production of TNF and other cytokines by blocking PKCor PKA-catalyzed activation of NFjB (Biswas et al., 1994). Cytokine-suppressing anti-inflammatory drugs (CSAIDs) (Young et al., 1994) have no significant effect on the transcription of TNF or other cytokines, but rather suppress TNF translation by inhibiting the activity of p38 MAP kinase (Lee et al., 1994). CNI-1493, a tetravalent guanylhydrazone that suppresses systemic and central inflammation (Meistrell et al., 1997; Tracey, 1998), inhibits TNF by preventing phosphorylation of p38 MAP kinase (Cohen et al., 1996; Denham et al., 2000). At the post-translational level, thalidomide selectively suppresses the release of TNF but not of other cytokines (Sampaio et al., 1991). IL-4 and IL-10 suppress TNF activity by inhibiting the biosynthesis of matrix-destructive metalloproteinases (Lacraz et al., 1992, 1995) and down-regulating the proteolytic processing of pro-TNF to its mature form. Furthermore, IL-4 inhibits the activation of the transcriptional factors NFjB and AP-1, providing yet another mechanism by which IL-4 can attenuate TNF activity. Like IL-4, IL-10 also inhibits TNF as well as other cytokines at multiple levels (Bogdan et al., 1992).

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RECEPTORS The pleiotropic effects of TNF are mediated through two distinct but structurally homologous TNF receptors, the 60-kDa receptor 1 (TNFR1, also known as p55 or p60) and the 80-kDa receptor 2 (TNFR2, also known as p75 or p80), which are both type I transmembrane glycoproteins and members of the TNF receptor superfamily. Members of this receptor family share sequence homologies within their extracellular domains, consisting of multiple cystine-rich repeats of about 40 amino acids in length (Mallett and Barclay, 1991). TNFR1 and TNFR2 are present on virtually all cell types except for red blood cells (Hohmann et al., 1989). TNFR1 is more ubiquitous, but TNFR2 is more abundant on endothelial cells and cells of hematopoietic lineage (Hohmann et al., 1989; Brockhaus et al., 1990). Both receptors can bind to TNF with high affinity; the dissociation constants (Kd) are 2–5 1010 and 3–7 1011, respectively. The precise roles of the TNF-induced receptor signaling in mediating the effects of TNF continue to be the focus of intensive study. TNFR1 is generally considered to be responsible for the majority of biologic actions of soluble TNF. Direct signaling through TNFR2 occurs less extensively and appears to be confined mainly to cells of the immune system, and may be especially important during cell–cell contact (Kriegler et al., 1988). Fibroblasts derived from TNFR1-knockout mice are deficient in leukocyte adhesion, and display reduced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD44. These cells also fail to up-regulate major histocompatibility complex (MHC) class I expression, cytokine secretion, cell proliferation, and NFjB activation. These activities appear to be specific attributes of the TNFR1 receptor, because activation of cells expressing only TNFR2 with exogenous TNF has no effect on these functions (Mackay et al., 1994). Similar observations were made with TNF mutants that selectively bind TNFR1. TNFR1-selective TNF mutants that bind TNFR2 poorly are equally cytotoxic and cytostatic to carcinoma and leukemic cell lines, but are less proinflammatory, than wild-type TNF. TNFR1-selective mutants also recapitulate the hypotensive and proinflammatory activities of wild-type TNF in vivo (Van Zee et al., 1994), whereas TNFR2 by itself does not

seem to be sufficient to stimulate these functions (Barbara et al., 1994). These observations suggest that the role of TNFR2 may be to potentiate the effects of TNFR1. A unique ‘passing model’ has been suggested to explain the role of TNFR2 in mediating TNF responses (Tartaglia et al., 1993b). The rapid rate of dissociation of the TNF–TNFR2 complex may facilitate the interaction of TNF with TNFR1 by recruiting TNF to the vicinity of the plasma membrane. In this scenario, TNFR2 can be envisioned as passing TNF to TNFR1, the main TNF signal transducer. In some cases, however, TNFR2 is also believed to mediate several TNF effects, such as proliferation of T and B cells (Tartaglia et al., 1991; Barbara et al., 1994), induction of NFjB, and cytotoxicity (Hohmann et al., 1990; Grell et al., 1994). TNF receptor activities are also subject to regulation by a wide variety of agents such as PKC and PKA modulators, LPS, retinoic acid, glucocorticoids and cytokines including TNF; IL-2, IL-4, IL-6, IL-8; IFNa, b, c; GM-CSF; and thyroid-stimulating hormone. Therefore, expression of either TNFR1 or TNFR2 is dependent upon cell type, as well as extracellular stimuli. Soluble receptors for TNF (sTNFR, also known as TNF-BP for TNF-binding protein) are derived from both TNFR1 and TNFR2 by proteolytic processing. Both sTNFRl and sTNFR2 are normally present in blood and urine (Seckinger et al., 1989), and may be produced by monocytes and macrophages (Gatanaga et al., 1991; Leeuwenberg et al., 1994). Increased levels of sTNFRs are found in many human diseases including rheumatoid arthritis, systemic lupus erythematosus, malignancy, sepsis, after surgery and chronic infection (Chikanza et al., 1993; Aderka, 1996). A number of stimuli, including TNF, IL-1, PMA and endotoxin, trigger proteolysis of the TNFRs, resulting in increased levels of sTNFRs in the circulation. The proteases responsible for this processing have not yet been identified, but may involve the same metalloproteinases that process the TNF precursor to its mature form, because metalloproteinase inhibitors block the shedding of both TNF receptors (Crowe et al., 1995; Mullberg et al., 1995). Recently, matrix metalloproteinase processing of both precursor TNF and the TNF receptors has been implicated in humans in vivo; administration of an MMP inhibitor prior to endotoxin infusion attenuated the release of both TNF and TNFRs into the circulation. This

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attenuation was accompanied by an increase in membrane-bound TNF and TNFRs in monocytes isolated from these patients (Dekkers et al., 1999). Soluble TNFRs bind and neutralize both circulating and membrane-bound TNF (Seckinger et al., 1989; Kohno et al., 1990). In vivo, administration of sTNFRs neutralizes or modulates TNF activity when sTNFRs are present in high concentrations; lower concentrations, however, stabilize the TNF trimer, providing a TNF reservoir (Mohler et al., 1993).

SIGNAL TRANSDUCTION TNF mediates or initiates diverse biologic responses by interaction with TNFR1 or TNFR2. Investigations into the signaling events activated by the ligand/receptor complex have been hampered by the diversity of cell-specific TNF responses, and a lack of

NSD

ASD

FAN

PC-PLC

similarities with other known cell surface receptor signaling pathways. X-Ray data suggest that the TNF homotrimer induces receptor trimerization, which activates receptor signaling. Several downstream signaling events initiated by the TNF receptor complex have been characterized, and signaling molecules that mediate the initial interaction with the ligandoccupied receptor have been identified.

TNFR1-mediated signaling pathway Multiple intracellular functional domains within TNFR1 transduce intracellular signals to coordinate the biological activity of TNF (Tartaglia et al., 1993a) by interacting with intracellular adaptor proteins (Figure 35.3). Three functional domains of TNFR1 – the C-terminal death domain (Tartaglia et al., 1993a), and the adjacent N-SMase (neutral sphingomyelinase) and A-SMase (acidic sphingomyelinase)

Death domain

TRADD

DAG

Death domain

N-terminus

N-SMase

A-SMase

PKC

TRAF2

RIP

Ceramide (membrane)

Ceramide (endosome)

IKK

NIK

RAIDD

MEKK1, 2

Caspase-2

CAPK NF-jB / I-jB complex RAF-1

ERK

Cell growth

TNFR1

JNK

FADD

PED/PEA-15

Caspase-8

Protease cascade

I-jB PLA2

NF-jB

Inflammation

AP-1 c-Jun

Cleavage of death substrates Activation of killer proteins

Anti-apoptosis

Apoptosis

FIGURE 35.3 The TNF receptor 1 (TNFR1)-mediated signaling transduction pathways. TNF triggers different signaling pathways via three functional domains of TNFR1 to mediate various activities. THE CYTOKINES AND CHEMOKINES

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activating domains (NSD and ASD) (Schutze et al., 1992; Wiegmann et al., 1994) – transfer signals from extracellular TNF to intracellular adaptor proteins that can contain additional death domains and/or caspase recruitment domains (CARD). TRADD (TNFR1-associated death domain protein) binds to the death domain of TNFR1 (Hsu et al., 1995) to mediate both apoptotic and antiapoptotic pathways. Proapoptotic effects of TNF are also mediated through activation of phosphatidylcholine-specific phospholipase c (PC-PLC) by the ASD domain (Wiegmann et al., 1994; Machleidt et al., 1996), which shares receptor regions and function with the death domain. The factor associated with N-SMase activation (FAN) binds the NSD domain of TNFR1 (AdamKlages et al., 1996) to mediate a number of inflammatory and cell proliferation responses. Together, it is apparent that TNFR1 signaling diverges into three functional branches at the level of the receptor itself: one that is mediated through the death domain to positively and negatively regulate apoptotic responses, and two that are processed through sphingomyelinases to modulate apoptotic and inflammatory pathways.

Death domain-mediated pathways The TNFR1-associated death domain protein (TRADD) mediates both apoptosis and antiapoptosis pathways by activating the interleukin-1 converting enzyme (ICE) pathway or the NFjB pathway, respectively (Hsu et al., 1995). The TRADD protein contains a death domain similar to the death domain of TNFR1. While overexpression of an isolated TRADD death domain can trigger cell death, overexpression of the full-length protein does not, suggesting that TRADD does not carry a caspase recruitment domain (CARD). This has led to one currently accepted view of TRADD as an adaptor protein that links TNFR1 to other death effectors. Once bound to TNFR1, TRADD recruits MORT1/FADD (Fas-associating protein with death domain) or RIP (receptor-interacting protein) through its C-terminal death domain to activate divergent apoptotic pathways (Grimm et al., 1996), and recruits TNF receptor-associated factor 2 (TRAF2) through its N-terminal TRAF-interacting domain (Hsu et al., 1996). Thus, TNFR1 death domain signaling diverges at the level of TRADD into three distinct

pathways: one that is dependent upon TRAF2 and activates inflammatory and antiapoptotic responses via NFjB and c-Jun; a second pathway that activates caspase 2 through the activity of RIP to induce apoptosis; and a caspase 8-dependent apoptotic pathway that is dependent upon FADD activity. The TNFR1–TRADD complex initiates apoptotic signaling through pathways dependent upon either FADD or RIP. Overexpression of FADD (Chinnaiyan et al., 1995; Boldin et al., 1996) or RIP (Stanger et al., 1995) results in apoptosis; however, a dominantnegative mutant of FADD inhibits TNF-induced apoptosis but not TNF-mediated NFjB activation (Hsu et al., 1996), demonstrating that the apoptotic and inflammatory cascades diverge at the level of the TRADD–FADD complex. Activated FADD recruits the FADD-homologous ICE/CED-3-like protease (FLICE), also referred to as pro-caspase 8 or MACH, which interacts with the caspase recruitment domains (CARD) of FADD, where it oligomerizes and autoproteolytic activity generates catalytically active capase-8 (Boldin et al., 1996; Muzio et al., 1996). The FADD–FLICE interaction is negatively regulated by PED/PEA-15, possibly by the dissociation of the complex (Condorelli et al., 1999). The death-associated protein (DAP) kinase is another death domaincontaining protein that operates downstream of FLICE but upstream of other caspases to activate apoptosis (Cohen et al., 1999). The FADD-activated caspase cascade induces apoptosis by proteolytically activating protein precursors, such as lamin, actin and polymerase, or by activating proteins that lead to cell death (Figure 35.3). TNFR1 can also induce apoptosis via the TRADD– RIP pathway. All three RIP family members (RIP, RIP2 [also known as Rick or CARDIAK] or RIP3) possess protein kinase and death domains, and transduce intracellular apoptotic signals. The death domain of RIP can mediate the death signal in the absence of the kinase domain, suggesting that RIP does not possess a CARD but rather induces apoptosis by recruiting other downstream effector molecule(s) through the RIP death domain. RIP binds preferentially to TRADD via protein–protein interaction between the death domains on both proteins (Hsu et al., 1996), but can also bind weakly to TNFR1 (Stanger et al., 1995). TNFR1–TRADD–RIP activate apoptosis via the intermediate effector, RAIDD (for RIP-associated Ich-1/

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CED-3 homologous protein with death domain), which binds RIP via a death domain (Duan and Dixit, 1997). The RAIDD/RIP complex recruits caspase-2 (Ich-1) to activate the protease cascade leading to apoptosis. RIP can also bind to TRAF2, which can be recruited to TRADD simultaneously with RIP. The significance of RIP binding to TRADD and TRAF2 as well as TNFR1 is still obscure, and it is possible that RIP may coordinate these signaling pathways. Overexpression of TRAF2 activates NFjB (Rothe et al., 1995) but TRAF2 is not required for the induction of apoptosis (Hsu et al., 1996). A serine–threonine kinase known as NFjB-inducing kinase (NIK) binds specifically to TRAF2 and mediates TRAF2-dependent activation of NFjB (Malinin et al., 1997). The binding between TRAF2 and NIK is mainly dependent on the C-terminus of TRAF2, but also involves the N-terminal of TRAF2. Because NIK can be autophosphorylated and has sequence similarity to several kinases that participate in MAP kinase cascades, it may act in a kinase cascade to activate NFjB. The cascade downstream of NIK that is responsible for NFjB activation, however, is still unclear. TRAF2-dependent activation of MEKK and c-Jun amino-terminal kinase (JNK)/stressactivated protein kinase (SAPK) through TNFR1 has been reported (Liu et al., 1996; Natoli et al., 1997). As MEKK has been shown to induce the phosphorylation and degradation of IjBa, resulting in the activation of NFjB (Lee et al., 1997), MEKK may function as a downstream factor of NIK and mediate TRADD– TRAF2-directed activation of NFjB to prevent TNFinduced apoptosis (Figure 35.3). It is plausible that this may be one of the mechanisms of TNF selftolerance. Because NFjB and JNK are major factors regulating the inflammatory response, this pathway may also play a role in the proinflammatory effect of TNF.

NSD-mediated pathway NSD (neutral sphingomyelinase domain) occupies a central role in mediating a number of TNF activities, including inflammatory responses and cell proliferation through the MAP kinase ERK and PLA2, respectively (Figure 35.3). The novel WD repeat ({X6–94[GH-X23–41-WD]}N4–8) protein FAN couples TNFR1 to N-SMase via protein–protein interactions between

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the C-terminal WD repeats of FAN and a cytoplasmic, nine-amino-acid binding motif (residues 310–318) of TNFR1 (Adam-Klages et al., 1996). FAN specifically activates N-SMase, and not A-SMase (see ASDmediated pathway below), which modulates the production of ceramide, a product of the sphingomyelin (SM) degradation. Ceramide is generated at several distinct subcellular locations that influence its action (Wiegmann et al., 1994). Activated N-SMase functions at the outer leaflet of the plasma membrane to trigger the degradation of sphingomyelin (SM) into ceramide, which in turn mediates further downstream events in NSD-mediated TNF response. Ceramide is also produced in endosomes by A-SMase, where ceramide mediates a distinct signaling pathway. On the plasma membrane, ceramide further activates ceramide-activated protein kinase (CAPK) (Winston and Riches, 1995), which phosphorylates cytoplasmic raf-1. Activated raf-1 can activate the MAP kinase cascade and activate MAPK/ERK (extracellular signal-regulated kinase), which in turn activates PLA2 (Lin et al., 1993). PLA2 may be responsible for the generation of the arachidonic acid metabolites, leukotrienes and prostaglandins, which contribute to TNF proinflammatory activities. MAPK/ERK also induces the expression of c-myc and other genes to regulate cell proliferation and differentiation. Activation of ERK may act as a negativefeedback loop to attenuate TNF-induced apoptosis, because inhibition of ERK is required for induction of apoptosis in some systems (Xia et al., 1995).

ASD-mediated pathway N-SMase and A-SMase are activated independently by different cytoplasmic domains of TNFR1, and elicit discrete signaling pathways (Wiegmann et al., 1994). Like the death domain, the ASD can induce activation of NFjB to protect against apoptosis (Van Antwerp et al., 1996; Wang et al., 1996), but is also involved in induction of apoptosis (Pena et al., 1997). The ASD maps to the same stretch of residues within the death domain, and may in fact be the same domain (Tartaglia et al., 1993a; Wiegmann et al., 1994). The ASD mediates activation of A-SMase via PC-PLC (Schutze et al., 1992). The connection between the ASD in TNFR1 and PC-PLC is still obscure, but appears to involve the activity of protein kinase C

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(PKC) (Müller et al., 1995). Activated PC-PCL produces 1,2-diacylglycerol (DAG), which activates ASMase and contributes to the activation of NFjB; TNF-induced A-SMase and NFjB activity is blocked by the specific PC-PLC inhibitor, D609, which effectively inhibits DAG production (Schutze et al., 1992). A-SMase produces ceramide in the acidic endosomal/lysosomal compartment (Spence, 1993). However, the precise role of A-SMase-induced ceramide is unclear, because cells that are deficient in ceramidase, and therefore accumulate intracellular ceramide, are equally responsive to apoptotic signals from TNFR1. In addition, TNF- or CD95-induced apoptosis does not induce the degradation of labeled sphingomyelin (Segui et al., 2000). A-SMase activity in TNF-induced NFjB may in fact be cell-type specific, because a recent study by Kouba et al. (2001) revealed that A-SMase activity can be used as a surrogate mediator of NFjB activation.

TNFR2-mediated signaling pathway Direct signaling through TNFR2 is less well characterized, and appears to be confined primarily to cells of the immune system. TNFR2 mediates specific TNF effects including proliferation of T and B cells (Tartaglia et al., 1991; Barbara et al., 1994), induction of NFjB and cytotoxicity (Hohmann et al., 1990a, 1990b; Grell et al., 1994), and may potentiate the effects of TNFR1. TNFR2 can down-regulate human and murine activated T cells by inducing apoptosis (Zheng et al., 1995). TNFR2 lacks an intracellular death domain, suggesting that TNFR2 uses distinct signaling pathway(s) to induce apoptosis. Downregulation of the anti-apoptotic protein Bcl-xL (Boise and Thompson, 1997) has been correlated with TNFR2-induced apoptosis (Lin et al., 1997), but the precise mechanisms triggering TNFR2-induced apoptosis remain to be elucidated. Whether TNFR2 alone can mediate the activation of NFjB is controversial. Although TRAF2 interacts with TNFR2, and TRAF2– TRAF1 (Rothe et al., 1995) and TRAF2–TANK (Cheng and Baltimore, 1996) heterocomplexes are able to mediate the activation of NFjB, the results of a recent study (Chainy et al., 1996) indicate that TNFR2 does not participate in the activation of NFjB in many cell types, including immune cells.

BIOLOGIC EFFECTS TNF was originally characterized as a protein inducing necrosis of methylcholanthrene (Meth A)-induced sarcomas in vivo (Carswell et al., 1975) and believed to be a growth modulatory agent that exhibits primarily antitumor activity. The discovery of the systemic inflammatory role of TNF in non-malignant disease (Tracey et al., 1986b) led to a focus on the pathogenic role of TNF in many immune-mediated diseases. The net effects of TNF are influenced by a complex array of cell- and tissue-specific factors. The diverse role of TNF in mediating cellular responses is summarized in Table 35.2.

Central nervous system Systemic TNF produces fever and anorexia via hypothalamic centers that regulate body temperature and appetite (Tracey et al., 1988; Plata-Salaman, 1991). The anorectic effect of TNF is attenuated by insulin and appears to be independent of serotonergic signals (Tracey and Cerami, 1990). Cells of the central nervous system (CNS), including microglia, astrocytes and neurons (Cheng et al., 1994), express TNF and high-affinity TNF receptors (Smith and Baglioni, 1992). Furthermore, elevated TNF levels have been detected in brain injury and certain CNS diseases including stroke (Liu et al., 1994), meningococcal meningitis (Leist et al., 1988), human immunodeficiency virus infection (Grimaldi et al., 1991), Alzheimer’s disease (Fillit et al., 1991) and multiple sclerosis (Hofman et al., 1989). These findings strongly suggest that TNF is involved in various biologic processes within the CNS. The inflammatory effects of TNF are pivotal to the development of cerebral inflammation and edema during meningitis. High levels of TNF in cerebrospinal fluid during meningitis are predictive of poor outcome (Leist et al., 1988; Waage et al., 1989; Saukkonen et al., 1990). The in vivo inflammatory effects of TNF have been implicated in the development of the plaques characteristic of multiple sclerosis, and in cerebral ischemia in experimental models of stroke, in which immunologic or pharmacologic inhibition of TNF activity can reduce the severity of brain damage (Saukkonen et al., 1990; Meistrell et al., 1997).

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TABLE 35.2 Selected list of biological effects of TNF Immune cells

Non-immune cells

In vivo

Monocytes/macrophages Autocrine induction of TNF Induction of IL-1, IL-8, GM-CSF, M-CSF, IFNc, NGF, TGFb, DGF and EG2 Chemotaxis Stimulation of metabolism Inhibition of differentiation Suppression of proliferation Internalization of complement-coated virus

Vascular endothelial cells Modulation of angiogenesis Increasing permeability Enhanced expression of MHC 1 Procoagulant and antifibrinolytic Increasing permeability of albumin Suppression of proliferation Rearrangement of cytoskeleton Induction of NO synthase, IL-1, IL-3 receptor, G-CSF, GM-CSF, ICAM-1, VCAM-1, P- and E-selectin, surface antigen, platelet-activating factor, prostacyclin Inhibition of integrin B30, thromomodulin, glutathione, protein S

Central nervous system Fever Anorexia Altered pituitary hormone secretion

Polymorphonuclear leukocytes Priming of integrin response Release of granule components Increasing phagocytic capacity Enhanced production of superoxide Increased adherence to extracellular matrix Suppression of chemotaxis to N-formyl-1-leucyl-1-phenylalanine Suppression of cell surface expression of sialophlorin CD43 Lymphocytes Induction of T-cell colony formation Induction of superoxide in B cells Activation of cytotoxic T-cell invasiveness Induction of apoptosis in mature T cells

Fibroblasts Induction of proliferation Induction of IL-1, 6, IFNb2, leukemia inhibitory factor, metalloproteinases (MMTs) Suppression of respiratory activity Inhibition of collagen synthesis, MMT inhibitor Adipocytes Enhanced release of free fatty acids and glycerol Suppression of lipoprotein lipase (LPL) Endocrine system Stimulation of adrenocorticotrophic hormone and prolactin Inhibition of thyroid-stimulating hormone Follicle-stimulating hormone and growth hormone Enhancing IL-1 inhibition of steroidogenesis

Although high levels of TNF play a pivotal role in tissue injury, accumulating evidence also indicates that low levels of TNF play critical roles in the repair and regeneration of damaged tissue in the CNS (Tracey et al., 1989). TNF prevents neuronal death following metabolic excitotoxic insults (Cheng et al., 1994), and activates CNS tissue repair by inducing proliferation of astrocytes (Selmaj et al., 1990) and neuronal progenitor cells (Mehler et al., 1993). The

Cardiovascular Shock ARDS Capillary leakage syndrome Gastrointestinal Ischemia Colitis Hepatic necrosis Inhibition of albumin expression Decreased catalase activity in liver Suppression of HBV gene expression Metabolic LPL suppression Net protein catabolism Net lipid catabolism Stress hormone release Insulin resistance Inflammatory Activation of cell cytotoxicity Enhanced NK cell function Mediation of IL-2 tumor toxicity Protective role in cutaneous leishmaniasis

proliferative effects of TNF may be dependent upon the production of nerve growth factor (NGF) (Gadient et al., 1990), which is protective in tissue damage (Mattson et al., 1993). Mice genetically deficient for both TNFR1 and TNFR2 are more sensitive to central ischemic insult and have more extensive cortical damage (Bruce et al., 1996), suggesting that TNF can be protective in ischemic brains. This contrasts with the overexpression of TNF during cerebral ischemia

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in normal rats subjected to focal infarction, where TNF significantly increases brain damage (Meistrell et al., 1997).

Cardiovascular system Endothelial cells serve as a semipermeable barrier in blood vessels, and mediate coagulation. During systemic inflammation, sepsis, multiple organ failure and other acute TNF-mediated diseases, TNF enhances tissue factor expression and suppresses co-factor activity for the anticoagulant protein C on endothelial cells (Bevilacqua et al., 1986; Nawroth et al., 1986). TNF also directly induces actin filament rearrangement, leading to endothelial cell damage and loss of tight junctions; this, in turn, causes capillary leakage syndrome as plasma proteins and water leak into tissues (Sato et al., 1986; Stephens et al., 1988; Brett et al., 1989). TNF exerts a negative inotropic effect on myocardial contractility through a mechanism that remains largely unknown. Evidence suggests that TNF induces the release of platelet-activating factor (PAF) (Alloatti et al., 1999), as well as stimulation of nitric oxide (NO) production (Finkel et al., 1992; Habib et al., 1996; Alloatti et al., 1999). Both epithelial cells and cardiac myocytes express TNF receptors and are capable of synthesizing TNF under certain forms of stress (Giroir et al., 1994; Kapadia et al., 1995). Elevated levels of TNF have been identified in patients with advanced heart failure (Moriarty et al., 1990; Katz et al., 1994). TNF may play a central role in the pathogenesis of heart failure, and the known biologic effects of TNF may contribute to the development of many of the clinical hallmarks of heart failure, including left ventricular dysfunction, cardiomyopathy, cachexia and pulmonary edema (Millar et al., 1989; Natanson et al., 1989; Suffredini et al., 1989; Hegewisch et al., 1990). The negative inotropic effects of TNF on isolated cardiac myocytes require TNF concentrations as low as 0.7–1.4 109 M (Yokoyama et al., 1993; Kapadia et al., 1995), which have been reported in congestive heart failure (Levine et al., 1990). Direct injection of TNF produces hypotension, metabolic acidosis, hemaconcentration and death within minutes, mimicking the cardiovascular response in endotoxin-induced septic shock (Tracey et al., 1986a). This is caused by several TNF-mediated

effects including a decrease in peripheral vascular resistance, falling cardiac output and loss of intravascular volume through capillary leakage (Tracey et al., 1986a, 1987b; Natanson et al., 1989). TNF-induced NO production in endothelium has been implicated in decreases of both peripheral vascular tone and cardiac function (Finkel et al., 1992), whereas TNF-induced rearrangement of actin filaments and loss of tight junctions are responsible for the capillary leakage syndrome (Sato et al., 1986; Stephens et al., 1988; Brett et al., 1989). TNF is extremely toxic to the lungs and is pivotal in the development of adult respiratory distress syndrome (ARDS), characterized by pulmonary edema, hypoxia and a high mortality rate. ARDS develops because of TNF-induced activation of pulmonary endothelium, margination of leukocytes with degranulation of granulocytes and capillary leakage, which precipitates the collection of edematous fluid in alveoli and prevents adequate perfusion (Stephens et al., 1988).

Human diseases Although TNF can preserve cellular and biochemical homeostasis thereby participating in beneficial tissue remodeling and host-defense responses, overproduction of TNF is harmful, even lethal, to the host (Tracey et al., 1989). Therefore, either insufficient or excessive production of TNF can be deleterious, and TNF has been directly implicated in the pathogenesis of many disease states (Table 35.3).

Septic shock TNF plays a pivotal role in septic shock, because it fulfils Koch’s postulates, a logical chain of evidence that can be modified here for considering cytokine biology. First, TNF is released systemically during overwhelming sepsis, and in some cases leads to lethality (Girardin et al., 1988; Waage et al., 1989). Persistent increases in TNF are associated with the development of multiple organ failure and mortality (Girardin et al., 1988; Calandra et al., 1990; van der Poll and Lowry, 1995). Second, administration of exogenous TNF causes shock and tissue injury that is physiologically, metabolically, hematologically and pathologically indistinguishable from septic shock syndrome (Tracey et al., 1986a, 1987a). Third, neutralization of

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TABLE 35.3 Selected TNF-associated diseases Infectious diseases

Autoimmune diseases

Cancer and others

Bacterial Meningococcal disease Leprosy Tuberculosis Septic shock syndrome Nocardia brasiliensis Cryptogenic fibrosing alveolitis Pneumocystis carinii pneumonia

Glomerulonephritis Guillain–Barré syndrome Inflammatory bowel disease Systemic lupus erythematosus Insulin-dependent diabetes mellitus Rheumatoid arthritis Dermatitis Celiac disease Myasthenia gravis Systemic sclerosis

Hairy cell leukemia T-cell leukemia Chronic lymphocytic leukemia Malignant lymphoma Colorectal cancer Lung cancer Cervical cancer

Viral AIDS HIV-related lung disease Measles Hepatitis Viral meningitis Murine retrovirus infection Parasitic Cerebral malaria Toxoplasma gondii Dysentery Shigella dysenteriae Children with pertussis

TNF with antibodies prevents septic shock syndrome during lethal bacteremia (Tracey et al., 1987b). In support of this view, TNFRl-deficient knockout mice are resistant to endotoxemic shock (Pfeffer et al., 1993). TNF induces other mediators of sepsis such as IL-1, and infusion of anti-TNF antibody attenuates the activity of these mediators (Tracey et al., 1987b; Fong et al., 1989). TNF and soluble receptors of TNF (sTNFRs) are liberated during sepsis (Girardin et al., 1992). As mentioned above, sTNFRs are inhibitors of TNF biologic activities but may also serve as a reservoir of TNF, and increased levels of sTNFRs are found in many TNFassociated diseases. In fact, increased levels of sTNFRs correlate with TNF levels and outcome in severe meningococcemia (Girardin et al., 1992). In general, sTNFR concentrations are associated with TNF levels when the TNF level is relatively low, but not when it is high. The imbalance between TNF and sTNFRs is probably pathophysiologically important, as reflected by the observation that their ratio on admission may

Euthyroid sick syndrome Hemorrhagic shock Disseminated intravascular coagulopathy Anemia Myocardial ischemia Obesity Shock Stroke Rejection of transplanted organs Pulmonary fibrosis Chronic osteomyelitis Graves’ disease Asthma Injury Inflammatory hyperalgesia Down’s syndrome Cachexia

be of predictive value for clinical outcome (Girardin et al., 1992).

Cancer TNF was originally characterized as an antitumor protein inducing necrosis of Meth A sarcomas in vivo (Carswell et al., 1975). TNF has been widely studied and explored as an antitumor drug; at present it is used clinically as a locally delivered intratumoral agent and has some efficacy (Lienard et al., 1992). The therapeutic application of TNF as a single and systemic antitumor agent is limited by toxic side-effects at tumoricidal doses (Blick et al., 1987). Several strategies have been proposed to avoid the limitations imposed by toxicity, including: (1) high-dose intraarterial administration of TNF directly into the tumor compartment (Ohkawa et al., 1989); (2) administration of non-toxic levels of TNF to tumors sensitive to its therapeutic effects; (3) administration of non-toxic doses of TNF in combination with other antitumor

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agents – synergistic effects have been identified (Zimmerman et al., 1989), but enhanced side-effects have also been reported (Schiller et al., 1992); (4) identification and development of TNF-like molecules with maximum efficacy as an antitumor agent, but minimal side-effects. In addition, chemical modification of TNF with compounds such as polyethylene glycol (PEG) has been found selectively to increase the antitumor potency of TNF while effectively reducing its systemic toxic side-effects (Tsutsumi et al., 1995). A few mechanisms have been suggested for the tumoricidal effect of TNF, including direct cytotoxicity against tumor cells, activation of immune antitumor response and selective damage of tumor blood vessels (North and Havell, 1988). Direct cytotoxicity to tumor cells has been supported by most in vitro studies (Duerksen-Hughes et al., 1992). TNF also activates other cytokines (Tracey and Cerami, 1994) and cytotoxic factors such as NO (Estrada et al., 1992), which in turn participate in immune antitumor responses and direct tumor cell killing. A study by Sato et al. (1996) reported that it might be feasible to use TNF gene-transduced tumor cells as a vaccine, since TNF is a powerful activator of the immune response. TNF selectively eradicates vascularized tumors but is much less effective in killing avascular implants (Yoshimura and Sone, 1987; Thomson, 1997). In contrast, TNF is produced by some tumors or tumor cells, and can act as an autocrine growth factor (Sugarman et al., 1985; Buck et al., 1990).

ASSOCIATED DISEASE SUSCEPTIBILITY The TNF locus with two tandemly arranged and closely linked genes, TNF and lymphotoxin-a, lies in the class III region of the MHC on the short arm of human chromosome 6. A striking feature of the MHC is the high degree of polymorphism of genes in this region. At least five polymorphisms (Partanen and Koskimies, 1988; D’Alfonso and Richiardi, 1994) and five microsatellites (Jongeneel et al., 1991; Udalova et al., 1993) have been described at the TNF locus. These polymorphisms have been linked to the variability of TNF production in different individuals (Molvig et al., 1988). Because TNF production has been implicated as an important factor in immune

regulation and the inflammatory response, and affects the outcome of human diseases, polymorphisms within the TNF locus are considered possible genetic bases for these diseases. Some studies have indicated that genetic variation of TNF and closely linked loci are important in determining susceptibility to a significant number of human diseases, including autoimmune diseases such as diabetes, rheumatoid arthritis and multiple sclerosis, parasitic, bacterial and viral infections and cancer. TNF responsiveness is controlled by variable genetic elements within the MHC region. A polymorphism located at 308 bp upstream of the TNF transcriptional start site, with two allelic forms referred to as TNF1 and TNF2, has been demonstrated directly to affect TNF production (Wilson et al., 1993, 1997); TNF2 is associated with higher constitutive and inducible transcriptional levels than the TNF1 allele (Wilson et al., 1994, 1997). There is also a correlation of human leukocyte antigen (HLA)-DR2 with low TNF production (Bendtzen et al., 1988; Molvig et al., 1988), and of HLA-DR3 and HLA-DR4 with high TNF production (Jacob et al., 1990; Abraham et al., 1993). These polymorphisms are associated with the susceptibility or severity of certain diseases. Thus, the TNF locus may contribute to the pathogenesis of diseases by influencing the TNF levels produced in those diseases.

Susceptibility to infectious diseases MHC alleles affect outcome in several infectious diseases. TNF2 homozygotes with associated higher TNF levels are at increased risk of cerebral malaria (McGuire et al., 1994) or post-traumatic sepsis (Majetschak et al., 1999). This is consistent with the phenotypic observation that Gambian children with cerebral malaria have higher TNF levels than children with mild malaria (Kwiatkowski et al., 1990), and that TNF levels are highest in children who die or develop neurologic sequelae due to cerebral malaria. In contrast, the HLA-B53 locus, which correlates with depressed levels of circulating TNF, appears to be protective against several forms of malaria (Hill, 1992). The TNFB2 allele, also associated with higher levels of TNF production, is linked to lower survival in patients with multiple organ failure secondary to severe sepsis (Stuber et al., 1996). Susceptibility to mucotaneous

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leishmaniasis, which is accompanied by high circulating TNF levels, is linked directly with the regulatory polymorphisms affecting TNF production (Cabrera et al., 1995). In addition, HLA-DQ and HLA-DP alleles are associated with generalized and localized forms of onchocerciasis (Meyer et al., 1994). As the understanding of allelic regulatory differences improves, it should be possible to more effectively translate our basic research advances in TNF mechanisms into clinically useful methods to prevent the pathologic sequelae of excessive TNF, and/or to improve the potential benefits of its controlled local production (e.g. cancer).

Susceptibility to cancer Alleles of HLA-A, -B and -DR loci are associated with resistance to lung cancer. Investigators have located one such element to the TNF locus and found that TNFB2 homozygosity is associated with disease resistance and a better prognosis in patients with lung cancer (Shimura et al., 1994), in keeping with a previous study that demonstrated a strong correlation between HLA-A1 and a lower 1-year survival rate (Markman et al., 1984). As TNFB2 is associated with higher levels of TNF production, these results are consistent with the original characterization of TNF as an antitumor factor. It has been reported that germline allelic frequencies for the TNF locus are significantly different in patients with colorectal cancer compared with those in normal controls. Most strikingly, the a3 allele in patients with colorectal cancer has an allelic frequency of nearly 30% but is not detected in the normal control group (Campbell et al., 1994). Other studies have shown that class II haplotypes such as DRB1*1501–DQB1*0602 are positively associated with the development of cervical cancer, but that DR13positive haplotypes are negatively associated (Apple et al., 1994).

Susceptibility to autoimmune diseases The outcome of a large number of studies suggests that TNF genetics is an important contributing factor to autoimmune disease susceptibility. Celiac disease (CD) is an immune disease triggered by the cereal antigen gliadin, resulting in villus atrophy in the small intestine. Susceptibility to the development of celiac

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disease is strongly influenced by the genes in MHC, and is associated with the HLA-A1–B8–DR3–DQ2 haplotype (Ahmed et al., 1993) and more strongly associated with the DQA*0501/DQB*0201 heterodimer (Sollid et al., 1989). Wilson and colleagues (1993) found that the TNF2 allele in the TNF locus is closely associated with HLA-A1–B8–DR3–DQ2, suggesting that the association of this haplotype with celiac disease and high TNF production may be related to polymorphisms within the TNF locus. At the same time, susceptibility to celiac disease is strongly related to TNF2 (Manus et al., 1996), supporting this speculation. The TNF2 allele appears to be involved in a variety of MHC-linked diseases including systemic lupus erythematosus, dermatitis herpetiformis and insulin-dependent diabetes mellitus (IDDM). Microsatellite polymorphisms (TNFa2 and TNFb3) close to the TNF genes are associated with celiac disease, an observation that could have functional significance because the TNFa2 allele correlates with high TNF production (McManus et al., 1996). Rheumatoid arthritis (RA) is a debilitating disease characterized by progressive joint dysfunction and chronic systemic inflammation (Pincus et al., 1994). TNF has been implicated in the pathogenesis of RA, although its precise mechanisms of action in the disease remain poorly understood. Genetic analyses have implicated the TNFcl allele as well as the 238 GG genotype, but not the TNFa allele, in rheumatoid arthritis (RA) susceptibility (Fabris et al., 2002). Elevated concentrations of TNF have been identified in the serum and synovial fluid of RA patients (Chu et al., 1991), and plasma TNF concentrations correlate with disease severity in terms of joint function and pain (Beckham et al., 1992). Because TNF induces the expression of endothelial cell adhesion molecules (Moser et al., 1989), TNF may recruit inflammatory cells into affected joints. TNF also induces the expression of matrix metalloproteinases from fibroblasts and chondrocytes, and may therefore accelerate joint degradation and dysfunction in RA (Shingu et al., 1993). Therapeutic interventions that inhibit the activity of TNF have proven efficacious for the treatment of both adult and juvenile RA (Maini et al., 1999; Weinblatt et al., 1999; Lovell et al., 2000). The role of various TNF alleles as susceptibility factors for systemic lupus erythematosus (SLE) is currently under intense investigation. The alleles TNFB1

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(Bettinotti et al., 1993) and TNF2 (Wilson et al., 1993) are associated with systemic lupus erythematosus (SLE), but the association is not independent of HLA-DR3 (Welch et al., 1988). The HLA-DR3 and TNF-308A loci appear to be independent susceptibility loci for SLE (Rood et al., 2000; van der Linden et al., 2001), but others challenge this view (Tsuchiya et al., 2001). In another study (D’Alfonso et al., 1996), 123 patients with SLE and 199 matched controls were analyzed. Three TNF238/A homozygous patients but no homozygous control were detected, suggesting that the 238/AA genotype is a marker of a particular clinical subtype; this has recently been confirmed in a cohort of 51 Mexican SLE patients (Zuniga et al., 2001). Because TNF has been genetically implicated as a mediator of SLE, several studies have attempted to neutralize TNF as a therapeutic approach to SLE. Although immunologic or pharmacologic inhibition of TNF production has reduced the clinical manifestations of experimental SLE in mice (Segal et al., 2001), aberrant TNF activity may promote humoral autoimmunity and allow the development of autoreactive B cells (Via et al., 2001). These findings suggest that, while abrogation of TNF activity may be therapeutically beneficial for the treatment of established SLE, depressed TNF levels may play a causative role in the development of this autoimmune disease. TNF has also been implicated in the pathogenesis of IDDM as a mediator in the immune-mediated destruction of pancreatic b cells (Mandrup-Poulsen et al., 1987). Studies of TNF genetics in patients with IDDM show a weak association between TNF2 and IDDM (Pociot et al., 1993b; Cox et al., 1994). However, another study demonstrated a higher frequency of the TNFa2 allele in DR3–DR4 heterozygotic patients with IDDM than in controls. As this marker is associated with higher TNF production independently of class II alleles, the TNF locus may play a direct role in susceptibility to IDDM (Pociot et al., 1993a).

Anti-TNF therapy Several strategies have been proposed to prevent TNF toxicity in sepsis (Tracey and Cerami, 1994). One approach is direct inhibition of TNF activity with antibodies or native and chimeric sTNFRs (Tracey et al.,

1987b; Ashkenazi et al., 1991; Peppel et al., 1991). Another is to suppress TNF synthesis using glucocorticoids, pentoxifylline, CNI-1493, spermine, thalidomide and cytokines such as IL-10 (Beutler et al., 1986; Lilly et al., 1989; Sampaio et al., 1991; Cohen et al., 1996; Zhang et al., 1997). The third approach is to induce TNF tolerance with repetitive administration of low-dose TNF. Inhibitors of receptor-mediated endocytosis of TNF decrease TNF-induced gene expression, thus providing yet another potential point of intervention (Bradley et al., 1993). In addition, pharmacologic inhibitors of metalloproteinases have been developed to inhibit TNF processing, although clinical efficacy has not been addressed (McGeehan et al., 1994). Finally, TNF cytotoxicity may be inhibited by targeting its secondary mediators such as IL-1 (McNamara et al., 1993). Some of these therapies have been demonstrated to have salutory effects in animal models of sepsis, yet preliminary clinical trials have not been as encouraging (van der Poll and Lowry, 1995). Strategies to directly inhibit TNF activity with antibodies or sTNFRs are effective in animal models (Mohler et al., 1993) (Table 35.2), and have been tested in clinical trials of Crohn’s diseases (Sandborn and Hanauer, 1999; Shanahan, 2000; Kam and Targan, 2000), rheumatoid arthritis (Taylor, 2001; Schwarz et al., 2000; Feldmann and Maini, 2001; Alldred, 2001), and sepsis (Dhainaut et al., 1995; Cronin et al., 1995; Abraham et al., 1997, 1998). For instance, a chimeric monoclonal anti-TNF antibody, infliximab, has shown efficacy in moderately to severely active Crohn’s disease, and has recently been approved by the U.S. Food and Drug Administration (FDA) (Sandborn and Hanauer, 1999). A recombinant human soluble TNF receptor, Etanercept (Enbrel), has recently been approved by the U.S. FDA for the treatment of rheumatoid arthritis, which has shown a statistically significant reduction in swollen and inflamed joint counts, and a significant improvements in quality of life measures (Schwarz et al., 2000; Taylor, 2001; Feldmann and Maini, 2001; Alldred, 2001). Although anti-TNF therapy has been successfully employed in the treatment of rheumatoid arthritis, a significant survival advantage has not been observed in the treatment of sepsis.

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REFERENCES

Regulation of TNF by the cholinergic anti-inflammatory pathway The cholinergic anti-inflammatory pathway is a neural pathway that rapidly and directly attenuates the production of TNF. In response to endotoxin or ischemia/ reperfusion injury, efferent parasympathetic signals through the vagus nerve increase heart rate, stabilize blood pressure, and attenuate serum and tissue TNF levels (Borovikova et al., 2000a, 2000b; Bernik et al., 2001, 2002). Acetylcholine, the primary neurotransmitter of the vagus nerve, binds alphabungarotoxin-sensitive nicotinic cholinergic receptors expressed on macrophages to inhibit the production of proinflammatory mediators, including TNF and IL-1 (Borovikova et al., 2000a, 2000b). Electrical stimulation of the vagus nerve can recapitulate or augment the innate central response to peripheral inflammation (Borovikova et al., 2000a, 2000b; Bernik et al., 2001, 2002); because vagus nerve stimulators are used clinically for the treatment of depression and epilepsy (Goodnick et al., 2001; Benbadis and Tatum, 2001), vagus nerve stimulation may provide a new approach for the treatment of inflammatory diseases, because it inhibits TNF and other proinflammatory mediators.

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36 TRAIL/Apo2L David H. Lynch1 and Avi Ashkenazi2 1

Amgen Corporation, Seattle, WA, USA Genentech Corporation, South San Francisco, CA, USA

2

Things which matter most should never be at the mercy of things which matter least Johann von Goethe

INTRODUCTION The process of apoptosis is critical to a vast array of physiologic events, including crucial events in fetal development, elimination of cells subsequent to DNA damage to DNA, death of transformed or virally infected somatic cells, and clonal down-sizing and deletion of lymphocytes to name a few. Tight control of this process is, obviously, critical to the survival of the organism. Failure of apoptotic mechanisms has been implicated in both the oncogenic process and certain autoimmune diseases, such as human autoimmune lymphoproliferative disorders (ALPS) (Fisher et al., 1995; Martin et al., 1999). In contrast, inappropriate apoptosis of certain neurons in the central nervous system may well contribute to Parkinson’s disease and Alzheimer’s disease (Nagata and Golstein, 1995; Steller, 1995; Thompson, 1995). Although there are many mechanisms that can ultimately result in cellular apoptosis, some of the best characterized are mediated by members of the tumor The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

necrosis factor (TNF) superfamily and their respective receptors. Of these, induction of apoptosis by TNF, LTa, FasL and TRAIL/Apo2L have received the lion’s share of attention, and are thus the best characterized.

TRAIL/APO2L AND THE TNF SUPERFAMILY The early members of the TNF superfamily (TNFSF) were isolated using expression cloning techniques, while some of the later members have been identified using bioinformatic approaches to screen EST databases. TRAIL/Apo2L was perhaps the first to have been identified via this latter approach and was discovered by screening an EST database for homologies to a consensus amino-acid sequence that was based on the most conserved portion of the b-pleated sheets of TNFSF members (LVVXXXGLYYVYXQVXF), which is found in the B and C regions in Figure 36.1 (Wiley

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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FIGURE 36.1 Alignment of predicted human and mouse TRAIL/Apo2L amino-acid sequences with TNF and LT. The complete predicted amino-acid sequences of human and mouse open reading frames are shown, and alignments with TNF, LTa and LTb are shown where significant homology begins. Solid shading of sequences depicts identity with at least one aligned residue in another sequence. The shaded bar labeled ‘TM’ indicates the predicted transmembrane region, and the solid bars designated ‘A’ to ‘H’ indicate b strands in the TNF crystal structure (modified from Wiley (1995) and used by permission). et al., 1995). Sequence analysis of the subsequently cloned human and mouse cDNAs revealed an open reading frame encoding a type II membrane protein in which the extracellular C-domains showed high homologies to the apoptosis-inducing ligands FasL, TNF and LTa (28%, 23% and 23% identity, respectively). Initial biologic studies using either the fulllength cell-surface form (expressed on fixed CV-1/ EBNA cells) or a recombinant soluble (Flag-tagged) form resulted in the apoptosis of a variety of transformed human tumor cell lines. Its clear homology to TNFSF members and the ability to induce apoptosis led to its being dubbed TRAIL (TNF-related apoptosis-inducing ligand). A similar approach (i.e. screening DNA databases for ESTs showing some homology to FasL and TNF) led to the independent cloning and demonstration of the apoptosis-inducing activity of this same molecule, which was dubbed Apo2-ligand (Apo2L) (Pitti et al., 1996). This ligand is also known as TNFSF-10 (http:// www.gene.ucl.ac.uk/nomenclature/genefamily/tnfto p.html). The cDNAs for human TRAIL/Apo2L predict a 32.5-kDa polypeptide of 281 amino acids (Wiley et al., 1995; Pitti et al., 1996), while the predicted polypeptide for the murine homolog (which is 65% identical) is 291 amino acids in length (Wiley et al., 1995). As noted earlier, the topology of this ligand is a type II membrane protein in that there is no signal sequence

and a hydrophobic transmembrane region (determined using various TM prediction algorithms) between residues 15–18 and 39 (Wiley et al., 1995; Pitti et al., 1996). Potential N-linked glycosylation sites were noted at positions 52 and 109. Interestingly, although TRAIL/Apo2L is highly homologous to FasL, TNF and LTa, its expression pattern is substantially different. Whereas mRNAs for FasL and TNF are rapidly induced upon activation, they are not constitutively expressed. In contrast, mRNA for TRAIL/Apo2L appears to be constitutively expressed on a wide variety of cells and tissues (Table 36.1). TABLE 36.1 Tissue expression pattern of mRNA for TRAIL/Apo2La Positive

Negative

Spleen Thymus Peripheral blood Prostate Ovary Small Intestine Colon Lung Kidney Placenta Pancreas Skeletal muscle Heart

Brain Liver Testis

a

Determined by Northern blot.

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PHYSIOLOGIC FUNCTIONS OF TRAIL / APO 2 L

MULTIPLE RECEPTORS FOR TRAIL/APO2L The receptor system that binds TRAIL/Apo2L is the most diverse of the TNF-receptor superfamily (TNFRSF), with at least four distinct, but closely related, receptors (TRAIL receptor-R1 (also known as DR4 (Pan et al., 1997a)); -R2 (Walczak et al., 1997; Schneider et al., 1997a) (also known as DR5 (Pan et al., 1997b; Sheridan et al., 1997; Chaudhary et al., 1997; MacFarlane et al., 1997), TRICK2 (Screaton et al., 1997) and KILLER (Wu et al., 1997)); -R3 (Schneider et al., 1997a; MacFarlane et al., 1997; Degli-Esposti et al., 1997a) (also known as TRID (Pan et al., 1997b), DcR1 (Sheridan et al., 1997) and LIT (Mongkolsapaya et al., 1998); and -R4 (Degli-Esposti et al., 1997a) (also known as DcR2 (Marsters et al., 1997) and TRUNDD (Pan et al., 1998)) that bind TRAIL/Apo2L at high affinity (Degli-Esposti et al., 1997a; 1997b). Using the TNFRSF nomenclature these receptors are designated as TNFRSF10A, -B, -C and -D, respectively (http:// www.gene.ucl.ac.uk/nomenclature/genefamily/tnftop. html). Osteoprotegerin (OPG) has also been reported to bind TRAIL/Apo2L, although with a lower affinity than the other receptors for TRAIL/Apo2L, especially at physiologic temperatures at which the affinity for is approximately 200 times lower than that of TRAIL-R2 (Truneh et al., 2000). OPG is produced solely as a secreted form (i.e. there is no cell-surface intermediate) and is found in serum of healthy normal adults at concentrations of approximately 1 ng ml1. However, OPG has been shown to be capable of acting as an inhibitor of TRAIL/Apo2L-induced apoptosis of Jurkat target cells in vitro (Emery et al., 1998) and has been associated with the resistance of some prostate cancer cell lines to TRAIL/Apo2L, also in vitro (Holen et al., 2002). The receptors for TRAIL/Apo2L are all expressed in a transmembrane cell-surface form and (with the exception of TRAIL-R3) are constitutively expressed in a wide variety of cells and tissues. Both TRAIL-R1 and -R2 contain so-called ‘death domains’ (DD) in the intracellular portions of the molecules, and ligation of either of these receptors is capable of inducing apoptosis. TRAIL-R3 is expressed as a GPI-linked cellsurface protein with no known signaling properties. TRAIL-R4 contains only a partial DD in the intracellular region of the molecule, and ligation of this recep-

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tor does not lead to apoptosis. Interestingly, the genes encoding these four receptors are all highly homologous (ranging from 54 to 70% identical). The genes encoding these receptors are also very tightly linked and map to human chromosome 8p21–23, suggesting that they arose by gene duplication in the recent evolutionary past.

PHYSIOLOGIC FUNCTIONS OF TRAIL/APO2L The normal physiologic function of TRAIL/Apo2L in vivo is, at present, only beginning to be elucidated. Chronic treatment of mice with a soluble form of one of the human receptors (sDR5/TRAIL-R2) for TRAIL/Apo2L has been reported to ameliorate both experimental autoimmune encephalomyelitis and rheumatoid arthritis (Song et al., 2000; Hilliard et al., 2001). Constitutively expressed TRAIL/Apo2L has also been identified on a subset of liver NK cells, and NK cell-mediated effects on reducing hepatic metastases of the RENCA renal cell carcinoma appears to be at least partially dependent upon TRAIL/Apo2L (Smyth et al., 2001). Additional blocking studies using neutralizing antibodies implicate TRAIL/Apo2L in IL-12 and interferon-c dependent antitumor function of NK and NKT cells (Smyth et al., Takeda et al., 2002). Indeed, activated NK cells (Johnsen et al., 1999; Kayagaki et al., 1999a), monocytes (Griffith et al., 1999), and CD4 or CD8 T cells (Thomas and Hersey, 1998; Jeremias et al., 1998; Mariani and Krammer, 1998a; Martinez-Lorenzo et al., 1998; Kayagaki et al., 1999b) express TRAIL/Apo2L and employ this ligand to trigger apoptosis in tumor target cells. TRAIL/ Apo2L may also be an important mediator of the apoptosis-inducing activity of type I interferons on renal cell carcinomas (Kayagaki et al., 1999c) and multiple myeloma cells (Chen et al., 2001), and of retinoids on leukemia cells (Altucci et al., 2001). Evolutionarily, the TRAIL/Apo2L pathway may have arisen as part of the immune defense against some as yet unspecified pathogen(s), as suggested by the presence of a transcriptional regulatory element in the TRAIL/Apo2L gene that is responsive to interferons (Gong and Almasan, 2000). Infection of cells with reovirus (Clarke et al., 2000) or herpesvirus (Secchiero et al., 2001) up-regulates TRAIL/Apo2L expression,

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and NK cells can use TRAIL/Apo2L, induced through type I interferons, to kill virus-infected cells (Sato et al., 2001). TRAIL/ mice have also been produced. Although these mice do not show any dramatic changes in phenotype, recent results indicate that TRAIL/Apo2L may play a key role in suppressing tumor induction (by mutagens) and metastasis (Cretney et al., 2002).

Recombination constructs for soluble TRAIL/Apo2L 1 18 38 TM Native Molecule N

84 TNF Homology region 95

Flag-tagged

CMV Flag

LZ-fusion

CMV Leucine zipper

95

114

FUNCTIONAL FORMS OF TRAIL/APO2L

poly-his tagged

CMV

281 C 281

281

281

poly-his

114

281

non-tagged

Lymphocytes express Apo2L/TRAIL as a transmembrane protein, and release the ligand in either a microvesicle-associated form (Monleon et al., 2001) or in a soluble form that is generated through enzymatic shedding of the protein’s extracellular, C-terminal portion (Mariani and Krammer, 1998b). Early studies into the structure of the extracellular regions of TNFRSF members identified characteristic pseudorepeat domains stabilized by multiple disulfide bridges (Banner et al., 1993). The structure of the complementary TNFSF ligands has been characterized as a ‘jelly roll’ structure formed by antiparallel b-pleated sheets, which in some cases is stabilized by a single disulfide bridge (Eck and Sprang, 1989; Karpusas et al., 1995). However, the structure of TRAIL/Apo2L appears to diverge from this model in that it contains an unpaired cysteine residue at a position 230 (Cys230), a position at which other ligands have a disulfide bridge. Crystallographic analyses of TRAIL/Apo2L, either alone or complexed with one of its cognate receptors (TRAIL-R2) indicate that Cys230 is not involved in creating a disulfide bridge. Instead, this amino acid appears to coordinate interaction with a zinc atom that stabilizes formation of TRAIL/Apo2L into its biologically active trimer configuration (Plate 36.2) (see plate section) (Hymowitz et al., 1999; Cha et al., 1999; Mongkolsapaya et al., 1999). The critical role of Cys230 in forming this zincchelate by site-directed mutational analysis has been recently demonstrated (Bodmer et al., 2000a). Various recombinant versions of human Apo2L/ TRAIL have also been produced (Figure 36.3). Initial versions contained either TRAIL/Apo2L amino acids 114–281 fused to an amino-terminal polyhistidine tag (Pitti et al., 1996) or amino acids 95–281 fused to an

FIGURE 36.3 Schematic representation of various recombinant protein constructs of human TRAIL/ Apo2L. amino-terminal ‘Flag’ epitope tag (Wiley et al., 1995); crosslinking of the Flag-tagged protein with anti-Flag antibodies enhances its activity against certain cell lines such as the Jurkat T leukemia (Wiley et al., 1995; Bodmer et al., 2000a). A third variant contains amino acids 95–281 fused amino terminally to a modified yeast Gal-4 leucine zipper (LZ) that promotes spontaneous oligomerization of the ligand into the biologically active trimer form (Walczak et al., 1999). A fourth recombinant version of the ligand that contains amino acids 114–281 of human Apo2L/TRAIL without any added exogenous sequences is also being evaluated (Ashkenazi et al., 1999). Owing to the lack of exogenous sequences this latter version is the least likely to be immunogenic in human patients. Production in bacteria of non-tagged recombinant TRAIL/Apo2L as a stable, soluble homotrimer has been optimized by the addition of zinc and reducing agents to the cell culture media and extraction buffers, and by formulation of the purified protein at neutral pH (Kelley et al., 2001; Lawrence et al., 2001). Recombinant soluble TRAIL/Apo2L induces apoptosis in cell lines from a broad spectrum of human cancers, including colon, lung, breast, prostate, pancreas, kidney, central nervous system and thyroid, as well as leukemia and multiple myeloma, suggesting that this ligand may be useful for treatment of many cancers (Griffith and Lynch, 1998; Rieger et al., 1998; Walczak et al., 1999; Ashkenazi et al., 1999; Gazitt, 1999; Keane et al., 1999; Mizutani et al., 1999; Yu et al.,

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REGULATION OF TRAIL / APO 2 L - MEDIATED APOPTOSIS

2000; Mitsiades et al., 2001). In athymic or SCID mice bearing human tumor xenografts derived from breast carcinoma (Walczak et al., 1999), colon carcinoma (Ashkenazi and Dixit, 1999; Kelley et al., 2001), multiple myeloma (Mitsiades et al., 2001), or glioma (Roth et al., 1999; Pollack et al., 2001), administration of recombinant soluble TRAIL/Apo2L exerts marked antitumor activity without systemic toxicity. Furthermore, combinations of TRAIL/Apo2L and certain DNA damaging drugs (Ashkenazi et al., 1999; Gliniak and Le, 1999) or radiotherapy (Chinnaiyan et al., 2000) have synergistic antitumor activity in several xenograft mouse models. TRAIL/Apo2L exhibits efficacy not only in cell linebased tumor models but also in models involving tumor cells taken directly from patients before transplantation into SCID mice (Naka et al., 2002). Most normal human cell types tested to date, including epithelial, endothelial, fibroblastic, and smooth muscle cells, are resistant to the non-tagged recombinant version of TRAIL/Apo2L (Ashkenazi et al., 1999; Lawrence et al., 2001). Some normal cell types, namely astrocytes (Walczak et al., 1999; Ashkenazi et al., 1999), hepatocytes (Jo et al., 2000; Lawrence et al., 2001), and keratinocytes (Qin et al., 2001), are resistant to the non-tagged, zinc-bound recombinant TRAIL/Apo2L and have shown sensitivity to apoptosis induction by the tagged and antibody-crosslinked recombinant variants. One potential explanation for this difference is that commitment of these normal cells to apoptosis may require high-order multimerization of TRAIL-R1 and TRAIL-R2. The tagged versions of the ligand, which are not optimized for zinc content, have a low solubility and tend to aggregate and/or precipitate at high concentrations, as does the antibody-crosslinked ligand (unpublished results). Therefore, some of these ligand preparations may over-multimerize death receptors, leading to a signal that surpasses the high threshold for apoptosis activation in normal cells. In contrast, the nontagged, zinc-bound TRAIL/Apo2L is highly stable and soluble as a trimer. This form of the ligand probably forms only trimeric death receptor complexes, which are not sufficient for triggering apoptosis in normal cells. This hypothesis is supported by the observation that polyhistidine-tagged TRAIL/Apo2L binds irreversibly to cultured hepatocytes, whereas non-tagged, zinc-bound TRAIL /Apo2L shows reversible binding

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(Jo et al., 2000; Lawrence et al., 2001). In tumor cells, however, the threshold for apoptosis induction is usually lower than in normal cells (Evan and Littlewood, 1998). Ligation of TRAIL-R1 and/or TRAIL-R2 with the non-tagged, zinc-bound trimeric TRAIL/Apo2L potently triggers apoptosis in numerous cancer cell lines (Ashkenazi et al., 1999; Lawrence et al., 2001), supporting the clinical potential of this form of the recombinant ligand. Given that certain versions of TRAIL/Apo2L can induce hepatocyte apoptosis (Jo et al., 2000; Lawrence et al., 2001), the preclinical assessment of the ligand as well as of other agents that target TRAIL-R1 or TRAIL-R2 must be carried out with the utmost diligence. Initial studies in non-human primates (i.e. cynomolgus monkeys and chimpanzees) showed that short-term intravenous administration of non-tagged, zinc-bound Apo2L/TRAIL is well tolerated even at high doses (Kelley et al., 2001; Lawrence et al., 2001). Further animal safety studies are needed to determine the impact of long-term administration of Apo2L/TRAIL as a single agent and in the combination with chemotherapy or radiotherapy.

REGULATION OF TRAIL/APO2L-MEDIATED APOPTOSIS Regulation of TRAIL/Apo2L-mediated apoptosis appears to be extremely complex and may be exerted at multiple levels in two related, but distinguishable, biochemical signaling cascades (Suliman et al., 2001) (see Plate 36.4). The initial intracellular events appear to be the formation of a death-inducing signaling complex (DISC) comprising the intracellular death domains (DD) of TRAIL-R1 or -R2 with FADD and cleavage of the pro-forms of both caspase-8 and -10 (Bodmer et al., 2000b; Sprick et al., 2000; Kischkel et al., 2000, 2001). High endogenous levels of FLIP may inhibit the critical biochemical events proximal to binding of TRAIL/Apo2L (or other TRAIL receptor agonists) to TRAIL-R1 or -R2 that result in apoptosis (i.e. cleavage of procaspase 8 (Irmler et al., 1997; Griffith et al., 1998; Algeciras-Schimnich et al., 1999) and very likely procaspase 10). The activated forms of caspase-8 and -10 can cleave

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pro-caspase-3 directly via the so-called ‘intrinsic’ pathway (Fernandes-Alnemri et al., 1996; Ozoren et al., 2000). Caspase-8 can also induce apoptosis via a second (‘extrinsic’) pathway through cleavage of Bid which, in association with Bax and/or Bak, leads to cytochrome c release from mitochondria, activation of procaspase-9 (via association with Apaf-1 and ATP) and ultimately cleavage of procaspase-3 to its active isoform. Studies have demonstrated the existence of a variety of interacting proteins that can either act to inhibit (Bcl-2, Bcl-XL, mutated Bax, XIAP, cIAP-1, cIAP2, NFjB) or enhance (Smac/DIABLO) the induction of apoptosis (Schneider et al., 1997b; Hinz et al., 2000; Munshi et al., 2001; Wei et al., 2001; Harper et al., 2001; Trauzold et al., 2001; Zhang et al., 2001; LeBlanc et al., 2002; Deng et al., 2002). Evaluation of expression levels of a panel of pro- and antiapoptotic factors in a panel of cell lines with varying levels of sensitivity to TRAIL/Apo2L indicates that no single apoptosis inhibitor is likely to be responsible for sensitivity or resistance of cells to this ligand (Mitsiades et al., 2002). Rather, the ultimate result is liable to be the ‘sum of the parts.’

POTENTIAL THERAPEUTIC USES FOR TRAIL/APO2L One of the interesting attributes of TRAIL/Apo2L is that it induced apoptosis in at least two-thirds of the tumor cell lines tested (Griffith and Lynch, 1998). In contrast, normal cells are resistant to the effect of TRAIL/Apo2L. This has been demonstrated to be the case both in vitro and in vivo, including non-human primates (Wiley et al., 1995; Pitti et al., 1996; Walczak et al., 1999; Ashkenazi et al., 1999). It was the differential in sensitivity of tumor cells and normal cells to the effects of TRAIL/Apo2L that led to studies which demonstrated that treatment of SCID mice bearing human tumor xenografts with TRAIL/Apo2L, whether as a soluble protein or by an adenoviral delivery vector, can lead to complete tumor remission in the absence of deleterious side-effects to the host (Walczak et al., 1999; Ashkenazi et al., 1999; Roth et al., 1999; Nagane et al., 2000; Kelley et al., 2001; Pollack et al., 2001; Griffith and Broghammer, 2001; Kagawa et al., 2001). In addition to demonstrating therapeutic activity against tumors as a single agent, the activity of

TRAIL/Apo2L in combination with other therapies (such as chemotherapy) is also enhanced in vivo (Ashkenazi et al., 1999; Gliniak and Le, 1999; Mitsiades et al., 2001). The therapeutic potential for TRAIL/Apo2L may not be limited to cancer. Data are beginning to emerge which indicate that TRAIL/Apo2L may also have a significant role in certain viral diseases. Infection of human foreskin fibroblasts or colonic epithelial cells by cytomegalovirus (CMV) induces sensitivity of these cells to apoptosis induced by TRAIL/Apo2L in vitro (Sedger et al., 1999; Strater et al., 2002). A similar result can be induced by IFNc and is associated with increased expression of TRAIL-R1 and -R2 and a decreased expression of NFjB. Reovirus infection has also been shown to induce increased susceptibility of various cell lines to TRAIL/Apo2L-induced apoptosis, and is associated with increased activity of caspase-8 and in some cases TRAIL/Apo2L itself (Clarke et al., 2001; Jelachich and Lipton, 2001). IFNa/b, a cytokine produced by virally infected cells, increases expression of TRAIL/Apo2L in NK cells via activation of an upstream IFN-stimulated response element (ISRE) (Sato et al., 2001). In addition, recent studies have demonstrated a therapeutic potential for TRAIL/ Apo2L in viral diseases, as in vitro treatment of PBL from HIV-infected donors resulted in the death of cells that produced viral RNA and p24 antigen (Lum et al., 2001).

CONCLUSION Among the apoptosis-inducing ligands of the TNFSF TRAIL/Apo2L is relatively unique. The receptor system for this ligand is clearly the most diverse of the members of the TNFRSF, and the observation that a high proportion of either transformed or virally infected cells, but not their normal counterparts, are sensitive to induction of apoptosis by TRAIL/Apo2L suggests a number of potential therapeutic uses. Understanding the regulation of signaling, the possibility for differential use of the extrinsic and intrinsic pathways in different cell types and the impact of ancillary regulatory signals (both pro- and antiapoptotic) and how these may be manipulated will clearly be critical to the continued development of this molecule for therapeutic use.

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37 RANKL (Receptor activator of NFjB ligand) Simon Blake and Ian James GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA

‘What’s the use of their having names’, the Gnat said, ‘If they won’t answer to them?’ ‘No use to them’, said Alice, ‘but it’s useful to the people that name them, I suppose. If not, why do things have names at all?’ Lewis Carroll

INTRODUCTION RANKL (receptor activator of NFjB ligand) is a member of the tumor necrosis factor (TNF) cytokine family, which to date includes 4-1BBL, APRIL, CD40L, CD30L, CD27L, FasL, LIGHT, LT-a, LT-b, OX40L, TNFa, TRAIL, and TWEAK (reviewed in Wong et al., 1999 and Kwon et al., 1999). RANKL was first described in 1997 (Anderson et al., 1997; Wong et al., 1999), initially as a dendritic cell (DC) survival factor and an important regulator of the interaction between T lymphocytes and dendritic cells (Wong et al., 1997). Subsequently, it was identified as being identical to the factor responsible for the formation of osteoclasts from osteoclast progenitors (Lacey et al., 1998; Yasuda et al., 1998) (see Plate 37.1). This diversity of function and its simultaneous discovery by a number of researchers has led to it being known by a variety of names. These include TRANCE (tumour necrosis factorrelated activation-induced cytokine) (Wong et al., 1998), ODF (osteoclast differentiation factor) (Yasuda The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

et al., 1998) and OPGL (osteoprotegerin ligand) (Lacey et al., 1998). RANKL can exist and exert its biologic activity in either a membrane-bound or soluble form (Lum et al., 1999; Ikeda et al., 2001). The biologic activities of RANKL are exerted through its specific receptor, RANK (Anderson et al., 1997; Wong et al., 1997; Lacey et al., 1998), which like its ligand shows a wide tissue distribution pattern. It is the interaction of RANKL with its receptor that has been demonstrated to be essential for both the differentiation and survival of resorbing osteoclasts from myeloid precursors and as a survival factor for mature DC (Wong et al., 1997; Lacey et al., 1998). Regulation of the activity of the interaction between receptor and ligand has been shown to occur through a secreted decoy receptor, OPG (osteoprotegerin) (Lacey et al., 1998; Tomoyasu et al., 1998; Yasuda et al., 1998). As with RANKL both the RANK receptor and OPG have been described in the literature using a variety of names (see Table 37.1 and individual sections for full description). For the purposes of this review the nomenclature used will be Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

TABLE 37.1 Summary of the members of the RANKL/RANK/OPG system, showing, alternative names, their MIM number designation, chromosome location and biologic activity Preferred designationa

Alternative names

MIM number designationb

Chromosome location

Biologic activity

RANKL (receptor activator of NFjB ligand)

TNFSF11 Osteoclast differentiation factor (ODF) Osteoprotegerin ligand (OPGL) TNF-related activation-induced cytokine (TRANCE)

602642

13q14

Exists in both membrane-bound and soluble forms Osteoclast formation, fusion, activation and survival Dendritic cell (DC)-survival factor Regulator of T-cell and DC interactions Lymph node organogenesis, B-cell maturation Mammary gland development Arterial calcification

RANK (receptor activator of NFjB)

TNFRSF11A Osteoclast differentiation factor receptor (ODFR) Osteoclast differentiation and activation receptor (ODAR)

603499

18q22.1

Specific receptor for RANKL through which the cytokine exerts its activity

OPG (osteoprotegerin)

TNFRSF11B Osteoclastogenesis inhibitory factor (OCIF) TNF-receptor-related molecule 1 (TR1) Follicular dendritic cell receptor 1 (FDCR-1)

602643

8q24

Soluble decoy receptor for RANKL Regulates the activity of RANKL by preventing its binding to RANK

a

Proposed by the American Society for Bone and Mineral Research President’s Committee on Nomenclature. where possible, the human genes referred to in this chapter can be found in the Mendelian Inheritance in Man (MIM, McKusick, 1998) catalog using the sixdigit entry number.

b

RANK - L

that proposed by the American Society for Bone and Mineral Research (The American Society for Bone and Mineral Research President’s Committee on Nomenclature, 2000). Despite RANKL being initially described as a regulator of the immune system, the majority of studies on its interaction with RANK and its regulation by OPG has focused upon its role in bone homeostasis which is reflected in this review.

RANK-L Gene structure of mouse RANKL Kodaira et al. (1999) isolated mouse RANKL genomic clones and sequenced them to determine their gene structure. They demonstrated mouse RANKL to be a single copy gene consisting of five exons which span approximately 40 kb of the mouse genome. They showed that the first exon encodes the intracellular and transmembrane domains, and that the extracellular region, containing the TNF homologous domain is encoded by exons 1 to 5. The translationtermination codon and six polyadenylation signal residues are present in exon 5. A major transcriptioninitiation site is present 143 nucleotide residues upstream of the initiation-ATG codon.

Chromosome location By analysis of somatic cell and radiation hybrids, Anderson et al. (1997) mapped the human RANKL gene to 13q14. Using an interspecific backcross, Wong and co-workers (1997) mapped the mouse RANKL gene to chromosome 14.

RANKL structure It has been shown that the TNF family of cytokines share a common structural scaffold and that individual receptor–ligand pairs associate with high specificity. Since there is a low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To address this, and to understand the elements of RANKL that mediate RANK activation, Lam et al.

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(2001) crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6Å. This group has shown that RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family members. Mutagenesis studies have shown that these loops modulate RANK activation, as evidenced by in vitro osteoclastogenesis studies (Lam et al., 2001). In these single amino acid substitutions in the DE loop of the molecule substantially reduced their ability to support the formation of morphologically normal osteoclasts. Xu et al. (2000) showed that the predicted amino acid sequence of rat RANKL (rRANKL) has 84% and 96% identity to human and mouse RANKL, respectively (Figure 37.2). RANKL transcripts were expressed abundantly in the thymus and bone tissues of ovariectomized rats. rRANKL has a single hydrophobic region between residues 53 and 69, which probably constitutes the transmembrane domain. The long C-terminal region containing b sheetforming sequences of the TNF-like core is considered the extracellular region.

Biological activity and expression of RANKL in normal tissues High levels of RANKL mRNA and protein expression have been demonstrated in T cells and lymphoid tissues (Anderson et al., 1997; Yasuda et al., 1998, 1998). Mature bone marrow and splenic DCs express high levels of RANK on their surfaces suggesting a central role for RANKL in the regulation of DC biology (Wong et al., 1997). The primary effect of RANKL is to increase the survival of mature DC, perhaps through up-regulation of Bcl-xL, a well-described apoptotic suppressor. Increased DC survival can in turn lead to enhanced T-cell proliferative responses by prolonging the stimulatory presentation of antigen/MHC complexes and co-stimulatory molecules such as B7-1 and B7-2. Stimulation of DC by RANKL is also known to induce transcription of several cytokine genes such as IL-12, IL-15, IL-1 and IL-6 (Wong et al., 1999; Josien et al., 2000). These cytokines regulate the intensity and type of immune response. In a CD40L knockout background, residual viral resistance is mediated by the RANKL–RANK pathway (Bachmann et al., 1999). Also, RANKL and RANK knockout mice are deficient in lymph node organogenesis and show some defects

THE CYTOKINES AND CHEMOKINES

Human RANKL Mouse RANKL Rat RANKLA Consensus

1 1 1

Human RANKL Mouse RANKL Rat RANKL concensus

80 81 81

Human RANKL Mouse RANKL Rat RANKL concensus

160 159 161

LEAQPFAHLTINATDIPSGSHKVSLSSWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLM PEAQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLYANICFRHHETSGSVPTDYLQLM PEAQPFAHLTINAADIPSGSHKVSLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLYANICFRHHETSGSVPADYLQLM .************..********.*****************.*****.**********************.....*****

Human RANKL Mouse RANKL Rat RANKL concensus

240 239 241

VYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID VYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISIQVSNPSLLDPDQDATYFGAFKVQDID VYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISVQVSNPSLLDPDQDATYFGAFKVQDID ***.**********.********.*********************.*****..*********************.***

MRRASRDYTKYLRGSEEMGGGPGAPHEGPLH-APPPPAPHQPPAASRSMFVALLGLGLGQVVCSVALFFYFRAQMDPNRI MRRASRDYGKYLRSSEEMGSGPGVPHEGPLHPAPSAPAPAPPPAASRSMFLALLGLGLGQVVCSIALFLYFRAQMDPNRI MRRANRDYGKYLRGSEEMGSCPGVPHEGPLHPAPSAPAPAPPPAASRFMFLALLGLGLGQVVCSIALFLYFRAQMDPNRI ****.***.****.*****..**.*******.**..***..******.**.*************.***.*********** SEDGTHCIYRILRLHENADFQDTTLESODTKLIPDSCRRGKQAFQGAVQKELQHIVGSQHIRAEKAMVDGSWLDLAKRSK SEDSTHCFYRILRLHENAGLQDSTLESEDT--LPDSCRRMKQAFQGAVQKELQHIVGPQRFSGAPAMMEGSWLDVAQRGK SEDSTRCFYRILRLSENTGLQDSTLESEDTEALPDSCRRMKQAFQGAVQPELQHIVGPQRFSGVPAMMEGSWLDVAPRGK ***.*.*.******.**...**.****.** .******.*********.*******.*.... .**..*****.*.*.* ▲

FIGURE 37.2 Alignment of the predicted amino-acid sequences of rat RANKL with those of the human and mouse RANKL. The predicted transmembrane regions are underlined. The metalloprotease cleavage site responsible for generation of soluble RANKL is marked by an arrow head. Identical amino acids are marked with a black block and asteroids on the consensus lines. Similar amino acids are indicated in grey color and dots on consensus lines. Reproduced from Xu et al. (2000) with permission from the American Society for Bone and Mineral Research.

THE RANK RECEPTOR

in early B- and T-cell development (Dougall et al., 1999; Kong et al., 1999; Li et al., 2000). RANK receptor and OPG have also been shown to be expressed in B lymphocytes (Anderson et al., 1997; Yun et al., 1998; Bachmann et al., 1999). More recently, in a study using knockout mice devoid of the soluble decoy receptor for RANKL, OPG, both B-cell maturation and the ability to mount efficient antibody responses was shown to be compromised (Yun et al., 2001) Thus, RANKL may play an important role in the development of the immune system and in the modulation of the quality and intensity of the immune response. The role of RANKL/RANK in the immune system has been somewhat overshadowed by its essential role in bone homeostasis. However, as will become clear later, there is now a well-described connection between the expression of RANKL on T cells and its role in bone turnover (Kong et al., 1999) (see section on rheumatoid arthritis).

The role of RANKL/RANK in osteoclastogenesis Osteoclasts are the cells which resorb bone. They are formed within bone marrow through the commitment, differentiation and fusion of mononuclear osteoclast precursors. This process along with the regulation of their functional activity including their termination via apoptotis has been the subject of intense study in the field of bone biology. Until the early 1990s no single cytokine or hormone had been identified which could support osteoclast formation and activity. It became increasingly clear that the formation and regulation of these cells occurred through the expression or production of one or more factors from cells of the osteoblastic lineage (for review see Suda et al., 1999). One major factor produced by osteoblasts/bone marrow cells which supports osteoclast formation is macrophage colony-stimulating factor (M-CSF) (Wiktor-Jedrzejczak et al., 1990; Yoshida et al., 1990). However, M-CSF is neither sufficient nor indeed essential for osteoclast formation or function in vivo. RANKL is now widely accepted as the essential differentiation factor for the development and activation of osteoclasts (see Plate 37.1) (see plate section). RANKL is expressed by osteoblasts and bone marrow stromal cells and has been shown to promote, in the presence of permissive amounts of M-CSF, the

875

differentiation of osteoclasts (Lacey et al., 1998; Matsuzaki et al., 1998; Quinn et al., 1998; Yasuda et al., 1998; Jimi et al., 1999). It has also been shown to induce osteoclast activation (Fuller et al., 1998; Burgess et al., 1999; Jimi et al., 1999) and attachment to the bone surface (O’Brien et al., 2000) and to promote their longevity (Lacey et al., 2000). Expression of RANKL by osteoblastic/bone marrow stromal cells can be up-regulated by several bone resorptive agents including PTH, 1,25(OH)2 vitamin D3, prostaglandin E2, interleukin-1 and tumor necrosis factor (Anderson et al., 1997; Lacey et al., 1998; Yasuda et al., 1998). Analysis of the RANKL gene promoter structure has revealed response elements for vitamin D and glucocorticoids (Kitazawa et al., 1999; Kodaira et al., 1999) as well as a binding site for the osteoblast-specific transcription factor Cbfa1 (Gao et al., 1998). Targeted deletion of either RANKL or its receptor RANK have served to illustrate dramatically the role of this cytokine/receptor pairing in the development and maintenance of the skeleton. Mice from both of these knockout lines were severely osteopetrotic (increased bone mass) and had impaired tooth eruption due to a complete absence of mature osteoclasts (Dougall et al., 1999; Kong et al., 1999; Li et al., 2000). The most complete study of the expression of RANKL, to date, has been performed by Kartsogiannis and colleagues (1999). In their study they investigated the expression at both the protein and mRNA level in various mouse, rabbit and human tissues. During neonatal and fetal mouse development, mRNA for RANKL was detected in the prehypertrophic and hypertrophic chondrocytes. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar distribution pattern for the protein in these tissues. In an intramembranous bone formation model in the rabbit, mRNA was detected in the mesenchymal cells and actively synthesizing osteoblasts but not in the flattened lining osteoblasts or late stage osteocytes. Variable expression of RANKL was noted in osteoclasts, with those within resorbing lacunae showing the highest expression of mRNA and protein. Studies on osteoclasts from human giant cell tumor of bone also indicated variable levels of RANKL

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876

RANKL ( RECEPTOR ACTIVATOR OF NF j B LIGAND )

expression (Atkins et al., 2000). More recently, the presence of RANKL mRNA has been demonstrated in normal human cancellous bone samples from autopsy specimens. Further analysis of these samples using histomorphometric techniques demonstrated a strong association between the ratio of RANKL to its naturally occurring decoy receptor OPG and bone turnover (Fazzalari et al., 2001). Given the essential role of RANKL in the differentiation and survival of the osteoclast and therefore its role in skeletal development and bone homeostasis, the expression patterns demonstrated for this cytokine in skeletal tissues were not surprising. It is of note, however, that RANKL expression has also been observed in several extraskeletal tissues in the developing mouse. These include brain, heart, kidney, skeletal muscle and skin. Embryonic mouse tissue also demonstrated expression in intestine, liver and lung which was absent in the adult. It has been postulated that RANKL may influence cell survival in a variety of cell types, although this has yet to be fully elucidated. RANKL has also been shown to be an essential regulator of mammary gland development. Mice lacking either RANKL or its receptor RANK failed to form lobulo–alveolar mammary structures during pregnancy (Fata et al., 2000), leading to a failure to form lactating breast tissue. Mobilization of calcium to the milk was also prevented in these mice due to a lack of bone-resorbing osteoclasts.

clasts. In addition, transcripts have been found on T and B lymphocytes, DCs and fibroblasts (Anderson et al., 1997; Hsu et al., 1999).

THE RANK RECEPTOR

The role of RANK and RANKL as important regulators of the interactions between T cells and DCs was demonstrated by the experiments of Anderson et al. (1997). This group demonstrated that NFjB/DNA complexes could be induced in RANK-positive T lymphocytes following incubation with RANK ligand, indicating RANK-specific activation of NFjB in human T cells. Furthermore, it was shown that RANK ligand augmented the ability of DCs to stimulate naive T-cell proliferation in a mixed lymphocyte reaction and increased the survival of RANK-positive T cells generated with IL4 and TGFb. Like other members of the TNFR superfamily, RANK contains four extracellular cysteine-rich pseudorepeats. The mouse and human RANK proteins are 70% identical. Northern blot analysis indicated that the 4.5-kb human RANK mRNA is expressed ubiquitously. In some human cell lines,

Anderson et al. (1997) identified cDNAs from DCs that encoded a protein with homology to the extracellular domain of tumor necrosis factor receptor superfamily members that they designated RANK (receptor activator of NFjB). They describe human RANK as a type I transmembrane protein of 616 amino acids that consists of a 28-amino-acid signal peptide, an N-terminal extracellular domain, a 21-amino-acid transmembrane domain and a large cytoplasmic C-terminal domain.

Tissue distribution The RANK receptor is expressed on cells of the monocyte/macrophage lineage which include preosteo-

RANK in bone The importance of RANK in bone homeostasis was realized because of a number of important experiments. Hsu et al. (1999) demonstrated that RANK mRNA was expressed by both immature and mature cells of the osteoclast lineage and that a severe osteopetrosis developed in transgenic mice expressing a soluble RANK–Fc fusion protein, a phenotype similar to that observed in the OPG transgenic mice. It was also shown that RANK knockout mice developed a severe osteopetrosis due to a lack of osteoclasts (Li et al., 2000) and that osteoclastogenesis could be recovered in these mice by the transfer of the RANK cDNA into hemopoietic precursors. Finally, Nakagawa et al. (1998) identified RANK as ODFR, the membrane-bound ODF receptor on osteoclast progenitors. In the presence of M-CSF, a polyclonal antibody against the extracellular domain of RANK induced osteoclastogenesis in spleen cells. Taken together, these data confirm that RANK is the signaling receptor essential for RANK ligand-mediated osteoclastogenesis.

RANK in T-lymphocyte and dendritic cell function

THE CYTOKINES AND CHEMOKINES

DISORDERS ASSOCIATED WITH THE RANKL / RANK / OPG SYSTEM

the authors noted additional transcripts were detected which were derived from the use of alternative polyadenylation signals in the RANK gene.

OSTEOPROTEGERIN (OPG) Regulation of RANKL activity Regulation of the interaction between RANKL and its receptor RANK is achieved at the molecular level by the decoy receptor, OPG. This molecule was identified in 1997 by three independent groups and as a result is also known as osteoclast inhibitory factor (Simonet et al., 1997; Tan et al., 1997; Tsuda et al., 1997). It is a 401-amino-acid propeptide which contains a short signal sequence of 21 amino acids and forms homodimers linked by a cysteine–cysteine disulfide bond at position 400. It acts by binding to both the soluble and cell-associated RANKL preventing its interaction with the receptor RANK. It has also been shown to be capable of binding weakly to the apoptosis-inducing TNF ligand family member, TRAIL (Emery et al., 1998). In vitro OPG has been demonstrated to inhibit osteoclastogenesis induced by various stimuli including 1,25(OH)2D3, parathyroid hormone, prostaglandin E2 and interleukin-1 in a co-culture of hematopoietic and stromal cells (Simonet et al., 1997). In addition, Tan et al. (1997) demonstrated that this molecule was capable of inhibiting resorption pit formation in vitro and inhibition of bone resorption in ex vivo cultures of fetal rat long bones. In vivo studies have further demonstrated the role of OPG in bone turnover. Transgenic mice overexpressing OPG in the liver resulted in high circulating levels of this glycoprotein and the development of a marked increase in bone density (osteopetrosis) of both femura and vertebrae. This was as a result of a significantly reduced osteoclast number (Simonet et al., 1997). Mice in which OPG has been inactivated by homologous recombination develop severe early onset osteoporosis, a significant increase in osteoclast number, increased cortical and trabecular bone porosity, decreased bone strength and an increase in fracture incidence (Bucay et al., 1998; Mizuno et al., 1998). These mice die at approximately 6 months of age due to complications associated with the skeletal abnormalities and calcification of the aorta and renal arteries. This latter

877

finding is especially interesting since calcification of the vasculature and osteoporosis correlate in aging women. It also suggests a normal role for this molecule in the prevention of calcification of the larger arteries. Indeed, recent data from rodent studies demonstrate that doses of OPG which inhibit bone resorption are able to potently inhibit warfarin or vitamin D-induced arterial calcification in rodents (Price et al., 2001).

Structure of OPG Morinaga et al. (1998) cloned and characterized OPG and demonstrated that human OPG is a single copy gene consisting of five exons spanning 29 kb of the genome. The translation-termination codon is located in exon 5 and a typical poly(A) addition signal resides 173 nucleotides downstream of the translation-termination codon. A major transcription-initiation site is present 67 nucleotides upstream of the initiation-ATG codon. A single intron divides a stretch that encodes four cysteine-rich motifs, implying diversity from the other members of the TNF receptor family. Two death domain homologous regions present in tandem in OPG are encoded separately by exons 4 and 5. The conservation of amino acid sequences suggests that exon 4 is produced by a duplication of a portion of exon 5.

Chromosome location By analysis of radiation hybrids, Tan et al. (1997) mapped the OPG gene to 8q24.

RANK/RANKL AND PATHOLOGIC BONE DISEASE The association of RANK/RANKL expression and pathologic conditions has focused primarily on their role in the pathogenesis of metabolic bone disease.

Disorders associated with RANK receptor The disorders most clearly related to alterations in the RANK receptor are familial expansile osteolysis and familial Paget’s disease. Both disorders appear to arise

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as a result of mutations in the signal peptide region of the RANK protein (Hughes et al., 2000). It has been postulated that these mutations may lead to an accumulation of defective RANK translation products in the secretion pathway that may lead to receptor selfassociation and increased constitutive RANK signal transduction (Khosla, 2001). More recently, expansile skeletal hyperphosphatasia has been added to the list of disorders related to alterations in the RANK gene.

Familial expansile osteolysis (FEO) and Paget’s disease FEO is also a rare autosomal dominant skeletal disorder (Hughes et al., 1994), characterized by early-onset hearing loss resulting from degeneration of middle ear ossicles. There is destruction of teeth and painful bony expansion, and in contrast to expansile skeletal hyperphosphatasia there is cortical thinning resulting from focal osteolytic lesions within major long bones. The histopathological appearance of the osteolytic lesions resemble those of Paget’s disease. The molecular basis for FEO was shown by Hughes et al. (2000) to be an 18-bp tandem duplication (84dup18) in exon 1 of the RANK gene. It was also shown that (Hughes et al., 2000) in the course of screening four families with Paget’s disease with evidence of possible linkage to 18q, that one had a duplication involving bases 75–101 (75dup27) in exon 1 of the RANK gene co-segregating with the disease. In both disorders these mutations were shown to result in an alteration in the signal peptide region of the RANK receptor and a consequent increase in RANK receptor signaling. Such an increase may result in uncontrolled osteoclastogenesis and osteoclast activity leading to the focal osteolysis which is a pathologic consequence of this disease.

Expansile skeletal hyperphosphatasia (ESH) Early reports suggest that ESH is allelic to FEO (Whyte et al., 2000; Whyte and Hughes, 2002). ESH is characterized by deafness in infancy or early childhood, premature loss of teeth, progressive hyperostotic expansion of long bones causing painful phalanges in the hands, accelerated bone remodeling and episodic hypercalcemia. ESH is caused by a 15-bp tandem duplication (84dup15) in RANK and probably reflects excessive RANK activity and NFjB effect within the skeleton.

DISORDERS ASSOCIATED WITH THE RANKL/RANK/OPG SYSTEM Osteoporosis Osteoporosis is a disease chiefly affecting postmenopausal women. It is characterized by a chronic loss in the skeleton’s mineralized mass and resiliency leading to an increased risk of fracture in affected individuals. It is now widely accepted that the upregulation of osteoclast formation is the main mechanism by which estrogen deficiency induces bone loss in postmenopausal osteoporosis. Estrogen has been shown to induce both OPG mRNA levels and protein secretion in human osteoblastic cell lines (Hofbauer et al., 1999; Shevde et al., 2000). Estrogen has also been shown to reduce the effects of RANKL on osteoclastic cells via repression of the c-Jun signaling pathway. Therefore, the loss of the controlling influence of estrogen in postmenopausal women may play a pivotal role in the increase in osteoclast differentation and function. However, data obtained with serum OPG levels and their relationship with markers of bone turnover are not so clear-cut. Two of these studies demonstrated elevated OPG levels in those individuals with the highest bone turnover. It is of note that these studies concentrate only on the levels of OPG. No relative measurement of RANKL was included. A relative ratio of RANKL to OPG would be expected to more accurately reflect the biologic influence of these molecules. The influence of estrogen on the RANK/RANKL system has also been studied in vivo. In a model of estrogen-deficient bone loss in mice, the production of TNF from the T cells was implicated as a factor which augmented osteoclastogenesis via RANKL (Cenci et al., 2000). Indeed, in mice deficient in T cells, estrogen deficiency failed to induce bone loss. These findings indicate that estrogen mediates its effects on osteoclast formation and function via the RANKL/RANK/OPG system. Osteoporosis can be modeled in both rats and mice by surgically ovariectomizing the animals. OPG delivered either systemically (Simonet et al., 1997) or by an adenoviral delivery system has been shown to have a protective effect against bone loss in these in vivo systems. The only clinical study so far reported was by

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Bekker and colleagues (2001). This study involved 52 postmenopausal women in a randomized doubleblinded placebo-controlled trial. A single subcutaneous administration of OPG resulted in a dosedependent rapid and sustained reduction in bone turnover as assessed by circulating levels of the bone turnover marker N-telopeptide/creatine levels. In women receiving the highest dose (3 mg kg1), marker levels were reduced up to 80% after 5 days and remained below baseline until the termination of the study at 29 days.

Rheumatoid arthritis (RA) The expression of RANKL by T cells and the association of these cells with the pathogenesis of RA has also been widely researched. This T cell-derived RANKL has been shown to be capable of inducing and supporting osteoclastogenesis in vitro (Horwood et al., 1999; Kong et al., 1999). In two rodent models of inflammatory joint disease, adjuvant arthritis in the rat and collagen type IIinduced arthritis in the mouse, RANKL expression has been demonstrated at sites of active bone erosion (Kong et al., 1999; Romas et al., 2000). Kong and coworkers (1999) demonstrated that administration of OPG to rats with adjuvant arthritis could prevent the bone destruction which is so characterisitic of this lesion. However, no effect on the inflammatory aspect of this model was seen with this treatment. Evidence from human studies also points to an involvement of RANKL in RA. Kotake and colleagues (1999) demonstrated that osteoclastogenesis induced by IL-17, a T-cell cytokine, could be inhibited by OPG. They also showed that IL-17 levels were significantly higher in synovial fluids from RA patients when compared with those from patients with osteoarthritis. Expression of RANKL on subsets of synovial T cells, fibroblasts and infiltrating macrophages has been demonstrated immunocytochemically (Kotake et al., 2001). The ratio of soluble RANKL to OPG was also significantly higher in the synovial fluids of RA patients compared with those with OA or gout in the same study. Furthermore, it has been shown that RA synovial T cells are capable of inducing osteoclastogenesis and that this can be inhibited by OPG (Gravallese et al., 2000; Itonaga et al., 2000; Takayanagi et al., 2000; Kotake et al., 2001).

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Tumor-associated osteolysis of bone Increased bone loss through resorption is a major pathologic consequence of several tumor-associated bone diseases. As a result of the identification of the RANKL/RANK/OPG a number of investigators have highlighted the key involvement of the cytokines RANKL and OPG in the pathogenic process.

Giant cell tumors of bone In giant cell tumors of bone, mesenchymal tumor stroma surrounds large multinucleated osteoclast cells (giant cells). The stroma from these tumors has been shown to express and produce large amounts of RANKL (Atkins et al., 2000; Huang et al., 2001). Furthermore, the giant cells themselves also express excessive levels of mRNA for the RANK receptor when compared with normal osteoclasts (Atkins et al., 2000; Huang et al., 2001). Therefore, the osteolysis associated with these epiphyseal tumors could be assigned to an unregulated overexpression of RANKL and RANK.

Multiple myeloma The osteolytic lesions, pathologic fractures and profound hypercalcemia associated with multiple myeloma have long been believed to occur as a result of interactions between myeloma cells, bone marrow stromal cells and osteoclasts. Two myeloma cellderived mediators which have been intimately implicated in this process are interleukin-6 and parathyroid hormone-related peptide (PTHrP) (Roodman, 1997; Firkin et al., 1998), both of which have been shown to induce RANKL and inhibit OPG (O’Brien et al., 1999; Thomas et al., 1999; Mancino et al., 2001) production. Multiple myeloma cells also express RANKL on their plasma membranes and secrete soluble RANKL in vitro (James et al., unpublished observations). Therefore, this environment can be seen as one in which the RANKL:OPG ratio is one which would promote both osteoclastogenesis and osteolysis.

Metastatic bone disease Osteoclast activity is intimately involved in both the osteolytic bone metastases and humoral hypercalcemia of malignancy, two frequently presented

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pathologic complications of breast carcinoma. As with myeloma, breast carcinoma cells are known to produce large amounts of PTHrP and as a consequence enhance RANKL mRNA expression (Thomas et al., 1999; Chikatsu et al., 2000; Mancino et al., 2001) and inhibit OPG production by osteoblastic-lineage cells (Thomas et al., 1999; Chikatsu et al., 2000). This pro-resorptive environment may be required for the survival and further expansion of the tumor.

Evidence from in vivo models In vivo studies also point to a key role for the RANKL/RANK/OPG system in mediating humoral hypercalcemia of malignancy and skeletal metastasis. Administration of OPG to rodents exposed to a variety of tumors which cause either hypercalcemia, skeletal metastasis or both, demonstrate a rapid and pronounced beneficial effect both on the prevention and reversal of hypercalcemia and bone resorption (Akatsu et al., 1998; Morony et al., 1999; Capparelli et al., 2000; Oyajobi et al., 2001). Using behavioral studies as an endpoint, Honore and colleagues (2000) showed that OPG could prevent cancer-induced skeletal pain as well as skeletal destruction and the neurochemical reorganization of the spinal chord in a murine model of osteosarcoma.

SUMMARY AND FUTURE PROSPECTS The observations highlighted in this chapter demonstrate the pivotal role of the RANKL/RANK system in a number of biologic systems, including the development and function of mammary tissue and in the maintenance of calcium metabolism and bone mass. Indeed, the discovery and elucidation of the RANKL/ RANK/OPG system as it relates to osteoclastogenesis has proven to be a groundbreaking finding for the field of bone biology. There is substantial evidence implicating a dysregulation of this system in a number of bone pathologies including osteoporosis, rheumatoid arthritis and metastatic bone disease. As we look to the future, therefore, it is assumed that the therapeutic potential for intervening with this system for the treatment of these diseases must be explored. The recent description by Lam et al. (2001) of the

crystal structure of RANKL, suggests exciting opportunities for pharmacologic intervention in a number of disorders outlined in this chapter. Proof of concept for this approach has already been achieved with recombinant OPG, which was shown to significantly reduce bone turnover in markers in postmenopausal women (Bekker et al., 2001). One may speculate therefore, that rational drug design of small molecular weight inhibitors, or OPG itself, may be useful in the treatment of disorders associated with increased osteoclast activity.

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38 CD95L/FasL and its receptor CD95 (APO-1/Fas) Marcus E. Peter, Bryan C. Barnhart and Alicia Algeciras-Schimnich University of Chicago, Chicago, IL, USA

Seeing death as the end of life is like seeing the horizon as the end of the ocean David Searls

INTRODUCTION In the mid 1980s, two groups initiated a screen for a monoclonal antibody that would inhibit the growth of tumor cells by binding to a cell surface antigen. This resulted in the identification of two monoclonal antibodies that induced programmed cell death, or apoptosis, in many tumor cells, anti-APO-1 (an IgG3 antibody; Trauth et al., 1989) and CH11 (an IgM antibody; Yonehara et al., 1989). The recognition that binding of an antibody to cells could trigger a cell death program was radical at the time and raised expectations for finding new antitumor drugs. This expectancy was mainly fueled by an experiment in which an injection of anti-APO-1 into nude mice harboring a human tumor resulted in complete regression of the tumor (Trauth et al., 1989). Cloning of the surface antigen to which these antibodies bound revealed a protein with homology to the tumor necrosis factor receptor type I (TNF-RI) (Itoh et al., 1991; Oehm et al., 1992) especially in a cytoplasmic region The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

which was essential for death induction, the death domain (DD) (Tartaglia et al., 1993). The molecule was termed Fas or APO-1 and is now known as CD95 or tumor necrosis factor receptor superfamily member 6 (TNFRSF6). The significance of CD95 for the function of the immune system became clear with the recognition that mice carrying the lymphoproliferation (lpr) mutation have a defect in CD95 (WatanabeFukunaga et al., 1992), which led to the discovery that mutations in CD95 in humans can cause the autoimmune lymphoproliferative syndrome (ALPS) (Fisher et al., 1995; Rieux-Laucat et al., 1995; Drappa et al., 1996). From experiments involving bone marrow transplantation (Allen et al., 1990) it had been predicted that another mouse strain homozygous for the gld (generalized lymphoproliferative diseases) (Roths et al., 1984) mutation had a defective CD95L gene and that lpr and gld represented two mutations in a corresponding receptor/ligand pair. The race was on to clone the ligand that bound to CD95, and induced apoptosis. This hunt was widely viewed as the ‘search

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for the holy grail’ with expectations of finding a new tumor-specific reagent. In 1993 the collaboration of two groups resulted in the cloning of this molecule (Suda et al., 1993). It was the rat homolog of CD95L which was cloned almost by accident from a cytotoxic T-cell hybridoma generated by fusing a murine cytotoxic T-cell clone (on which the CD95 ligand was suspected) with a rat myeloma fusion partner cell line. Shortly after both mouse and human CD95L were cloned (Lynch et al., 1994; Takahashi et al., 1994a, 1994b) and it was confirmed that gld mice had a defect in CD95L (Lynch et al., 1994; Takahashi et al., 1994b). CD95L is also known as FasL or tumor necrosis factor ligand superfamily member 6 (TNFSF6). CD95 was shown to be expressed on almost all normal tissues with highest expression in the colon and the liver (Moller et al., 1994; Galle et al., 1995). Injection of anti-mouse CD95 antibodies resulted in early death of mice due to liver failure dampening expectations for the use of CD95L as an antitumor reagent (Ogasawara et al., 1993). In the following years the signaling pathway of CD95 was studied intensively (Peter and Krammer, 1998; Wallach et al., 1999) as were many other aspects of the role of the CD95/CD95L pair in the immune system (Siegel et al., 2000a) and in various diseases (Peter et al., 1997). The concept of a tumor-specific death ligand returned with the cloning of the CD95L-related molecule TRAIL and its receptors (see Chapter 36 of this book) which kills the majority of tumor cells without significant toxicity (Walczak et al., 1999). Many of the aspects of signaling through CD95 can be applied to the TRAIL receptors (Walczak and Krammer, 2000). This chapter will summarize the structure, function and roles of the death receptor CD95 and its ligand in physiology and disease.

THE CD95 LIGAND Structure, regulation of expression and functions of CD95L As are all members of the TNF superfamily, CD95L is a type II transmembrane protein; the C-terminus is expressed extracellularly. The 281-amino-acid protein is located on human chromosome 1q23, a four-exon gene within 8 kb (Takahashi et al., 1994b).

The mature membrane-bound protein contains an extracellular domain which binds to CD95, a transmembrane domain and a 77-amino-acid long intracellular domain. Human CD95L shows 76.9% amino acid identity with the mouse protein and mouse CD95L kills human cells and vice versa indicating crossreactivity (Takahashi et al., 1994b).

Regulation of CD95L expression CD95L is widely expressed in different tissues with high expression in activated T lymphocytes and thymocytes (Suda et al., 1993). However, CD95L is also expressed in several non-lymphoid tissues such as testis. As a death ligand expression of CD95L must be tightly regulated in different tissues. This regulation is achieved by a diverse set of transcription factors and promotor elements. One mediator of CD95L expression is Nur77 (N10/NGFI-B/Nak1) an orphan steroid receptor (Davis and Lau, 1994). Nur77 is highly up-regulated in immature thymocytes and T-cell hybridomas upon TCR stimulation (Chan et al., 1998). Antisense Nur77 (Liu et al., 1994) or transfection of a dominant-negative Nur77 (Woronicz et al., 1994; Zhou et al., 1996) has been shown to block antigeninduced cell death (AICD) of peripheral T cells by down-regulating expression of CD95L. However, the role of Nur77 in AICD is unclear as Nur77-deficient mice do not have an apparent defect in this process (Lee et al., 1995). This could be due to redundancy of this protein with other factors such as Nor-1 (Cheng et al., 1997). c-myc has been implicated in regulating AICD and therefore expression of CD95L in T cells. Both antisense c-myc (Shi et al., 1992) and expression of a dominant-negative c-myc-binding protein Max (Bissonnette et al., 1994) blocked CD95L expression and AICD. Recent data suggested direct regulation of CD95L expression by c-myc (Brunner et al., 2000) and CD95L up-regulation as a mechanism to explain apoptosis induction by c-myc (Hueber et al., 1997). Recent data have also demonstrated that other transcription factors and signaling pathways regulate expression of CD95L in various tissues. Two sites in the CD95L promotor bind the nuclear factor of activated T cells (NF-AT) (Latinis et al., 1997). Although both sites seem to be important for optimal expression of CD95L the distal site plays a more significant role in CD95L up-regulation in activated T cells and muta-

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tion of this region substantially decreases inducible promotor activity (Latinis et al., 1997; Norian et al., 1998). Another family of transcription factors shown to regulate CD95L expression are the early response genes (EGR) 1–3. Egr3 was reported to bind to a specific site in the CD95L promotor and induce CD95L expression in a cyclosporin A-insensitive manner (Mittelstadt and Ashwell, 1998). Overexpression of Egr3 even induced expression of CD95L in nonlymphoid cells that did not normally express CD95L. Another report demonstrated that Egr1, Egr3 and NF-AT bound cooperatively to a novel Egr/NF-AT composite site regulating expression of CD95L in response to T-cell activation (Li-Weber et al., 1999). However, this finding was not supported by another report which showed that activated T cells in the presence of IL-2 regulate CD95L promotor activity through SP1 and NF-AT without a role for Egr2 or Egr3 (Xiao et al., 1999). Recently, it was shown that the CD95L promotor in TH2 T cells but not in TH1 cells recruits the transcription factor SP1 to a composite Egr/NF-AT site suggesting a differential regulation of CD95L expression in TH1 and TH2 cells (Dzialo-Hatton et al., 2001). Furthermore, it was shown that in TH1 cells Egr1, 2 and 3 bound to a known Egr site in the CD95L promotor where no binding of Egr3 was detected in TH2 cells, suggesting a potential mechanism for the differential expression of CD95L in distinct T-cell lineages (Dzialo-Hatton et al., 2001). Thus the transcription factor that regulates CD95L expression appears to depend upon both the conditions/ stimulation and the subset of T cells. NFjB has also been well studied as a regulator of CD95L expression. Early after cloning the CD95L promotor it was recognized that a stretch of 300 bp with high homology between mouse and human in the CD95L promotor contained a putative binding site for this ubiquitously expressed transcription factor (Takahashi et al., 1994b). Subsequently, two functional NFjB sites (designated FasL–jB1 and FasL–jB2) have been identified in the promotor (approximately 700 bp) of CD95L (Matsui et al., 1998). NFjB appears to bind to the FasL–jB1 site resulting in up-regulation of CD95L. Two other studies demonstrated that NFjB in conjunction with AP-1 (a transcription factor comprising the proteins Fos and Jun) are crucially involved in upregulation of CD95L during drug-induced apoptosis (Kasibhatla et al., 1998; Harwood et al., 2000). A 1.2-kb

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fragment of the promotor responded to DNA damage and mutations of both the NFjB and the AP-1 binding sites eliminating these responses. Another AP-1 site was recently identified in the CD95L promotor which binds Jun–Fos heterodimers resulting in upregulation of CD95L during anticancer drug-treated hepatocarcinoma cells (Eichhorst et al., 2000). Although it was reported that activation of NFjB is essential for up-regulation of CD95L in T cells (Chan et al., 1999; Hsu et al., 1999a), it was later shown that T-hybridoma cells deficient for the I-kB kinase c, an essential NFjB signaling component had a defect in interleukin-2 gene induction but no defect in upregulating CD95L (Rivera-Walsh et al., 2000), suggesting that NFjB is not always required to induce CD95L expression. CD95L expression is also a target for viral interference in apoptosis regulation. For example, the HIV-1 gene product Tat has been shown to accelerate CD95L expression in uninfected peripheral T cells by interfering with the redox state of T cells (Westendorp et al., 1995a, 1995b), activating NFjB (Li-Weber et al., 2000). Other transcription factors and signaling pathways that have been implicated in the regulation of CD95L expression are p38 (Hsu et al., 1999b) and IFN regulatory factor 1 and 2 (Chow et al., 2000). In summary, regulation of CD95L expression is a highly complex process regulated by a number of different transcriptional activators.

Membrane-bound (mCD95L) versus soluble (sCD95L) CD95 ligand Most of the members of the TNF superfamily are expressed as membrane and soluble proteins. The membrane-bound form of TNFa was shown to stimulate TNF-RII preferentially whereas soluble TNFa was active enough to induce apoptosis through TNF-RI (Grell et al., 1995). TNFa is processed from membranebound TNFa by a metalloproteinase, the TNFaconverting enzyme (TACE) (Black et al., 1997; Moss et al., 1997). CD95L is similarly regulated. All CD95L is first expressed as a 40-kDa membrane-bound form which is then processed by a metalloproteinase to release the soluble 27-kDa form (Mariani et al., 1995; Kayagaki et al., 1995). The metalloproteinase that processes CD95L is as yet not known, however, it can be inhibited by known metalloproteinase inhibitors such as phenathroline, which improves staining for

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CD95L on cells (Mariani et al., 1995). The CD95L protease is distinct from TACE (Itai et al., 2001). Suspected candidates are matrilysin/MMP-7 (Powell et al., 1999; Mitsiades et al., 2001) and stromelysin1/MMP-3 (Matsuno et al., 2001). There has been some controversy about the differential function of sCD95L versus mCD95L. Originally, it was shown that the supernatants of some cytotoxic killer cells and of COS cells overexpressing full-length CD95L were cytotoxic to CD95-sensitive cells (Suda et al., 1994; Takahashi et al., 1994b). Analysis of the CD95L that killed these cells revealed that it was mainly the full-length protein shedded from the cells. Later analyses confirmed that highly purified sCD95L has a significantly lower cytotoxic activity than mCD95L (Schneider et al., 1998). Some data even suggested that sCD95L was an antiapoptotic cytokine functioning to counteract the effect of mCD95L (Suda et al., 1997; Tanaka et al., 1998; Hohlbaum et al., 2000). Overall, it was demonstrated that the cytotoxic activity of CD95L depends on its ability to aggregate. Soluble CD95L can therefore be made highly active by either adding a FLAG tag and an anti-FLAG antibody (Schneider et al., 1997) or by fusing it with a trimerizing form of a leucine zipper domain (Walczak et al., 1999). Data describing an inactive, antiapoptotic sCD95L are difficult to reconcile with some of the earlier data which demonstrated that the soluble untagged CD95L has cytotoxic activity (Tanaka et al., 1995). Furthermore, the extracellular domain of mouse CD95L, again in untagged form (called WX1), aggregates similarly to mCD95L and is highly active in inducing apoptosis (Suda et al., 1996). Finally, it was demonstrated that CD95L is involved in AICD of human T cells and it was shown that T cells committed autocrine suicide on the single cell level. The supernatant of these T cells, which was active in induction of apoptosis after transfer on to CD95-sensitive target cells (Dhein et al., 1995), contained the 27-kDa sCD95L (H. Walczak, personal communication). Recently, it was demonstrated that sCD95L, under certain circumstances, gained significant activity as an apoptosis-inducing factor by binding to extracellular matrix proteins (Aoki et al., 2001). In summary, all data confirm that mCD95L is much more active than sCD95L. However, a number of reports support the view that sCD95L can be an active cytotoxic factor in vivo, consistent with data which showed that injection of sCD95L into mice can

cause apoptosis of tumor and liver cells (Rensing-Ehl et al., 1995; Tanaka et al., 1997).

Signaling through CD95L CD95L has been reported to be a signaling receptor as well as a death ligand. Compared with other members of the TNF superfamily CD95L has a relatively long intracellular tail that contains a unique region rich in proline residues and thus suspected as a docking site for SH3-containing proteins (Hahne et al., 1995). Indeed, a number of SH3 domains from various intracellular signaling proteins were found to bind to this region in vitro (Hahne et al., 1995). More recently, the proline-rich region in CD95L was implicated in regulating the sorting of CD95L into secretory lysosomes (Blott et al., 2001). It was additionally shown that for cytotoxic T lymphocytes to gain full proliferative and killing activity a reverse signaling through CD95L is required (Suzuki and Fink, 1998). In contrast, CD4positive T cells seem to be negatively affected by surface CD95L. A reverse signaling through CD95L in these cells has been demonstrated to result in cell cycle arrest, preventing expansion of CD4 T cells (Desbarats et al., 1998). CD95L engagement therefore has a potent anti-inflammatory effect specific for CD4 T cells.

THE CD95 RECEPTOR Structure and regulation of expression of the CD95 receptor CD95 (APO-1/Fas) is the best-studied member of the death receptor family (Schulze-Osthoff et al., 1998; Peter et al., 1999). It consists of 335 amino acids, containing an extracellular region with three cysteinerich domains, a single transmembrane region and a relatively long cytoplasmic tail which contains a death domain (DD). CD95 is a 35-kDa type I transmembrane protein which is heavily glycosylated with an apparent molecular weight of the mature protein of 48–54 kDa (Itoh et al., 1991; Oehm et al., 1992). Sialylation, which accounts for most of its mostly N-linked carbohydrates, regulates the CD95 apoptosis sensitivity of a number of different tumor cells (Peter et al., 1995). The gene for CD95 maps to human

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chromosome 10q24.1 (Lichter et al., 1992; Inazawa et al., 1992) and the receptor is coded for by nine exons that cover 26 kb and which are differentially utilized to produce both membrane-bound and soluble forms of CD95 (Cheng et al., 1994; Cascino et al., 1995). It has been shown that splice forms that lack a transmembrane domain can act as dominantnegative inhibitors of CD95-induced apoptosis (Cascino et al., 1995; Papoff et al., 1999; Jenkins et al., 2000) by either binding CD95L or directly binding and inhibiting membrane CD95. Elevated levels of soluble CD95 receptors, all of which lack exon 6 (which codes for the transmembrane region), have been found in a number of different diseases (Mountz et al., 1994; Owen-Schaub et al., 2000; Andrikoul and Tsatsoulis, 2001). Another CD95L-binding protein which is upregulated in certain tumor cells has been reported and termed decoy receptor 3 (DcR3/TR6) (Pitti et al., 1998; Roth et al., 2001). The physiologic function of this molecule as a CD95L-regulating protein, which is more related to TRAIL and OPG receptors than to CD95, has not been established. However, death receptor expression and activity must be tightly regulated and this is no exception in the CD95/CD95L system. CD95 is constitutively expressed at high levels of up to 50 000 epitopes a cell in the liver, the colon and other organs such as lung, ovary and heart (Itoh et al., 1991; Oehm et al., 1992). Low constitutive expression is found in most other tissues. In some tissues expression of CD95 is induced either by transcriptional activation of the gene or by transport to the cell surface. For example, T cells strongly upregulate CD95 during stimulation through the T-cell receptor (TCR) (Klas et al., 1993) and during many other forms of stimuli. Additionally, preformed CD95 is transported from the Golgi complex to the cell surface in human vascular smooth muscle cells in a p53dependent fashion (Bennett et al., 1998a). p53 also regulates expression of CD95 and its ligand on the transcriptional level. An intronic element in intron 1 and three putative p53-binding sites in the human CD95L promotor were identified (Muller et al., 1998) and it has been shown that CD95 is up-regulated during drug treatment of various tumor cells in a p53-dependent fashion (Muller et al., 1997, 1998). Recently, an equivalent p53-binding site in intron 1 of the mouse CD95 gene was identified (Munsch et al., 2000). It was demonstrated that both wild-type and

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p53 mutants that do not induce apoptosis caused upregulation of CD95 suggesting that like Bax, CD95 is a direct target for p53 but unlike Bax its up-regulation does not directly result in induction of apoptosis but rather sensitizes cells for CD95-mediated apoptosis. Another mechanism of CD95 up-regulation is through activation of PKC which appears to involve TDAG51 (Wang, Z.G. et al., 1998), a protein related to the tumor suppressor genes TSSC3 (Lee and Feinberg, 1998) and IPL (Qian et al., 1997). Expression of CD95 can also be suppressed under physiologic circumstances. It was shown that Egr1 could down-regulate CD95 in B-cell lines resulting in CD95 apoptosis resistance (Dinkel et al., 1997). In T cells the transcription factors YB-1 and Puralpha were identified as proteins that negatively regulate CD95 expression (Lasham et al., 2000). In melanoma cells it was shown that activation of an Ask1/MKK6/p38 pathway protects cells from UV radiation-induced apoptosis by down-regulating expression of CD95 (Ivanov and Ronai, 2000). Also in melanoma cells, it was shown that STAT3 cooperates with c-Jun to down-regulate CD95 expression (Ivanov et al., 2001).

The role of CD95 and CD95L in the immune system Apoptosis induced by CD95/CD95L plays a crucial role in the immune system through its participation in maintenance of T-cell homeostasis, induction of peripheral tolerance, lymphocyte-mediated cytotoxicity against virally infected and tumor cells and in maintenance of ‘immune privilege’ sites (Singer and Abbas, 1994; Ju et al., 1995, Van Parijs et al., 1996; Griffith and Ferguson, 1997). The first evidence for the crucial role of CD95 and CD95L in the immune system came from two mouse models of systemic autoimmunity. The lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease) mice harbor spontaneous autosomal recessive mutations in the CD95 and CD95L gene (Watanabe-Fukunaga et al., 1992; Takahashi et al., 1994a; Ramsdell et al., 1994), respectively. Two mutations on the CD95 gene can lead to the lpr phenotype: an insertion of a transposable element, which prevents normal processing of mRNA (in the lpr mice) (Adachi et al., 1993; Kobayashi et al., 1993; Wu et al., 1993); and a point mutation (T→A) in the intracellular region of CD95

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causing a substitution of asparagine to isoleucine in the death domain (in the lpr cg mice) (Matsuzawa et al., 1990), abolishing the death signal transmission. The gld mice have a point mutation near the C-terminus of the CD95L coding region (Takahashi et al., 1994a). This mutation causes the substitution of a phenylalanine for a leucine in the extracellular region and abolishes the binding of CD95L to CD95. All three mouse strains exhibit lymphadenopathy and splenomegaly, due to the accumulation of aberrant CD220 CD4CD8 T cells, as well as excessive production of IgG and IgM antibodies, including autoreactive anti-DNA antibodies and rheumatoid factor. These mutations cause various autoimmune diseases depending on the mice genetic background similar to systemic lupus erythematosus (SLE) and rheumatoid arthritis. In both lpr and gld mice the pathological manifestations are due to impaired peripheral clonal deletion of activated T lymphocytes (GilletteFerguson et al., 1994; Ramsdell et al., 1994; Reap et al., 1995. In humans, mutations in CD95 are responsible for the ALPS type IA (Fisher et al., 1995; Rieux-Laucat et al., 1995; Drappa et al., 1996). ALPS patients generally have non-malignant adenopathy and hepatosplenomegaly, manifest lymphocytosis, including accumulation of single positive T cells and increased numbers of double-negative T cells, and they also develop hypergammaglobulinemia with generation of multiple autoantibodies. In addition, singlepositive T lymphocytes from these patients fail to undergo apoptosis following CD95 receptor stimulation (Fisher et al., 1995). Mutations in human CD95L have also been described. In a patient with SLE who showed lymphadenopathy, a deletion of 84 bp within the CD95L gene resulted in a frame-shift deletion (Wu et al., 1996). Peripheral blood mononuclear cells from this patient show decreased CD95L activity, decreased antigen-induced cell death and increased T-cell proliferation after activation. This mutation in CD95L is classified as ALPS type IB. In addition, ALPS patients with defective CD95 signaling but without mutations on CD95 or CD95L have been identified. Mutations in caspase 10 (Wang et al., 1999) have recently been shown to be responsible for the defect in these patients and have been classified as ALPS type II. Cytotoxic T lymphocytes kill target cells by two different ways either involving perforin/granzyme or

CD95L (Plate 38.1a) (see Plate section) (Berke, 1995). In the immune system the CD95/CD95L system plays a critical role in regulating T-cell numbers and in the induction of peripheral T-cell tolerance to self antigens (Lenardo et al., 1991; Radvany et al., 1993; Russell, 1995; Van Parijs and Abbas, 1998). AICD might serve as a second checkpoint to eliminate autoreactive T cells in the periphery and prevent autoimmunity. Restimulation of previously activated T cells through the TCR induces apoptosis in the majority of these cells by AICD (Plate 38.1b). T cells sensitive to AICD include T-cell hybridomas, transformed Tcell lines and in vitro-activated peripheral T cells (Ucker et al., 1989; Takahashi et al., 1989; Wesselborg et al., 1993). Blocking of the CD95-signaling pathway with soluble CD95 and antibodies against CD95L prevents AICD (Brunner et al., 1995; Ju et al., 1995; Dhein et al., 1995). In addition, AICD is defective in T cells derived from lpr mice and in T cells from ALPS patients, confirming the role of CD95/CD95L in AICD (Fisher et al., 1995; Cui et al., 1996). AICD was first characterized in transformed T-cell hybridomas (Ucker et al., 1989; Shi et al., 1989, 1990). Stimulation of transformed T cells via the TCR results in induction of CD95L expression and subsequent apoptosis (Dhein et al., 1995; Brunner et al., 1995; Ju et al., 1995; Van Parijs et al., 1996). AICD was described to occur by both fratricide and suicidal mechanisms (Brunner et al., 1995; Dhein et al., 1995). Death by fratricide occurs by interaction of membrane-bound CD95 with membrane-bound CD95L on neighboring T cells that results in their apoptosis. In addition, it has been shown that TCR ligation can induce apoptosis on the single-cell level, suggesting that CD95/ CD95L interaction can induce cell death in a cellautonomous manner (‘suicide’ death) (Brunner et al., 1995; Dhein et al., 1995). Freshly isolated lymphocytes, which express CD95, are resistant to AICD triggered through the TCR (Lenardo et al., 1991; Owen-Schaub et al., 1992). Regulation of T cell fate, i.e. proliferation versus death, may be regulated at various levels. For example, it has been shown that the strength of the TCR signal delivered by a specific MHC peptide complex may be decisive and may determine whether the cell survives or proliferates (Combadiere et al., 1998). This is important since the death inducer CD95 itself has no inherent antigen specificity. Efficient death induc-

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tion therefore requires a ‘competency’ signal from the TCR delivered at the same time as CD95L binding (Hornung et al., 1997; Combadiere et al., 1998). AICD in non-transformed T lymphocytes requires stimulation by two mitogenic signals: first, antigenic stimulation by the TCR and second, a cytokine stimulation such as IL-2 (Lenardo et al., 1991; Ucker et al., 1992; Boehme et al., 1993). TCR stimulation increases the expression of CD95 and induces the synthesis of CD95L. However, increases in CD95 and CD95L expression levels are not sufficient for a T cell to undergo CD95-mediated apoptosis. These observations suggest that activation must induce intracellular modifications which make an otherwise resistant T cell sensitive to CD95-mediated apoptosis. Production of IL-2 is necessary for responding T cells to become sensitive to CD95-induced death (Fournel et al., 1996; Refaeli et al., 1998; Algeciras-Schimnich et al., 1999) and for up-regulation of CD95L following activation of STAT5 (Van Parijs et al., 1999). The importance of IL-2 in AICD is supported by the fact that IL-2-deficient mice show diminished AICD following antigen stimulation and accumulate T cells with memory phenotype (Kneitz et al., 1995). In the thymus T lymphocytes go through various stages of development before exiting into the periphery as mature T cells. Approximately 95% of thymocytes are eliminated by apoptosis which prevents autoreactive T cells from exiting the thymus. High expression levels of CD95 in thymocytes prompted the idea that the CD95/CD95L system was involved in negative selection (Castro et al., 1996). However, because negative selection is not defective in lpr or gld mice it was concluded that CD95 does not play a significant role in this process (Herron et al., 1993; Singer et al., 1994). It was recently shown that negative selection of thymocytes, induced by the superantigen staphylococcal enterotoxin B or by injection of an ovalbumin peptide in TCR transgenic mice, occurs normally under low to moderate doses of antigen in the absence of CD95 (Kishimoto et al., 1998). In contrast, when high doses of antigen were used, negative selection failed to occur in lpr mice. These findings suggest that negative selection is CD95 independent at low concentrations of antigen, but CD95 dependent at high doses (Kishimoto et al., 1998). At this point the requirement of CD95 during thymic-negative selection is still controversial. Further studies are

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necessary to delineate the role of CD95 and other apoptotic systems during this process. The CD95/CD95L system is also involved in preventing the autoantibody production that occurs in lpr and gld mice and in ALPS patients (Ishigatsubo et al., 1988). CD95-induced apoptosis is involved in the induction of B-cell tolerance, prevention of misguided T-cell help, and in selection of high-affinity B cells in the germinal centers (Rothstein et al., 1995; Rathmell et al., 1996). Analysis of different B-cell subsets revealed that CD95 expression is low on resting B cells and highly increased on activated B cells and germinal center B cells, suggesting that CD95 might be involved in the negative selection in the germinal centers (Mandik et al., 1995; Lagresle et al., 1996). It has recently been shown that CD95 is indeed required for B-cell clonal selection in the germinal centers and for the establishment of the memory B-cell repertoire (Takahashi et al., 2001). CD95-induced apoptosis in germinal-center B cells is regulated by the caspase 8 inhibitor c-FLIP. In these cells, apoptosis correlates with down-regulation of c-FLIP (van Eijk et al., 2001; Hennino et al., 2001). Interestingly, formation of the CD95 death-inducing signaling complex (DISC) and activation of caspase 8 seem to occur independently of CD95L since CD95L message was not detected in germinal-center B cells, inhibition of CD95/CD95L interaction does not prevent apoptosis, and CD95L was not part of the DISC in these cells (Hennino et al., 2001). CD95-induced apoptosis in B cells appears to be regulated by the type of stimulus and the developmental stage. Stimulation through the B-cell receptor (BCR) and CD40 can determine the fate of B cells. BCR stimulation has been reported to induce both proliferation and apoptosis (Lam et al., 1997, 1998). It is independent of CD95 and requires the apoptogenic activity of mitochondria. Stimulation through CD40 can determine whether activation of the BCR causes apoptosis or activation. In naive B cells CD40 engagement rescues cells from BCR-induced apoptosis whereas in mature B cells CD40 stimulation upregulates CD95 levels and sensitizes to CD95-induced apoptosis (Garrone et al., 1995; Schattner et al., 1995; Lagresle et al., 1996). In contrast, stimulation of the BCR in non-tolerant B cells induces resistance to CD95-mediated apoptosis by the up-regulation of the caspase 8 inhibitor c-FLIP (Wang et al., 2000). In

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addition, CD95-induced apoptosis might function to eliminate the autoreactive B cells following their interaction with T cells. Interaction of autoreactive B cells with T cells results in the activation of CD40 in B cells which then induces CD95 up-regulation and sensitivity to CD95 (Garrone et al., 1995; Schattner et al., 1995; Lagresle et al., 1996). Subsequently, ligation of CD95 by CD95L on the T cells results in the elimination of autoreactive B cells (Rathmell et al., 1996).

The role of CD95/CD95L on nonlymphoid cells The CD95/CD95L system not only regulates the function of the immune system but also plays an important role in the liver. CD95 is present in both developing (French et al., 1996) and mature (Leithauser et al., 1993) liver of both mouse and human. Hepatocytes are sensitive to CD95-induced apoptosis in vitro and in vivo (Ni et al., 1994; Galle et al., 1995; Lacronique et al., 1996; Nishimura et al., 1997). Moreover, CD95 seems to regulate liver homeostasis as mice deficient for CD95 develop increased cellularity and liver hyperplasia (Adachi et al., 1995). CD95-induced apoptosis is involved in liver injury in various diseases such as in hepatitis C virus infection, alcoholic liver cirrhosis, Wilson disease and sepsis (Hayashi and Mita, 1997; Kondo et al., 1997; Strand et al., 1998; Pianko et al., 2001; Chung et al., 2001). CD95L-induced liver damage is in some systems mediated by CD95L-induced IL-18 secretion which augments the cytotoxic activity of NK and T cells resulting in death of hepatocytes through CD95-induced apoptosis (Tsutsui et al., 1999). Although CD95L was originally found to be mainly expressed in activated lymphocytes, it was later shown to be expressed in non-lymphoid cells as well. CD95L is expressed in the eyes and the testis and it is implicated in the maintenance of immune privilege in these organs (Griffith et al., 1995; Bellgrau et al., 1995). It has been proposed that CD95L protects these organs by inducing apoptosis of infiltrating inflammatory cells (Plate 38.1c). After viral infection of the anterior chamber of the eye infiltrating lymphocytes are eliminated by apoptosis. Additionally, apoptosis of lymphocytes was defective following

viral infection of the gld mice resulting in inflammation and destruction of the ocular tissue (Griffith et al., 1995). The observation that CD95L could cause specific immune suppression prompted the idea that expression of CD95L on allografts may prevent rejection of transplants (Bellgrau et al., 1995). However, when this theory was tested it gave contradictory results. In some cases the model could be confirmed, as in the case of experimental liver, kidney, pancreatic islet allografts and cornea transplantation (Lau et al., 1996; Stuart et al., 1997; Li et al., 1998a; Swenson et al., 1998). However, in many studies both CD95L-expressing tumors as well as many transplants engineered to express CD95L were rejected more rapidly than control transfected cells (Allison et al., 1997; Kang et al., 1997). Rapid rejection of transplants was due to inflammation mediated by neutrophil infiltration into the graft (Allison et al., 1997). Neutrophil infiltration was mediated by the release of proinflammatory cytokines, mainly IL-1b, from apoptotic cells which then resulted in chemotaxis of neutrophils into the graft (Miwa et al., 1998). In addition, it was shown that CD95L itself acts as a chemoattractant for neutrophils (Seino et al., 1997, 1998; Ottonello et al., 1999). It was then demonstrated that TGFb can regulate whether CD95L acts as a death ligand or as a chemoattractant (Chen et al., 1998). The benefits of CD95 expression in allografts therefore requires further study. Expression of CD95L by tumor cells may also provide a growth advantage by enabling them to escape the host’s immune system and preventing tumor rejection (Strand et al., 1996; Hahne et al., 1996; O’Connell et al., 1996). A few years ago it was noted that certain tumors that express high amounts of CD95L when incubated with CD95-sensitive cells induced apoptosis of the target cells (O’Connell et al., 1996; Muschen et al., 2000a). This led to the ‘the tumor strikes back’ (Walker et al., 1997) or ‘the tumor counterattack’ theory in which tumor cells kill activated cytotoxic T lymphocytes causing a specific immune suppression (Plate 38.1d) (O’Connell et al., 1996). Expression of CD95L in tumor cells has been shown to kill activated T cells in vitro and in vivo (Strand et al., 1996; Hahne et al., 1996; O’Connell et al., 1996, 1998). In addition, it has been shown that increased expression of CD95L in cancer cells contributes to tissue damage and metastatic spread

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(Peduto Eberl et al., 1999; Yoong et al., 1999). The tumor counterattack theory has been supported in vivo in some models. Human esophageal cancer expressing CD95L shows a significant decrease in the number of in situ tumor-infiltrating lymphocytes (TIL) when compared with CD95L-negative tumors. The decrease in TILs correlated with increased apoptosis in those cells (Bennett et al., 1998b). Additionally, in colon and ovarian cancer a correlation between CD95L expression in the tumor and decreased number of TILs was observed (Okada et al., 2000). However, recently it has become clear that the ‘tumor strikes back’ model does not apply to all cases (Restifo, 2000) and requires further evaluation. Up-regulation of CD95L is usually accompanied by down-regulation of CD95 in many tumors. Downregulation of CD95 by tumor cells may protect them from being rejected by cytotoxic T cells and also enables the CD95-positive tumor cells to kill other cells through CD95 without the risk of ‘suicide’. Downregulation of CD95 has been detected in breast cancer, squamous cell carcinoma and colon cancer (Keane et al., 1996; Butler et al., 1998; Moers et al., 1999). Another mechanism that confers resistance to CD95 in tumor cells involves somatic mutations in CD95. Somatic CD95 mutations that impair the transduction of the death signal were first described in cancers from the lymphoid system including acute lymphoblastic leukemia, multiple myeloma, and nonHodgkin’s lymphomas (Xerri et al., 1995; Landowski et al., 1997; Beltinger et al., 1998). In B cell-derived lymphomas, mutations of CD95 might have been acquired during somatic hypermutation. Muschen et al. (2000b) recently showed that human B cells can acquire somatic mutations of the CD95 gene during the germinal-center reaction conferring resistance to apoptosis. Mutations of the CD95 gene have been reported in approximately 60% of non-Hodgkin’s lymphomas derived from postgerminal-center B cells (Xerri et al., 1995; Landowski et al., 1997) and somatic mutations of CD95 have also been found in solid tumors including bladder cancer (Lee et al., 1999a), malignant melanoma (Shin et al., 1999), and nonsmall cell lung cancer (Lee et al., 1999b). More recently, it was also reported that germline mutations in CD95 cause a 15-fold greater predisposition to lymphoma (Straus et al., 2001). Another mechanism of immune escape by tumors

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is by up-regulation of the inhibitor of CD95 signaling c-FLIP. Overexpression of c-FLIP in tumor cells results in escape from cytotoxic T cells in vivo (Medema et al., 1999). Moreover, high expression of c-FLIP in tumor cells results in the development of aggressive tumors showing high survival rate and vigorous growth (Djerbi et al., 1999; Medema et al., 1999). Increased expression of c-FLIP correlates with resistance to CD95-induced apoptosis in melanomas (Irmler et al., 1997; Griffith et al., 1998), colon adenocarcinomas (Ryu et al., 2001), adult T-cell leukemia (Kamihira et al., 2001), non-Hodgkin’s lymphoma (Irisarri et al., 2000) and cholangiocarcinomas (Que et al., 1999). Loss of sensitivity to CD95-induced apoptosis has also been observed in chronic myelogenous leukemia cells that express the Bcr-Abl fusion molecule (McGahon et al., 1995). Inhibition of Abl kinase restores induction of CD95-induced apoptosis in these cells. How Bcr-Abl confers resistance to CD95 remains, however, unknown.

CD95/CD95L in the pathology of diseases The list of diseases with suspected or established involvement of CD95/CD95L is long and still growing and we cannot cover the entire spectrum. We will therefore highlight a few representative examples.

Apoptosis in AIDS Depletion of CD4 T cells is a hallmark of the pathogenesis of AIDS. In HIV-infected individuals low numbers of T cells are productively infected with HIV suggesting that T-cell depletion in HIV comprises both infected and non-infected T cells. Multiple theories have been proposed to explain the mechanism of T-cell depletion and it appears that T-cell depletion is indeed likely to be a multifactorial process. Here, we will discuss some of the findings that suggest a role for CD95 in HIV-induced T-cell depletion. Accumulating data suggest that depletion of CD4 T cells in HIVinfected individuals is secondary to enhanced lymphocyte apoptosis (Meyaard et al., 1992; Gougen et al., 1993; Finkel et al., 1995). CD4 T cells from HIV-infected individuals show increased CD95 expression and are highly sensitive to CD95-induced apoptosis when compared with HIV-seronegative

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CD 95 L / FASL AND ITS RECEPTOR CD 95 ( APO - 1/ FAS )

controls (Katsikis et al., 1995; McCloskey et al., 1995; Badley et al., 1997), suggesting that CD95L/CD95 interactions are important in CD4 T-cell depletion. In addition, membrane-bound and soluble CD95L have been shown to be elevated in HIV-infected patients and to correlate with HIV viral burden (Mitra et al., 1996; Silvestris et al., 1998; Hosaka et al., 1998). The direct cytotoxic effects of HIV on T cells seems to be independent of CD95 since HIV-induced death of T cells from ALPS patients with a mutation in CD95 is indistinguishable from controls (Ghandi et al., 1998). However, accumulating evidence suggests that various HIV viral proteins have the potential to sensitize T cells to CD95-induced apoptosis (Westendorp et al., 1995a; Oyaizu et al., 1997; Algeciras et al., 1998; Zauli et al., 1998). One potential mechanism leading to enhanced susceptibility to apoptosis includes the interaction of HIV gp120 with CD4 and the chemokine receptors CXCR4 and CCR5. This is achieved by gp120 crosslinking of CD4, resulting in the increased expression of CD95 and enhanced sensitivity to apoptosis (Oyaizu et al., 1994; Accornero et al., 1997; Algeciras et al., 1998). In addition, crosslinking of CD4 has been shown to induce CD95L expression in monocytes and T cells (Westendorp et al., 1995a; Oyaizu et al., 1997; Algeciras et al., 1998). In HIV-infected individuals, the chance of gp120 interaction with CD4 is very high. In addition to the gp120 that is physically associated with intact HIV virions, gp120 is present on the surface of cells infected with HIV. HIV gp120 has also been shown to circulate in free form, complexed with anti-gp120 antibodies, and as part of defective, noninfectious virions (Ellaurie et al., 1990; Oh et al., 1992; Feijoó et al., 1995). Because CD4 T cells may interact with gp120 without becoming infected, this mechanism might explain depletion of CD4 T cells that are not infected with HIV. Recent evidence suggests that chemokine receptors may play a role in the induction of apoptosis by gp120. However, the mechanism of chemokine receptor-induced apoptosis is controversial: it appears that CXCR4 signals a caspaseindependent (Berndt et al., 1998; Vlahakis et al., 2001) whereas CCR5 triggers a caspase-dependent form of apoptosis (Vlahakis et al., 2001). Other HIV proteins implicated in the induction of apoptosis include Tat, Nef, Vpr and the HIV protease. Tat and Nef have been implicated in CD95-induced apoptosis as both can

up-regulate CD95L expression (Westendorp et al., 1995a; Zauli et al., 1998). HIV Tat can enhance apoptosis of uninfected cells by inducing a pro-oxidative state in the affected cells and by rendering them sensitive to TCR-induced apoptosis which is mediated by a CD95/CD95L interaction (Li et al., 1995; Westendorp et al., 1995a, 1995b). In addition Nef, Vpr and the HIV protease have been implicated in CD95-independent apoptosis of infected T cells. For example, HIV protease can cleave the antiapoptotic protein Bcl-2 (Strack et al., 1996), while vpr can trigger apoptosis through the mitochondrial pathway (Jacotot et al., 2000). In summary, activation of CD95L and susceptibility to CD95 by some HIV viral proteins may be responsible for cell death of uninfected T cells resulting in CD4 T-cell depletion and HIV pathogenesis.

CD95/CD95L system in autoimmune diseases Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease of the CNS that causes prolonged and severe disability in young adults. Lack of T-cell apoptosis as well as damage to myelin, oligodendrocytes and neurons caused by immune cells are pathogenic mechanisms in MS (Martin et al., 1992). T cells specific for myelin antigens are believe to initiate and perpetuate the immune process of the disease. There is direct and indirect evidence that T-cell apoptosis is impaired in MS patients (Macchi et al., 1999; Zang et al., 1999; Comi et al., 2000). Therapeutic studies suggest that induction of T-cell apoptosis by AICD could be a treatment for the disease since in vitro studies indicated impaired deletion of myelinspecific T cells in MS patients as well as defective deletion of polyclonal T cells (Critchfield et al., 1994; Zhang et al., 1994; Macchi et al., 1999; Zang et al., 1999). Recently, it was shown that the expression of c-FLIP is elevated in activated T cells from patients with clinically active MS when compared with naive T cells from MS patients or control individuals (Sharief, 2000; Semra et al., 2001). High levels of c-FLIP in active MS correlated with resistance to CD95-induced apoptosis. In addition, some studies have reported high levels of soluble CD95 in the serum of patients with relapsing remitting and active MS (Inoue et al., 1997; Zipp et al., 1998; Sakai et al.,

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1999). This soluble CD95 is able to block apoptosis induced by CD95L on susceptible cells. However, other authors did not observe differences in sCD95 levels in sera from MS patients when compared with controls (Bansil et al., 1997; Ciusani et al., 1998). Whether sCD95 is responsible for decreased T-cell apoptosis in MS requires further investigation. Apoptotic cell death of oligodendroglial cells has been confirmed in MS lesions by morphology and DNA laddering (Dowling et al., 1996). It has been suggested that CD95-induced apoptosis may contribute to oligodendroglial cell loss in MS (Dowling et al., 1996). Enhanced CD95 and CD95L expression is detected in brain lesions of MS patients when compared with brain derived from patients with other neurological or non-neurological diseases (D’Souza et al., 1996). CD95L expression has been detected mainly in lymphocytes, microglial cells, astrocytes and oligodendroglial cells, whereas CD95 expression has been shown to be increased in oligodendroglial cells in MS (Dowling et al., 1996; D’Souza et al., 1996; Bonetti et al., 1997; Huang et al., 2000). In vitro studies indicated that oligodendroglial cells are indeed sensitive to CD95-induced death, however, they did not exhibit the classical morphologic features of apoptosis (D’Souza et al., 1996). Further studies are necessary to confirm whether the CD95/CD95L pathway contributes in vivo to oligodendroglial cell loss characteristic for MS. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model for human MS. In EAE, the CD95/CD95L system plays a role in disease pathogenesis. Mice harboring the lpr and gld mutations show ameliorated clinical signs of EAE (Sabelko et al., 1997; Waldner et al., 1997), suggesting that CD95 and CD95L are involved in the development of EAE by possibly inducing death within the target tissue. Adoptive transfer experiments demonstrated that transfer of CD95L-deficient lymphocytes into normal myelin basic protein TCR transgenic mice resulted in diminished disease (Dittel et al., 1999; SabelkoDownes et al., 1999). Furthermore, CD95 expressed in the recipient animals is important for the progression of EAE, since minimal clinical signs of EAE were detected after transfer of either wild-type or lpr T cells. In addition, CD95-mediated apoptosis has been shown to be important in the spontaneous remission after the initial acute inflammatory

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response in EAE (Suvannavejh et al., 2000; SabelkoDownes et al., 1999). CD95/CD95L also appears to play a role in other autoimmune diseases such as autoimmune diabetes and Hashimoto’s thyroiditis (HT). The CD95/CD95L system has been suggested to contribute to the development of insulin-dependent diabetes mellitus (IDDM) by inducing islet b cell destruction. Some of the findings that support this hypothesis include data that show CD95-deficient non-obese diabetic (NOD) mice fail to develop diabetes (Itoh et al., 1997), and transfer of splenocytes from NOD mice cells into NOD-lpr mice did not result in diabetes (Itoh et al., 1997; Kim et al., 2000). It was then shown that lpr mice express high levels of CD95L and inhibition of diabetes was overcome by treatment with antiCD95L antibodies, suggesting that CD95L-expressing lymphocytes of the lpr mice induced apoptosis of the injected T cells, thereby preventing development of diabetes (Itoh et al., 1997; Kim et al., 2000). Additionally, NOD heterozygous gld (/gld) mice, which are partially deficient in CD95L, do not develop diabetes despite the presence of T-cell islet infiltrates (Su et al., 2000). In humans, analysis of pancreas sections from patients with type I diabetes showed that CD95 is expressed on lymphocytes and most islet b cells whereas CD95L was exclusively detected on isletinfiltrating lymphocytes (Moriwaki et al., 1999), suggesting that the CD95 system plays a role in the development of autoimmune diabetes in mouse models and in humans. The role of CD95 in HT is more controversial. Normal thyrocytes express very low levels of CD95 but expression of the receptor is elevated in thyrocytes from HT patients (Giordano et al., 1997). Although very few CD95L-expressing lymphocytes have been detected in HT thyroids (Stassi et al., 1999), it has been shown that CD95L is constitutively expressed in normal thyrocytes and it is up-regulated during the active phase of HT (Mitsiades et al., 1998). It has been suggested that co-expression of CD95 and CD95L on thyrocytes may lead to selfdestruction of thyroid cells (Giordano et al., 1997; Mitsiades et al., 1998). However, the expression of CD95L in thyrocytes has been a matter of debate due to the use of different anti-CD95L antibodies and the detection methods. In vivo data will be necessary to corroborate the role of CD95-induced apoptosis in HT.

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CD 95 L / FASL AND ITS RECEPTOR CD 95 ( APO - 1/ FAS )

CD95 receptor signaling and inhibition The enormous profusion of publications describing CD95 signaling in recent years is a testament to both the importance of the pathway transduced by this receptor as well as to the complexity of its signaling. Since the cloning of CD95 in 1991 over 10 000 publications involving this receptor have been produced. The majority of these publications describe the apoptotic pathway of CD95, which has been well demonstrated. However, a number of publications have implicated the CD95 receptor in alternative signaling pathways. These include the induction of proliferation, and co-stimulation of T cells during TCR signaling. Additionally, numerous inhibitory mechanisms have been described in CD95 signaling, which are capable of blocking cell death. The physiologic importance of the different inhibitors of apoptosis has been the subject of much controversy. Overall, the prodigious publication record of this receptor has highlighted its central role in apoptosis, even as recent and forthcoming publications begin to elucidate the increasing evidence for the role of the receptor in alternate pathways. The role of the CD95 receptor in signaling for apoptosis is the best characterized of its functions and CD95 is the best characterized member of the TNF superfamily of receptors (Schulze-Osthoff et al., 1998). CD95 is expressed on the surface of cells as preassociated homotrimers (Papoff et al., 1999; Siegel et al., 2000b). A similar association was described for the several members of the TNF superfamily, including the TNF receptor itself, CD40 and the TRAIL receptor I (Chan et al., 2000). These interactions were found to be mediated by a domain in the N-terminus, within the first of three cysteine-rich domains (Papoff et al., 1999; Siegel et al., 2000b). This association was described by binding of in vitro-generated proteins to cells transfected with CD95, and by the use of chemical crosslinking reagents that allowed visualization of the associated homotrimers present on the cell surface (Papoff et al., 1999; Siegel et al., 2000b). Additionally, the use of the fluorescence resonance energy transfer technique, in which the emission wavelength of one spectral variant of the green fluorescent protein (GFP) is used to excite a second spectral variant of GFP when the two are in close proximity (approximately 100 Å), supported these observations. Using

this method Siegel et al. (2000b) demonstrated that a deletion or mutation in certain regions of the first cysteine-rich domain of CD95 led to the disruption of these interactions. They termed this domain the PLAD for pre-ligand association domain. Functionally, disruption of the association mediated by this domain led to reduced apoptotic potency using agonistic anti-CD95 antibodies or CD95L. Although the precise molecular role of this pre-association is not known, the physiologic importance of the domain has been characterized. Mutations in this domain have been shown to result in the autoimmune disorder ALPS (Siegel et al., 2000b), indicating the importance of pre-association for signaling physiologically. This finding gains additional importance physiologically, due to the requirement for heterotrimerization for efficient CD95 signaling. Heterozygous mutations in the CD95 gene, which are found in ALPS patients, could therefore act as dominant-negative mutations, interrupting signaling, because CD95 functions as a trimer; a single mutant protein could significantly disrupt signaling by CD95. It has been suggested that superclustering of CD95 may be required for efficient signaling (Siegel et al., 2000a), a molecular configuration that would be especially susceptible to disruption by a single mutant protein. Pre-association of CD95 was observed to be independent of the expression of the intracellular domain of CD95 (Siegel et al., 2000b). The exact molecular mechanism of the initiation of signaling through CD95 awaits further investigation, however, the general aspects of this initiation are well known. CD95 contains a protein–protein interaction domain in its cytoplasmic region termed the death domain (DD), a characteristic region of the death receptor family of the TNF superfamily which includes CD95 as well as TNF receptor I (TNF-RI), DR3/APO-3/TRAMP, TNFrelated apoptosis inducing ligand (TRAIL) receptor 1/DR4 and TRAIL-R2/DR5 (Peter et al., 1998). When the pre-associated receptor is ligated, CD95 becomes competent to recruit the adaptor molecule FADD (Fas-associated death domain containing protein) through homotypic interactions of the DD of CD95 and the DD of FADD (Plate 38.2) (Kischkel et al., 1995; Chinnaiyan et al., 1996). FADD contains in addition to its DD, another protein–protein interaction domain at its N-terminus, termed the death effector domain

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(DED). This domain is required for the recruitment of caspases containing these domains. Both the DD and DED enable the interaction with other proteins containing these same domains. Caspase 8 is the best described and one of the two known DED-containing caspases (Muzio et al., 1996; Boldin et al., 1996). Caspases are cysteine proteases which cleave after a loosely specific series of four amino acids and which absolutely require the presence of an aspartate in the P1 position of their substrate (Earnshaw et al., 1999). These proteases are responsible for performing the labor of apoptosis. Various caspases are involved in both the initiation of the apoptotic process and the execution of the final apoptotic program. The apoptotic caspases are therefore segregated according to their role. The effector caspases, which include caspases 3, 7 and 6 are responsible for most of the cleavage of proteins characteristic of apoptosis, and are therefore responsible for the cleavage of the proteins which induce the major morphological changes observed during programmed cell death (Earnshaw et al., 1999). The initiator caspases transduce the first signals of apoptosis. In apoptosis induced by CD95, the initiator caspases contain tandem DED which homotypically interact with the DED of FADD, once this adaptor has been recruited to the CD95 receptor. The best studied of these caspases is caspase 8. Caspase 8 is expressed as two isoforms, caspase 8/a and 8/b which are both recruited to the activated CD95 receptor (Scaffidi et al., 1997). The association of CD95, FADD and caspase 8 (and potentially other associated molecules, see below) comprises the death-inducing signaling complex, or DISC (Plate 38.2), which is responsible for the generation of active caspase 8 (Boldin et al., 1996; Kischkel et al., 1995; Muzio et al., 1996). Caspase 8 resides primarily in the cytoplasm (or as we have recently observed, on the mitochondria) (Stegh et al., 2000, 2002) as an inactive zymogen, and is recruited to the receptor upon binding of FADD. Once caspase 8 associates with FADD, the high local concentration of caspase 8 leads to what is believed to be its autoproteolytic cleavage and activation (Salvesen and Dixit, 1999). Aside from its two DED, caspase 8 contains a protease domain consisting of two subunits. Following the autoproteolytic cleavage of the enzyme, caspase 8 is released from the DISC as an active heterotetramer containing two p18 and two p10 subunits (Plate 38.2). This active

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caspase 8 is then released into the cytoplasm of the cell as an active protease and initiates the apoptotic program. A second caspase has recently been shown to associate with FADD through the homotypic association with its DED, and to be activated in the DISC. Caspase 10 was cloned several years ago as a homolog of caspase 8 and is the only other caspase known to contain tandem DED. Association with FADD has been demonstrated in vitro, as has the ability of caspase 10 to induce apoptosis when overexpressed. We and others (Kischkel et al., 2001; Wang et al., 2001; Chang et al., 2002) have isolated caspase 10 associated with the DISC and Wang et al. have demonstrated that caspase 10 is expressed, recruited to the DISC and proteolytically processed, and is processed by drug induced dimerization in vitro, a behavior identical to that of caspase 8. The substrate specificity of caspase 10 may be different from caspase 8, however, making it attractive to speculate that the two caspases may play different roles in signaling through the CD95 receptor (Wang et al., 2001). Another study has implicated the lack of functional caspase 10 in one form of the autoimmune disease ALPS type II (Wang et al., 1999). This finding suggests that caspase 10 may indeed have a role in signaling apoptosis. The lack of a caspase 10 ortholog in the mouse has complicated the study of this caspase, however, although functional expression appears to be important in the human system. Following the activation of caspase 8 in the DISC, the protein can initiate the apoptotic program. Our laboratory has described two pathways of CD95 apoptosis signaling which are centered around the production of caspase 8 in the DISC (Scaffidi et al., 1998). In type I cells, a fulminant production of caspase 8 at the DISC is able to process the effector caspase, caspase 3 directly, leading to its activation and the ultimate apoptosis of the cell (Plate 38.2). In type II cells, however, a very small amount of caspase 8 is produced in the DISC, indeed, the DISC in these cells is quite poorly formed, with little recruitment of FADD and little active caspase 8 induction. Apoptosis in these cells is dependent, at least in part, on the cleavage of the BH3 domain containing Bcl-2 family member BID (Li et al., 1998b; Luo et al., 1998), whose cleavage results in the proapoptotic fragment termed tBID. This fragment induces the proapoptotic

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CD 95 L / FASL AND ITS RECEPTOR CD 95 ( APO - 1/ FAS )

functions of the mitochondria by causing aggregation of Bax or Bak (reviewed in Korsmeyer et al., 2000). This is followed by the loss of cytochrome c from the mitochondrial intermembrane space and the induction of caspase 9 activation, with the assistance of the adaptor molecule Apaf-1 and dATP (Li et al., 1997). Caspase 9 then activates caspase 3 resulting in the apoptosis of the cell. Another hallmark of CD95mediated apoptosis in type II cells is the effect of the expression of the antiapoptotic members of the Bcl-2 family. Expression of either Bcl-2 or Bcl-xL renders type II cells resistant to CD95-mediated apoptosis (Scaffidi et al., 1998). Type I cells, however, are not able to overcome the production of such large amounts of caspase 8 at the DISC and therefore are not protected from CD95-mediated apoptosis even by expression of very high levels of Bcl-2 or Bcl-xL (Scaffidi et al., 1998). The physiologic importance of the two pathway models has recently been validated by the description of mice which forcibly express members of the apoptosis pathway, or which are deficient in various molecules involved in apoptosis. Mice deficient in BID have thymocytes which are not protected from CD95-mediated apoptosis by the loss of this molecule, whereas their hepatocytes are protected from CD95-induced apoptosis (Yin et al., 1999). Bax/Bak doubly deficient mice demonstrate resistance to CD95-mediated apoptosis in their hepatocytes as well, whereas their thymocytes and T cells are affected similarly to control mice (Lindsten et al., 2000; Wei et al., 2001). The hepatocytes of mice expressing Bcl-2 as a transgene are also protected from CD95-induced apoptosis, whereas their T cells and thymocytes from mice expressing Bcl-2 as a transgene in T cells are not protected from apoptosis by CD95 (Strasser et al., 1995; Lacronique et al., 1996; Rodriguez et al., 1996). Thus, thymocytes and T cells are type I cells and hepatocytes type II. Several other proteins have been shown to be recruited to the DISC by direct interaction with DISC proteins. The role of many of these proteins is at present unclear, although others have been more completely described, even though they remain controversial. The viral cell death inhibitor v-FLIP (viral-FLICE-like inhibitory protein) was identified as a protein, expressed by c-herpesviruses, capable of blocking CD95-mediated apoptosis through association with the receptor in the DISC (Hu et al., 1997a;

Bertin et al., 1997; Thome et al., 1997). This protein contains two tandem DED, which are highly homologous to the N-terminus of caspase 8. Two cellular homologs of the viral protein were quickly identified (Irmler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997; Inohara et al., 1997; Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997b), termed c-FLIPS (short) and c-FLIPL (long). c-FLIPS contains only the tandem DEDs and is quite similar to v-FLIP. c-FLIPL contains not only the tandem DEDs, but a protease-like domain, homologous to caspase 8 in which several amino acids important for protease activity are mutated, including the active site cysteine. This inactive homolog was found to be cleaved in the DISC by caspase 8 (Scaffidi et al., 1999), and has been reported to be either an inhibitor of apoptosis signaling or an inducer of apoptosis (presumably by oligomerizing caspase 8 and inducing its autoproteolytic activation). The inhibition of apoptosis has been shown to be through the recruitment to and cleavage in the DISC, but because c-FLIPL is not an active protease, the cleavage is not reciprocated, resulting in the generation of two p10 subunits (one from caspase 8, the other from c-FLIPL) followed by no further cleavage and therefore an inactive caspase 8 molecule (Krueger et al., 2001). c-FLIPL was shown to be down-regulated in T cells following treatment of IL-2 and was suggested to be responsible for the CD95 apoptosis sensitivity of long-term-activated peripheral T cells (Refaeli et al., 1998; Algeciras-Schimnich et al., 1999). In contrast, we recently reported that CD95 sensitivity in peripheral T cells is regulated by differentiation of T cells from a type II to a type I phenotype without any changes in the expression levels of c-FLIPL (Scaffidi et al., 1999). In fact, we recently showed that c-FLIPL, depending on its expression level, can either activate caspase 8/10 in the DISC (at low concentrations) or block it (at high concentrations) (Chang et al., 2002), so even an increase of c-FLIPL expression does not necessarily mean that cells are more resistant to CD95-mediated apoptosis. The situation is different for c-FLIPS, which only seems to have antiapoptotic activity similar to the v-FLIP proteins. Both restimulated and co-stimulated T cells were shown to specifically up-regulate c-FLIPS (Kirchhoff et al., 2000a, 2000b). A large number of studies concerning the function of c-FLIP have been published, implicating c-FLIP in

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numerous aspects of apoptosis. c-FLIP-deficient mice display a phenotype similar to that of caspase 8deficient animals, and the mice are not viable past day E10.5 (Yeh et al., 2000). This has complicated the study of this molecule in vivo but does underscore its apparent importance, at least in development. Recently, c-FLIP has been implicated in signaling alternative pathways, linking the CD95 receptor to the NFjB, JNK and MAPK pathways. Several studies have shown that c-FLIP is involved in the transmission of the NFjB pathway (Hu et al., 2000; Chaudhary et al., 2000; Kataoka et al., 2000). One study found that c-FLIP recruited not only TRAF1 and 2 but RIP and Raf-1 in addition, which activated the extracellular signal-regulated kinase (Erk) pathway (Kataoka et al., 2000). These studies link the CD95 signaling pathway to survival pathways in addition to its well-known role in apoptosis. Several of these molecules are wellestablished mediators of TNF receptor signaling (reviewed in Wallach et al., 1999), although their role in CD95 signaling is still controversial. These studies do raise the interesting possibility that through c-FLIP, CD95 may be induced to signal in pathways that are distinct from those leading to the apoptosis of the cell. The physiologic role of CD95 in these pathways is by no means clear however, and requires more study to determine its significance. Several other proteins have been shown to associate with the CD95 receptor, aside from those described above as mediators of death receptor-induced apoptosis. Daxx was identified as a CD95-binding protein which could link the receptor to the JNK signaling pathway (Yang et al., 1997). Overexpression of Daxx led to apoptosis, and Daxx was shown to interact with the C-terminal portion of CD95, which contains the DD. It was suggested that Daxx induces CD95-mediated apoptosis through an alternate pathway, which involves the induction of apoptosis through JNK activation followed by activation of an unknown caspase. The role of Daxx physiologically is not known, however, as Daxx has not been shown directly to associate with the DISC. It is interesting, however, that Daxx was also shown to be an integral part of nuclear PML bodies (Torii et al., 1999; Li et al., 2000a; Zhong et al., 2000) and PML-deficient mice have a defect in CD95-mediated apoptosis (Wang, R. et al., 1998). At present the molecular basis for this cytosolic/nuclear connection in the CD95 pathway is unknown.

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FAP-1, or Fas-associated phosphatase-1, was identified in a yeast two-hybrid screen as a phosphatase that associates with the extreme C-terminus of the CD95 receptor (Sato et al., 1995), indeed only the final three amino acids of the CD95 receptor are necessary for the binding of one of the FAP-1 domains. In that study, it was suggested that this phosphatase was a negative regulator of CD95 signaling, and studies suggest that FAP-1-transfected cells are resistant to CD95-mediated apoptosis and that the activation of caspase 8 and thereby caspase 3 are inhibited in these cells (Li et al., 2000b). Additionally, down-regulation of FAP-1 has been suggested to contribute to the IL-2induced sensitivity of activated T cells, one study found a decrease in FAP-1 mRNA in cells by competitive RT-PCR (Zhou et al., 1998a). It is interesting to note that the three amino acids in human CD95 involved in binding to FAP-1 are not found in mouse CD95 (Yanagisawa et al., 1997). The physiologic role of the binding of FAP-1 to CD95 has therefore not been sufficiently established. The protein FLASH (FLICE-associated huge protein) was also suggested to associate with the CD95 receptor (Imai et al., 1999), and was shown to be recruited to the DISC of the activated receptor and actually required for the activation of caspase 8 there, but recent studies have cast serious doubt on the validity of these data and subsequent claims (Koonin et al., 1999). RIP was described as a DD-containing protein which could interact with the CD95 receptor (Stanger et al., 1995). The role of this kinase is, however, not completely clear. A recent study implicates RIP in the initiation of a caspase 8independent death signaling pathway through the CD95 receptor (Holler et al., 2000) resulting in a necrotic cell death. It was suggested that this kinase binds to the CD95 receptor, phosphorylates an unknown protein and leads to the CD95-mediated, necrotic death of the cell. This pathway was suggested to occur in primary human T cells treated with the caspase inhibitor z-VAD-fmk, which in this study resulted in equal levels of cell death upon induction of AICD. The role of these molecules in cell death is suggestive, although they require much more study before their physiologic roles are fully characterized. Several modes of inhibition of caspase signaling through CD95 have been characterized in detail. The cowpox virus expresses a serpine-like protease inhibitor, CrmA. This caspase inhibitor has been

THE CYTOKINES AND CHEMOKINES

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CD 95 L / FASL AND ITS RECEPTOR CD 95 ( APO - 1/ FAS )

shown to inhibit activity of caspase-1 potently, primarily involved in inflammatory cytokine processing, as well as caspase 8 (Zhou et al., 1997). The inhibition of caspase 8 by this viral inhibitor has been linked to the inhibition of the apoptosis of virally infected cells. Another mechanism of inhibition of CD95 signaling by viral infection is by forced internalization and/or degradation of the CD95 molecule from the cell surface. Removal of CD95 leads to the loss of apoptotic potency, and to the survival of the cell. This mode of receptor inhibition was described for the adenovirus, through the activity of the protein RID (E3/10.4K–14.5K), which results in the forced internalization and degradation through a lysosomally mediated pathway, inhibitable by bafilomycin A, which blocks the transition of endosomes into lysosomes and thereby degradation of the receptor (Tollefson et al., 1998; Elsing and Burgert et al., 1998). The activation of CD95 receptor signaling has been described in the absence of CD95 ligand-mediated stimulation. In these pathways, induced by the drug thymidine kinase/ganciclovir (TK/GCV) (Beltinger et al., 1999) or by exposure to ultraviolet light (UV) (Aragane et al., 1998), CD95 ligand-independent induction of activation of the DISC and apoptosis of the cells occur. TK/GCV was demonstrated to lead to the aggregation of the CD95 receptor and the recruitment of FADD, forming an active DISC which was able to recruit and activate caspase 8. Additionally, this activity was inhibited by the expression of a dominant-negative form of FADD and by the caspase 8-selective inhibitor z-IETD-fmk, in a manner similar to ligand-induced apoptosis (Beltinger et al., 1999). UV light was also shown to induce the aggregation of the CD95 receptor, which was able to recruit FADD, leading to the apoptosis of the cell (Aragane et al., 1998). Whether this is a physiological relevant pathway in apoptosis of UV-exposed cells is questionable, however, although the expression of a dominantnegative form of FADD did decrease the apoptosis of cells, suggesting that these cells may utilize the CD95 ligand-independent CD95 pathway to apoptosis. However, expression of this molecule did not fully block apoptosis, suggesting other apoptotic mechanisms are functioning (Aragane et al., 1998). Clearly, the role of DNA damage in this system is critical, although CD95 aggregation and DISC formation was suggested to be a possible secondary, though not

mutually exclusive pathway involved in UV-induced apoptosis. We and others have recently described the stimulation-induced clustering of CD95 (Cremesti et al., 2001; Grassme et al., 2001; Algeciras-Schimnich et al., 2002). We found that ligation of CD95 with soluble ligand or agonistic antibody resulted in the aggregation of the receptor followed by its internalization into an endosomal pathway. This occurred in a variety of cell lines including one that did not subsequently undergo apoptosis due to the protective expression of Bcl-xL (Stegh et al., 2002). This clustering and internalization was dependent on the actin cytoskeleton as treatment of cells with latrunculin A, an inhibitor of the formation and stability of actin filaments led to the inhibition of this phenomenon. Furthermore, treatment with this drug led to the inhibition of CD95mediated apoptosis, implicating the actin filament network in the induction of CD95-mediated apoptosis. Additionally, the clustering and internalization of CD95 was dependent on the activation of caspases, specifically, of caspase 8. This is the first study to implicate the involvement of caspases in the internalization of surface receptors. Several other studies have described the stimulation-induced capping of the CD95 receptor. These studies implicated the involvement of ceramide released by acid sphingomyelinase, which translocated to the extracellular surface of stimulated cells, enriched in lipid rafts, and induced the release of ceramide which led to the clustering of CD95 and subsequent apoptosis (Grassme et al., 2001). Another study suggests that ceramide is essential for the efficient signaling of CD95 and that the signaling is preceded by the capping of CD95 (Cremesti et al., 2001). In our studies, the clustering and internalization of CD95 were independent of lipid rafts suggesting that these structures are not required for signaling in our systems (Algeciras-Schimnich et al., 2002). The physiologic implications of CD95 internalization are not yet known, but we have speculated that this phenomenon could play a role in the protection of cells from receiving an apoptotic signal from neighboring cells which have a CD95 receptor that has bound soluble CD95 ligand, and may play a role in the evasion of some tumor cells from CD95mediated apoptosis. The CD95 receptor has recently been implicated in the stimulation of T cells. It has been known for some

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time that the CD95 receptor can transduce activation signals to T cells (Alderson et al., 1993). This study has gained support recently with reports on the requirement for active caspases in T-cell proliferation. Some of the first indications for this possibility arose with the observations that T cells from mice deficient in or expressing a transgene for the dominant-negative form of FADD had a defect in proliferation (Newton et al., 1998; Zhang et al., 1998). Additionally, mice expressing a Bcl-2 transgene had a deficiency in proliferation (O’Reilly et al., 1996). Two recent studies have suggested that the activation of caspases occurs following stimulation through the TCR and that treating cells with caspase inhibitors led to a loss of proliferative capacity that was not due to apoptosis (Kennedy et al., 1999; Alam et al., 1999). Furthermore, one study determined that T cells stimulated with suboptimal levels of anti-CD3 and CD95 ligand responded with more robust proliferation than cells stimulated with anti-CD3 alone, and that treatment of cells with a CD95–Fc fusion which blocked CD95 signaling led to a decrease in the proliferative response of T cells (Alam et al., 1999). Interestingly, these studies implicate CD95 in the co-stimulation of T cells and suggest that there may be a role for caspases in other non-apoptotic pathways. Perhaps in cells in which the balance between proliferation and apoptosis is tipped toward proliferation, caspases are important in aiding this proliferative pathway by cleavage of certain substrates that may inhibit progression through the cell cycle. Indeed, one study implicating the activation of caspases in proliferation described the cleavage of a protein that inhibits cell cycle progression, Wee1, an inhibitor of the Cdc2 cyclindependent kinase which is required for the G2/M transition (Zhou et al., 1998b). Thus, caspase activity induced by stimulation through the CD95 receptor may play a critical, non-apoptotic, and indeed, proliferative role. The exact pathway that is initiated in CD95-co-stimulated T cells requires further study. Additionally, the stimulation of memory T cells results in significantly less apoptosis than in naive T cells (Inaba et al., 1999). In this study, TCR transgenic mice were used to produce memory cells which were much less susceptible to CD95-mediated apoptosis than activated naïve T cells. This resistance was found to be at least in part due to the increase in c-FLIP expression in these restimulated cells. This study suggests

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that CD95 signaling is differently regulated in restimulated T cells compared with naïve T cells. Additionally, it was demonstrated that the regulation of c-FLIP was dependent on the cell cycle status of T cells (Algeciras-Schimnich et al., 1999). In T cells stimulated through the TCR, this study found that T cells in the G1 phase expressed high levels of c-FLIP, whereas in S phase c-FLIP levels were lower. These results may help to explain the observation that T cells are more susceptible to TCR-mediated apoptosis in the S phase of the cell cycle (Dao et al., 1997). Another study using T-cell clones and the cell line Jurkat found that in these cells there was no difference in apoptosis susceptibility dependent on the cell cycle (Karas et al., 1999). The differences in these studies are probably due to the model system used.

Areas of active research The critical in vivo role of the CD95 receptor is evident in mice and humans which harbor defects in functionally important regions, resulting in defective CD95 signaling. The 10 years during which this receptor has been studied have demonstrated its profound importance. Research on CD95 is still very active, as is evident from the above descriptions of current and recent data. Functions of the receptor distinct from apoptosis have begun to be described, and it is clear that despite the tremendous amount of work that has been performed in the characterization of the signaling properties in apoptosis, there are many more avenues to investigate than were originally imagined. Included in this active research is the examination of CD95L as a co-stimulatory or proproliferative cytokine, the physiologic relevance of the distinction between soluble and membrane-bound ligand, and the potential exploitation of the CD95/CD95L system in tumor therapies and transplantation tolerance. Determining the nature of the block in the apoptosis pathway in many tumor cells is both an attractive method of tumor therapy and an area of active research. Additionally, c-FLIP has generated much interest recently, as this molecule is certainly involved in the regulation of DISC activity, and has recently been suggested to link the CD95 receptor to several signaling pathways distinct from those which lead to apoptosis. The molecular differences between type I and type II cells is still unknown as is the mechanism

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CD 95 L / FASL AND ITS RECEPTOR CD 95 ( APO - 1/ FAS )

of the differentiation between these two types of CD95-mediated apoptosis. The recently described clustering and internalization of CD95 awaits further study as well, especially in the determination of the role of this process physiologically, and its role in tumor cells, as well as the involvement of lipid rafts in the process. The role of nuclear events in CD95mediated apoptosis awaits further characterization; for example, current research has led to an interest in PML in apoptosis. The role of several proteins in the regulation of CD95 signaling at the plasma membrane additionally is not well characterized and further study of this process may lead to an understanding of the molecular mechanisms that regulate the signaling of this receptor. These are but a few of the many areas of current research on this receptor–ligand pair. Because the study of this receptor is still very active, many of the current areas of research are highlighted in the above sections. A deeper understanding of this receptor–ligand pair, especially in light of the knowledge of its importance in such a wide variety of diseases, will undoubtedly lead to numerous effective treatments for a vast number of human disorders.

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39 HMGB-1 Haichao Wang,1,2 Christopher J. Czura1 and Kevin J. Tracey1 1

North Shore-LIJ Research Institute, Manhasset, NY, USA 2

North Shore University Hospital, Manhasset, NY, USA

And the end of all our exploring Will be to arrive where we started And know the place for the first time T.S. Elliot

INTRODUCTION Sepsis, endotoxemia and hemorrhagic shock are lethal systemic inflammatory diseases, mediated primarily by proinflammatory cytokines such as TNF, IL-1b, and IL-6. However, death frequently occurs many hours to days after the initial activity of these and other cytokines, suggesting that late-acting, downstream mediators may also play critical roles in the lethality associated with systemic inflammation. HMGB-1 is a candidate late-acting cytokine, because it is released from activated macrophages and reaches peak serum levels 20–30 hours after the initial inflammatory response to endotoxin or peritonitis in animals. Inhibition of HMGB-1 activity confers significant protection against murine sepsis even when HMGB-1 antagonists are administered as late as 24 hours after disease onset, suggesting that targeting HMGB-1 may broaden the therapeutic window for acute inflammation. In this chapter, we review the cytokine biology of HMGB-1 as a late mediator of system inflammation. The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

HISTORY OF HMGB-1 DISCOVERY TNF is an early mediator of systemic inflammation, but animal models of lethal systemic inflammation (e.g. endotoxemia) indicate that the onset of death is often long after the early TNF response (Figure 39.1). In an effort to identify late-acting mediators of systemic inflammation that may be targeted late in disease progression, we initiated a search for other macrophage-derived, putative mediators released relatively late after TNF. We stimulated Murine macrophage-like RAW 264.7 cells were stimulated with endotoxin, and recovered conditioned media recovered 16 hours later. Protein gel electrophoresis revealed the presence of 30-kDa protein with an N-terminal amino acid sequence identical to murine HMGB-1 (Wang et al., 1999a), a member of the high mobility group-1 non-histone chromosomal protein family (Paonessa et al., 1987; Ferrari et al., 1994).

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FIGURE 39.1 Early versus late mediators of endotoxin lethality. Vertebrates treated with lethal doses of LPS succumb at latencies of up to several days, long after serum TNF has returned to basal levels. The kinetics of HMGB-1 release is delayed, and parallels the onset of lethality. Approximately 30 years ago, a group of proteins copurified with histone from thymus nuclei were termed ‘high mobility group’ (HMG) protein because of rapid mobility during electrophoresis (Johns, 1982). Mammalian HMG proteins are divided into three groups based on their size, sequence similarities and DNA-binding properties: (a) the HMGB-1/2 family, (b) the HMGN1/2 family and (c) the HMGA-1 family (Bustin et al., 1990; Sparrow and Wells, 1992; Maher

and Nathans, 1996). Non-mammalian HMG proteins include HMG T1 and T2 isolated from trout (Johns, 1982; Sparrow and Wells, 1992; Maher and Nathans, 1996), HMG-X from Xenopus (Ner et al., 1993; Ner and Travers, 1994; Kinoshita et al., 1994), and HMG D and Z from Drosophila (Johns, 1982). HMGB-1 and its homolog, HMGB-2, are closely related, often colocalized in cells, and exhibit 80% sequence homology to one another (Sparrow and Wells, 1992). HMGB-1 proteins are among the most ubiquitous, abundant and evolutionarily conserved proteins in eukaryotes. As a highly conserved protein, HMGB-1 shares 100% identity in amino acid sequence between mouse and rat, and a 99% amino acid identity between rodent and human (Figure 39.2) (Bustin et al., 1990). In mice, theHmgb1 gene resides on chromosome 5 (Gariboldi et al., 1995), while in humans the gene lies on chromosome 13 (Ferrari et al., 1996). HMGB-1 is produced by nearly all cell types, but cellular levels vary with development and age (Prasad and Thakur, 1988a).

CELLULAR LOCALIZATION OF HMGB-1 Nuclear HMGB-1 As a non-histone chromosomal protein, HMGB-1 has been implicated in diverse cellular functions, includ-

1 MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWK 50 HuHMGB-1 1 MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWK 50 MuHMGB-1 A Box 51 TMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPS 100 HuHMGB-1 51 TMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPS 100 MuHMGB-1 101 AFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAK 150 HuHMGB-1 101 AFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAK 150 MuHMGB-1 B Box 151 LKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEE 200 HuHMGB-1 151 LKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEDDEEDEEDEEEE 200 MuHMGB-1 201 EDEEDEDEEEDDDDE 215 HuHMGB1 (ACCESS # AAB08987) 201 EEEEDEDEEEDDDDE 215 MuHMGB1 (ACCESS # AAB20508)

FIGURE 39.2 Amino acid sequence of murine and human HMGB-1. The C-terminal portion of HMGB-1 contains a continuous sequence of between 35 and 40 aspartic and glutamic acid residues, and the N-terminal portion of HMGB-1 comprises two internal repeats of a positively charged domain of about 80 amino acids (termed ‘HMG boxes’) (shown by bold text). Two conservative amino acid substitutions in the human C terminus are underlined. THE CYTOKINES AND CHEMOKINES

STRUCTURAL FEATURES OF HMGB - 1

ing determination of nucleosomal structure and stability, and binding of transcription factors to their cognate DNA sequences (Bustin, 1999). The HMG boxes provide the structure-specific binding sites for DNA with secondary structures such as supercoiled DNA (Stros and Reich, 1998), cruciform DNA (Bianchi et al., 1989), four-way junctions (Hill and Reeves, 1997; Hill et al., 1999), and cisplatin-damaged DNA (Locker et al., 1995). These observations suggested a possible role for HMGB-1 in DNA recombination, repair, replication and gene transcription (Bustin, 1999). HMGB-1 binds the minor groove of doublestranded DNA in a sequence-independent manner, and covers approximately 14 bases or pairs (Butler et al., 1985; Bustin and Reeves, 1996). Although HMGB-1 has not been associated with gene transcription in vivo (Landsman and Bustin, 1991), it can stimulate transcription in vitro (Aizawa et al., 1994; Ge and Roeder, 1994; Zappavigna et al., 1996). HMGB-1 bends DNA and facilitates binding of various transcription factors to their cognate sequences, including the steroid/nuclear hormones progesterone (Onate et al., 1994; Zhang et al., 1999) and estrogen (Verrier et al., 1997; Zhang et al., 1999), HOX proteins (Zappavigna et al., 1996), p53 (Jayaraman et al., 1998), and transcription factor II B (Sutrias-Grau et al., 1999). Similarly, a Drosophila homolog of HMGB-1, termed DSP-1, can bind to a homolog of NFjB (i.e. Rel65) (Brickman et al., 1999), and participate in regulation of gene expression (Carballo et al., 1984). Hmgb1deficient mice are viable only for a few hours after birth, although parenterally administered glucose can prolong survival for a few days (Calogero et al., 1999). The lack of chromosomal HMGB-1 protein does not disrupt cell growth, but may affect the transcriptional regulation of certain genes, such as those activated by the glucocorticoid receptor (Calogero et al., 1999).

Cytoplasmic HMGB-1 Cellular localization studies have revealed that HMGB-1 is a cytoplasmic protein that can migrate between the cytoplasm and nucleus in a cell cycledependent fashion. Lymphoid cells contain significantly elevated levels of HMGB-1 in both cytoplasm and nucleus (Landsman and Bustin, 1993), whereas cells in liver and brain tissues contain HMGB-1 predominantly in the cytoplasm (Mosevitsky et al., 1989).

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The localization of HMGB-1 has been studied recently in immune cells under both in vivo (Kokkola et al., 2002) and in vitro (Rendon-Mitchell et al., 2003) conditions. For instance, Kokkola et al. reported that HMGB-1 immunostaining was predominantly found in the nucleus of synoviocytes and chondrocytes in biopsy specimens from normal rats (Kokkola et al., 2002). In inflammatory synovial tissue of both experimentally induced arthritis and clinical RA, however, HMGB-1 staining was found in both the nuclei and cytoplasmic regions of many mononuclear cells (Kokkola et al., 2002). We observed that quiescent cultures of murine macrophage-like RAW264.7 cells constitutively expressed HMGB-1 and maintained an intracellular ‘pool’ of HMGB-1 in the cytoplasmic and nuclear regions (Rendon-Mitchell et al., 2003). Following stimulation with IFNc, cellular HMGB-1 was observed predominantly in the cytoplasm as numerous aggregated granules, raising the possibility that nuclear HMGB-1 translocates to the cytoplasm before release into the extracellular milieu (Rendon-Mitchell et al., 2003).

STRUCTURAL FEATURES OF HMGB-1 HMGB-1 contains a continuous stretch of negatively charged (aspartic and glutamic acid) residues in the C-terminus, and two internal repeats of a positively charged domain (termed the ‘HMG box’) in the N-terminus (Figure 39.2) (Cary et al., 1983, 1984; Landsman and Bustin, 1993). The ‘HMG box’ is composed of approximately 80 amino acid residues, and forms three a-helixes that fold into an ‘L’-shaped structure (Einck and Bustin, 1985; Broadhurst et al., 1995; Bustin and Reeves, 1996). The two HMG boxes are designated A and B box, and share more than 30% homology in amino acid sequence (Weir et al., 1993). HMG boxes have been found in 120 proteins, ranging from protozoans to humans (Laudet et al., 1993; Landsman and Bustin, 1993). Proteins bearing HMG boxes are divided into two groups based on the number of HMG boxes in the protein. The first group contains two or more HMG boxes, and includes the founding members, HMGB-1 (also known as HMG-1 or amphoterin) and HMGB-2, the RNA polymerase I transcription factor UBF (Mosevitsky et al., 1989), its

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Xenopus homolog, xUBF (van de Wetering et al., 1991), the lymphoid-specific factor (LEF-1) (Love et al., 1995), the T-cell-specific transcription factor (TCF-1) (van de Wetering et al., 1991), and members of the V(D)J recombinase, and the mitochondrial transcription factors mtTF and ABF2 (Baxevanis et al., 1995; Baxevanis and Landsman, 1995; Bustin and Reeves, 1996). The second group contains proteins bearing a single HMG box, and includes DNA structure-specific binding proteins, non-histone chromatin-binding proteins, a number of transcription factors (Agrawal et al., 1998; Bustin et al., 1995), proteins involved in sex-type or mating-type determination such as the mammalian testis-determining factor (SRY) (Sinclair et al., 1990), and three fungal proteins (mat-1, ste11, Mc1) (Staben and Yanofsky, 1990; Sinclair et al., 1990; Sugimoto et al., 1991; Nasrin et al., 1991). HMG boxes provide the structural basis for DNA binding, and for recognition of distorted DNA structures as occurs in four-way junctions, cisplatinDNA adducts, and cruciform DNA (Landsman and Bustin, 1993).

POSTTRANSLATIONAL MODIFICATION OF HMGB-1 After translation, HMGB-1 undergoes posttranslational modifications including acetylation (Dimov et al., 1990), phosphorylation (Johns, 1982; Kimura et al., 1985; Wisniewski et al., 1999), methylation (Boffa et al., 1979), ADP-ribosylation (Johns, 1982; Tanuma et al., 1983), and glycosylation (Chao et al., 1994). Acetylation of HMGB-1 occurs on internal lysine residues (Elton and Reeves, 1986), and can be influenced by spermine, dexamethasone and hormones (Prasad and Thakur, 1988b; Pasqualini et al., 1989). Acetylation of HMGB-1 in the HMG box has been implicated in influencing chromatin structure (Johns, 1982), and may be required for participation in DNA replication and transcription (Sterner et al., 1979; Dimov et al., 1990; Wong et al., 1991). Phosphorylation of HMG proteins may also affect DNA affinity (Johns, 1982; Wisniewski et al., 1999). For instance, the Drosophila homolog of HMGB-1 is constitutively phosphorylated at multiple serine residues by casein kinase II (Wisniewski et al., 1999); dephosphorylation weakens binding of HMG protein to four-way

junction DNA without changing the strength of binding to linear DNA (Wisniewski et al., 1999).

EXTRACELLULAR HMGB-1 AS A MEDIATOR OF LETHAL SYSTEMIC INFLAMMATION Release of HMGB-1 after stimulation with endotoxin HMGB-1 is released from endotoxin-stimulated macrophage cultures in a time-dependent manner, with significant HMGB-1 accumulation first detectable 8 hours after lipopolysaccharide (LPS) stimulation (Wang et al., 1999a). The inducible nature of HMGB-1 release has also been observed in primary peritoneal macrophages, primary peripheral blood mononuclear cells (Wang et al., 1999a) and murine pituitary cells after stimulation with proinflammatory cytokines (e.g. TNF, IL-1b) (Wang et al., 1999b). Since TNF- and IL-1b-mediated inflammatory effects are often highly synergistic (Conti et al., 1988; Wankowicz et al., 1988; Elias and Lentz, 1990), it is plausible that such a synergistic interaction between TNF and IL-1b may be involved in the regulation of HMGB-1 release during systemic inflammation. Human primary blood mononuclear cell cultures stimulated with recombinant HMGB-1 release TNF in a dose-dependent manner (Andersson et al., 2000). Notably, up-regulation of TNF mRNA and protein following HMGB-1 stimulation is biphasic, with an early peak appearing 4 hours after addition of rHMGB-1, and a subsequent peak beginning at 10 hours post stimulation. The biphasic TNF response to HMGB-1 is inconsistent with the mono-phasic, rapid kinetics of TNF typically observed after endotoxin stimulation. Trypsin digestion completely abolishes the cytokine-stimulating activity of HMGB-1, but does not affect the cytokine-stimulating activity of endotoxin (Andersson et al., 2000), suggesting that the observed cytokine-stimulating activity is not due to endotoxin contamination. Structurally, HMGB-1 has two conserved DNAbinding domains, referred to as HMGB-1 boxes ‘A’ and ‘B’. Structure/function analyses revealed that B box recapitulated the cytokine activity of full-length HMGB-1, and efficiently activated macrophages to

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EXTRACELLULAR HMGB - 1 AS A MEDIATOR OF LETHAL SYSTEMIC INFLAMMATION

release TNF (Yang et al., 2003a). Further truncation of the B box revealed that the TNF-stimulating activity localizes to the first 20 amino acids of the B box (HMGB-1 amino acids 89–108) (Yang et al., 2003a). Exposure of Caco-2 cells to HMGB-1 and B box resulted in an increase in the production of nitric oxide and the production, but also the permeability, of Caco-2 monolayers in a time- and dose-dependent manner (Sappington et al., 2002).

HMGB-1 correlates with inflammatory responses in vivo To examine the kinetics of HMGB-1 accumulation in vivo, mice were challenged with sublethal doses of endotoxin or subjected to cecal ligation and puncture, and serum was assayed for HMGB-1 levels by immunoblot analyses. Serum HMGB-1 was first detectable 8 hours after administration of an LD50 dose of endotoxin, and increased to a prolonged plateau level from 16 to 32 hours after endotoxin infusion (Wang et al., 1999a). Similarly, serum HMGB-1 levels are increased significantly in a standardized model of murine sepsis (i.e. cecal ligation and puncture), beginning 18 hours after induction of peritonitis (Yang et al., 2002b). The increase of HMGB-1 levels in the serum of endotoxemic and septic mice correlates with lethality (Figure 39.1). The late appearance of HMGB-1 distinguishes it from TNF and other previously described early-acting mediators of LPS lethality (Wang et al., 2001). Based on the late and prolonged kinetics of HMGB1 accumulation in mice, we reasoned that it might be possible to detect increased serum HMGB-1 levels in critically ill patients with sepsis, as clinical signs of sepsis typically develop long after the early cytokine response to the acute infection. In the serum of 25 critically ill patients with surgical sepsis and sepsisinduced organ dysfunction (i.e. hypotension, lactic acidosis, disseminated intravascular coagulation, hypoxemia or decreased urine output) secondary to infection, HMGB-1 levels were significantly elevated as compared with the serum of healthy volunteers, which expressed no detectable HMGB-1 (Wang et al., 1999a). Moreover, serum HMGB-1 levels were significantly higher in septic patients who did not survive as compared with survivors (Wang et al., 1999a). Similarly, hemorrhagic shock, in the absence of infection,

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can stimulate the systemic release of HMGB-1. In a clinical case, serum HMGB-1 levels increased significantly within 24 hours after the onset of hemorrhagic shock, and returned towards basal levels as the clinical condition improved (Ombrellino et al., 1999). The mechanisms of HMGB-1 release in the absence of infection remain elusive, but it is plausible that HMGB-1 is released from both activated immunocytes, and from damaged and dying cells. Indeed, recent reports demonstrate that necrotic or damaged cells can passively release HMGB-1 (Degryse et al., 2001; Scaffidi et al., 2002). Interestingly, HMGB-1 is required to trigger inflammation, because necrotic cells derived from HMGB-1 knockout mice fail to induce an inflammatory response; these observations suggest that the release of HMGB-1 can signal the demise of a cell to its neighbors (Scaffidi et al., 2002). Notably, HMGB-1 is not released by apoptotic cells even after subsequent secondary necrosis and partial autolysis (Scaffidi et al., 2002), and therefore fails to promote inflammation even if not cleared promptly by phagocytic cells. Thus, it is likely that cells undergoing apoptosis are programmed to withhold the signal that is broadcast either by activated immune cells or cells that have been damaged or killed by tissue injury, shock and trauma (Scaffidi et al., 2002).

HMGB-1 causes tissue injury and sickness behavior Intratracheal administration of purified HMGB-1 to endotoxin-resistant C3H/HeJ mice induced neutrophil filtration into the lungs in a time- and dosedependent manner, and significantly increased tissue levels of proinflammatory cytokines (e.g. IL-1b, TNF and MIP-2) and acute lung edema. Histological examination of lung tissue sections revealed evidence of an acute diffuse inflammatory response including accumulation of neutrophils in the interstitial and intraalveolar areas, interstitial edema, and protein exudation into the alveolar space (Abraham et al., 2000). These pathological changes are similarly observed in response to acute lung injury mediated by endotoxin, TNF and other proinflammatory stimuli; these responses could not be attributed to trace amounts of endotoxin co-administered with the HMGB-1, because C3H/HeJ mice do not respond to low doses of endotoxin. When administered

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intracerebroventricularly, HMGB-1 induced brain TNF and IL-6 production, and induced sickness behaviors including anorexia and taste aversion (Agnello et al., 2002). Passive immunization with antiHMGB-1 antibodies attenuated the development of LPS-induced hypophagia, indicating that HMGB-1 is a mediator of sickness behavior associated with endotoxemia (Agnello et al., 2002). Administration of HMGB-1 B box to mice increased both ileal mucosal permeability and bacterial translocation to mesenteric lymph nodes (Sappington et al., 2002). Thus, the release of HMGB-1 by activated macrophages, its causative role in lethal endotoxemia, and its activity as a macrophage-stimulating agent (Table 39.1), reveal that HMGB-1 is a late cytokine mediator of systemic inflammation.

HMGB-1 antagonists protect against lethal systemic inflammation Neutralizing anti-HMGB-1 antibodies significantly protect mice from lethal doses of endotoxin when the first dose of anti-HMGB-1 antibodies is administered after circulating TNF reaches peak levels (Wang et al., 1999a). Furthermore, anti-HMGB-1 antibodies significantly protect against lethal sepsis (induced by cecal ligation and puncture), even when antibody administration is delayed for 24 hours after surgery (survival in non-immune IgG-treated controls  28% versus survival in the anti-HMGB-1 group  72%, p  0.03) (Yang et al., 2003b). Passive immunization with B box-specific antibodies conferred protection against lethality caused by endotoxemia and cecal perforationinduced sepsis (Yang et al., 2003b). Administration of anti-HMGB-1 antibodies before or after intratracheal administration of endotoxin significantly decreased endotoxin-induced neutrophil accumulation into the lungs and attenuated the severity of lung edema (Abraham et al., 2000). Despite the protective effects of anti-HMGB-1 antibodies on the development of lung injury and neutrophil accumulation, this treatment did not affect endotoxin-induced increases in tissue levels of cytokines such as IL-1b, TNF or MIP-2, further implicating HMGB-1 as a critical late mediator of acute lung injury. Structure/function analyses of HMGB-1 revealed that the A box acts as a specific antagonist of fulllength HMGB-1. In vitro, the A box inhibits HMGB-1-

induced release of TNF and IL-1b, but fails to prevent IL-1b-induced TNF release; moreover, the A box significantly displaced saturable 125I-labeled HMGB-1 binding to macrophage cultures, suggesting that the A box acts as a competitive inhibitor of HMGB-1. Systemic administration of the A box significantly increased survival after sepsis, even if the treatment was delayed 24 hours (28% survival in mice treated with GST control protein versus 68% in animals receiving A Box, P  0.03) (Yang et al., 2003b). These data suggest that compounds targeting HMGB-1 activity or expression may effectively inhibit the progression of lethal systemic inflammation, and may prove efficacious in a wider treatment window than is available for anti-TNF strategies. Consistent with this hypothesis, we have recently demonstrated that ethyl pyruvate, a non-toxic food additive, protects against lethal systemic inflammation by inhibiting the release of HMGB-1. Ethyl pyruvate specifically inhibited activation of p38 MAP kinase and NFjB, components of two signaling pathways that are critical for cytokine release. As with the A box, ethyl pyruvate administration to septic mice prevented accumulation of HMGB-1 in serum, and significantly increased survival (vehicle survival  30% versus ethyl pyruvate survival  88%; P  0.005), even when administered 24 hours after cecal puncture (Ulloa et al., 2002). Thus, antagonizing endogenous HMGB-1 with either antibodies or specific HMGB-1 antagonists (A box or ethyl pyruvate) may offer a clinically relevant treatment to reverse the course of established lethal sepsis.

HMGB-1 RECEPTORS HMGB-1 localizes to the nucleus and cytoplasm of most cells, to the cell membrane of neuronal (neuroblastoma) cells (Merenmies et al., 1991), and to the filopodia of the advancing plasma membrane of neurites (Merenmies et al., 1991; Rauvala et al., 2000), where it co-localizes and interacts with tissue plasminogen activator (t-PA). Local acceleration of t-PAcatalyzed plasminogen activation by HMGB-1 at the leading edge of the cytoplasmic membrane has been implicated in cell migration (Fages et al., 2000), differentiation and regeneration (Parkkinen et al., 1993; Hori et al., 1995; Huttunen et al., 1999). HMGB-1 binds to the receptor for advanced glycation endproducts

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TABLE 39.1 Release and extracellular roles of HMGB-1 Release of HMGB-1

Administration of HMGB-1

Administration of anti-HMGB-1

Released by erythroleukemia cells after stimulation with hexamethylenebisacetamide (Melloni et al., 1995) by pituicytes after TNF/IL-1 stimulation (Wang et al., 1999b) by macrophages/monocytes after LPS stimulation (Wang et al., 1999a) by necrotic (but not apoptotic) or damaged cells (Degryse et al., 2001; Scaffidi et al., 2002)

Stimulated the release of TNF and other proinflammatory cytokines in macrophage/monocyte cell cultures (Andersson et al., 2000)

Not determined

antibodies Cell Culture

Animal

Increased in mouse serum at 16–24 hours after LPS administration (Wang et al., 1999a) or cecal ligation and puncture (Yang et al., 2002), and remains elevated for 36 hours

Induced chemotaxis and cytoskeleton reorganization in smooth muscle cells (Degryse et al., 2001) Increased the permeability of Caco-2 monolayers (Sappington et al., 2002) Intratracheal injection: Caused acute lung injury in LPS-resistant C3H/HeJ mice (Abraham et al., 2000) Intraperitoneal injection: Caused lethality in both LPS-sensitive (Balb/C and C3H/HeN) and LPS-resistant (C3H/HeJ) mice (Wang et al., 1999a)

Protected against LPS induced acute lung injury (Abraham et al., 2000), hypophagia (Agnello et al., 2002), and lethality in mice (Wang et al., 1999a)

Peri-sciatic administration: Produced a unilaterial and bilateral allodynia (decreased mechanical response threshholds to thermal stimulation) (Chacur et al., 2001) Intracerebroventricular administration: Resulted in anorexia and taste aversion (Agnello et al., 2002) Human

Elevated levels in serum of patients with surgical sepsis (Wang et al., 1999a), and hemorrhagic shock (Ombrellino et al., 2000)

Not determined

Not determined

Elevated levels in synovial fluid of patients with rheumatoid arthritis (Kokkola et al., 2002). (RAGE) in a concentration-dependent manner (Hori et al., 1995). RAGE is a member of the immunoglobulin superfamily, and is expressed on mononuclear phagocytes and vascular smooth muscle cells

(Schmidt et al., 2000; Schmidt and Stern, 2001). RAGE interacts with a range of ligands, including AGEs, amyloidpeptide,amphoterin(HMGB-1),andmembers of the S100 family. Engagement of RAGE with ligands

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(such as advanced glycation end products (AGE), transthyretin or HMGB-1), activates the NFjB signaling pathway (Thornalley, 1998; Sousa et al., 2000; Sappington et al., 2002), and the mitogen-activated protein kinase (MAPK) pathways (Degryse et al., 2001). Extracellular HMGB-1 can activate mitogenactivated protein kinases (e.g. p38, ERK1/2), and induce migration of rat smooth muscle cells (Fages et al., 2000; Degryse et al., 2001). Interestingly, the HMGB-1-mediated chemotaxis and cytokine production can be partially inhibited by antibodies directed against RAGE, implicating RAGE as a receptor mediating the HMGB-1-dependent migratory responses (Degryse et al., 2001; our unpublished observations) (Figure 39.3). The HMGB-1/RAGE interaction may also be important in tumor formation, because blocking this interaction can decrease the growth and metastasis of both implanted and spontaneous tumors (Taguchi et al., 2000).

HMGB-1

RAGE

MAPK (ERK1/2, p38. JNK)

NF-jB

TNF DNA

mRNA

TNF Protein

FIGURE 39.3 Potential signal transduction pathways of HMGB-1-mediated TNF production in macrophage/monocytes.

SUMMARY

HMG PROTEINS IN AUTOIMMUNE DISEASE HMGB-1 has been detected in most (12 out of 14) synovial fluid samples from RA patients (Kokkola et al., 2002). Anti-HMG antibodies have been detected in the sera of patients with drug-induced autoimmunity (Ayer et al., 1994a), juvenile rheumatoid arthritis (JRA) (Wittemann et al., 1990; Jung et al., 1997), autoimmune hepatitis (Sobajima et al., 1999), systemic lupus erythematosus (SLE) (Bustin et al., 1982), and systemic sclerosis (Ayer et al., 1994b). A similar pattern of antibody response to HMG-14/17 was reported in drug-induced autoimmunity (Ayer et al., 1994a); in 42 patients who developed lupus (drug-induced lupus), 67% showed antibodies to HMG-14/17, while 21% of the patients had auto-antibodies to HMGB-1/2. Similarly, in 126 JRA patients, 39% had antibodies against HMGB-1/2 (Wittemann et al., 1990). Fine epitope mapping followed by immunoblotting analysis revealed that JRA sera reacts with the linker region between the HMG box domains A and B of the HMGB-1/2 proteins (Jung et al., 1997). The high prevalence of antibodies to the non-histone nucleosomal HMG proteins in these patients suggests that nucleosomal HMG proteins are immunogens in autoimmune diseases.

Unknown

The identification of TNF as an early mediator of inflammation led to clinical therapies which have improved the clinical course of many patients suffering from chronic inflammatory disease. In contrast, anti-TNF strategies have met with little success in diseases of acute systemic inflammation such as sepsis. It is hoped that the discovery and characterization of new cytokines and pathological mechanisms will be important to the development of future therapies for inflammation. HMGB-1, first identified as a nuclear chromosomal protein, is now known as a proinflammatory cytokine that mediates delayed lethality in systemic inflammation. The discovery of HMGB-1 as a monocyte-derived, late-acting cytokine mediator of endotoxin lethality has initiated a new field of investigation for development of experimental therapeutics. The downstream or ‘late’ action of HMGB-1 is a marked departure from the early activities of TNF and other classical proinflammatory cytokines, and has significant implications for understanding and manipulating innate immune responses. Emerging data also suggest that therapeutics that inhibit HMGB-1 activity are effective in preventing death in murine sepsis. The insights gained from these and other studies of HMGB-1 may lead to the development

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of new compounds which provide a wider therapeutic window for treatment of endotoxemia, sepsis and septic shock.

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Ferrari, S., Finelli, P., Rocchi, M. and Bianchi, M.E. (1996). The active gene that encodes human high mobility group 1 protein (HMG1) contains introns and maps to chromosome 13. Genomics 35, 367–371. Gariboldi, M., De Gregorio, L., Ferrari, S. et al. (1995). Mapping of the Hmg1 gene and of seven related sequences in the mouse. Mamm. Genome 6, 581–585. Ge, H. and Roeder, R.G. (1994). The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATA-binding protein. J. Biol. Chem. 269, 17136–17140. Hill, D.A. and Reeves, R. (1997). Competition between HMGI(Y), HMG-1 and histone H1 on four-way junction DNA. Nucleic Acids Res 25, 3523–3531. Hill, D.A., Pedulla, M.L. and Reeves, R. (1999). Directional binding of HMG-I(Y) on four-way junction DNA and the molecular basis for competitive binding with HMG-1 and histone H1. Nucleic Acids Res. 27, 2135–2144. Hori, O., Brett, J., Slattery, T. et al. (1995). The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. J. Biol. Chem. 270, 25752–25761. Huttunen, H.J., Fages, C. and Rauvala, H. (1999). Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways. J. Biol. Chem. 274, 19919–19924. Jayaraman, L., Moorthy, N.C., Murthy, K.G. et al. (1998). High mobility group protein-1 (HMG-1) is a unique activator of p53. Genes Dev. 12, 462–472. Johns, E.W. (1982). History, Definitions and Problems. In: Johns, E.W. (ed.) The HMG Chromosomal Proteins. London: Academic Press, pp. 1–8. Jung, F., Neuer, G., and Bautz, F.A. (1997). Antibodies against a peptide sequence located in the linker region of the HMG-1/2 box domains in sera from patients with juvenile rheumatoid arthritis. Arthritis Rheum. 40, 1803–1809. Kimura, K., Katoh, N., Sakurada, K. and Kubo, S. (1985). Phosphorylation of high mobility group 1 protein by phospholipid-sensitive Ca2-dependent protein kinase from pig testis. Biochem. J. 227, 271–276. Kinoshita, M., Hatada, S., Asashima, M. and Noda, M. (1994). HMG-X, a Xenopus gene encoding an HMG1 homolog, is abundantly expressed in the developing nervous system. FEBS Lett. 352, 191–196. Kokkola, R., Sundberg, E., Ulfgren, A.-K. et al. (2002). High mobility group box chromosomal protein 1 (HMGB-1) – a novel proinflammatory mediator in synovitis. Arthritis Rheum. 46, 2598–2603. Landsman, D. and Bustin, M. (1991). Assessment of the transcriptional activation potential of the HMG chromosomal proteins. Mol. Cell. Biol. 11, 4483–4489. Landsman, D. and Bustin, M. (1993). A signature for the HMG-1 box DNA-binding proteins. Bioessays 15, 539–546. Laudet, V., Stehelin, D. and Clevers, H. (1993). Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 21, 2493–2501. Locker, D., Decoville, M., Maurizot, J.C. et al. (1995). Interaction between cisplatin-modified DNA and the

HMG boxes of HMG 1: DNase I footprinting and circular dichroism. J. Mol. Biol. 246, 243–247. Love, J.J., Li, X., Case, D.A. et al. (1995). Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature 376, 791–795. Maher, J.F. and Nathans, D. (1996). Multivalent DNA-binding properties of the HMG-1 proteins. Proc. Natl Acad. Sci. USA 93, 6716–6720. Melloni, E., Sparatore, B., Patrone, M. et al. (1995). Extracellular release of the ‘differentiation enhancing factor’, a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation. FEBS Lett. 368, 466–470. Merenmies, J., Pihlaskari, R., Laitinen, J. et al. (1991). 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane. J. Biol. Chem. 266, 16722–16729. Mosevitsky, M.I., Novitskaya, V.A., Iogannsen, M.G. and Zabezhinsky, M.A. (1989). Tissue specificity of nucleocytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions. Eur. J. Biochem. 185, 303–310. Nasrin, N., Buggs, C., Kong, X.F. et al. (1991). DNA-binding properties of the product of the testis-determining gene and a related protein. Nature 354, 317–320. Ner, S.S. and Travers, A.A. (1994). HMG-D, the Drosophila melanogaster homologue of HMG 1 protein, is associated with early embryonic chromatin in the absence of histone H1. EMBO J 13, 1817–1822. Ner, S.S., Churchill, M.E., Searles, M.A. and Travers, A.A. (1993). dHMG-Z, a second HMG-1-related protein in Drosophila melanogaster. Nucleic Acids Res. 21, 4369–4371. Ombrellino, M., Wang, H., Ajemian, M.S. et al. (1999). Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet 354, 1446–1447. Onate, S.A., Prendergast, P., Wagner, J.P. et al. (1994). The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences. Mol. Cell. Biol. 14, 3376–3391. Paonessa, G., Frank, R. and Cortese, R. (1987). Nucleotide sequence of rat liver HMG1 cDNA. Nucleic Acids Res. 15, 9077. Parkkinen, J., Raulo, E., Merenmies, J. et al. (1993). Amphoterin, the 30-kDa protein in a family of HMG1type polypeptides. Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation. J. Biol. Chem. 268, 19726–19738. Pasqualini, J.R., Sterner, R., Mercat, P. and Allfrey, V.G. (1989). Estradiol enhanced acetylation of nuclear high mobility group proteins of the uterus of newborn guinea pigs. Biochem. Biophys. Res. Commun. 161, 1260–1266. Prasad, S. and Thakur, M.K. (1988a). Age-dependent effects of sodium butyrate and hydrocortisone on acetylation of high mobility group proteins of rat liver. Biochem. Int. 16, 375–382. Prasad, S. and Thakur, M.K. (1988b). In vitro acetylation of the liver HMG non-histone proteins and its modulation by spermine and dexamethasone during aging of rats. Mol. Biol. Rep. 13, 221–224. Rauvala, H., Huttunen, H.J., Fages, C. et al. (2000). Heparinbinding proteins HB-GAM (pleiotrophin) and ampho-

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40 Macrophage Colony Stimulating Factor [CSF-1] Michael T. Lotze1 and John A. Hamilton2 1 Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2

Inflammation Research Centre, Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Australia

There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes. Stimulate the phagocytes. Drugs are a delusion. George Bernard Shaw; The Doctor’s Dilemma, preface, 1913

INTRODUCTION Since this Shavian insight 90 years ago, we have come to know that the large phagocyte, the macrophage, is delineated from other hematopoietic precursors by its unique cytokine requirement. Macrophage colonystimulating factor (M-CSF) is a survival factor for cells expressing the c-fms CSF-1 receptor which include macrophages as well as osteoclasts and immature dendritic cells (DCs). Interaction of colony stimulating factor-1 (CSF-1) or (M-CSF) with its receptor is associated with important biologic effects (Kawasaki et al., 1985; Stanley, 1985; Lee, 1991; Sherr, 1991; Vairo and Hamilton, 1991). Human M-CSF exists as an 85-kDa homodimer. In murine cells only a 552-aminoacid form encoded by 1.4 and 4.5 kb messages has been found thus far (Rajavashisth et al., 1987). Liver expresses a 1.4-kb species, heart and lung express the 4.5-kb species, and brain expresses high levels of both transcripts, while intestine lacked detectable transcripts. The CSF-1 gene is present as a single The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

copy in the mouse genome. All forms of M-CSF have a 32-amino-acid signal peptide and a 23-amino-acid hydrophobic transmembrane domain. Much of the carboxy-terminus, including the transmembrane domain, is not found in the mature protein. Thus, M-CSF is a prepropolypeptide, with processing occurring at both the amino- and carboxy-termini. Only the first seven of nine cysteines in the mature molecule are required for biologic activity. Glycosylation found in the native protein is not important for the biologic activity. The M-CSF gene as well as the gene encoding its receptor are tightly linked on the long arm of chromosome 5 and the syntenic murine chromosome 11. GM-CSF, IL-3, ECGF and the receptor for PDGF are also found in this region. Up to 20 000 receptors per cell exist for the mature M-CSF (Metcalf, 1985). Most recently M-CSF has been identified as important in the biology of several neoplasms, notably ovarian carcinoma and as a possible therapeutic agent in fungal infections.

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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GENE STRUCTURE AND REGULATION Gene structure Gene organization and location The human CSF-1 gene contains 10 exons and nine introns, spanning 20 kb (Ladner et al., 1987). Alternative use of exon 6 splice acceptor sites and 3 non-coding sequence exons occurs. Two alternatively spliced transcripts encode either a 224- or a 522amino acid protein. At Immunex, human cDNA clones were subsequently isolated from a pancreatic carcinoma cell line (Cosman et al., 1988). Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length), designated M-CSF alpha, beta, and gamma were predicted to be encoded by these cDNAs. All three share a 32-amino-acid signal sequence as well as the first 149 amino acids of the mature protein. Subsequently, M-CSF beta and gamma insertions of 298 and 182 amino acids, with the first 182 amino acids of these insertions shared, are noted when compared with M-CSF alpha. A Cterminal 75 amino acids encodes transmembrane and cytoplasmic domains in all three splice variants.

Soluble and cell surface M-CSF Soluble M-CSF activity is associated with proteins of 44 kDa (beta and gamma) or 28 kDa (alpha). M-CSF is also expressed on the cell surface, suggesting that it is processed to a soluble form following proteolytic digestion by a cellular converting enzyme. The cleavage site is determined by the amino acid sequence of the juxtamembrane region, the distance of the site from the transmembrane domain and steric accessibility of the protease dictated by glycosylation (Deng et al., 1998). The cell surface form of human CSF-1 is biologically active, and can functionally interact with receptor-bearing cells by direct cell–cell contact (Stein and Rettenmier, 1991). The ectodomain cleavage regulates in part the biologic activity of membraneanchored CSF-1 since mutants incapable of cleavage are more active (Deng et al., 2000). Human M-CSF can also be exfoliated in association with surface membrane-derived vesicles which are likewise active (Tuck et al., 1994). Myeloma cells appear uniquely

to express a 3.5-kb M-CSF transcript (Nakamura et al., 1989). Proteoglycan macrophage colonystimulating factor (PG-M-CSF) is a high molecular type of M-CSF, biologically active and containing substitution with the chondroitin sulfate form of glycosaminoglycan (Kimura et al., 1994). The predominant form of secreted M-CSF (Price et al., 1992) is indeed this highly sulfated species of 375and 250-kDa with a smaller amount of the 100-kDa species. The three predominant CSF-1 species secreted are the 80-kDa homodimer, an 80-kDa/ 50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contains a single 18-kDa chondroitin sulfate chain, absent from the 50-kDa subunit. The chondroitin sulfate binding site in the 150-200-kDa subunit is Ser277. Conversion of Ser277 to an alanine results in complete loss of the substitution. PG-MCSF does not bind to types I, II, III, and IV collagen or to fibronectin but does bind to collagen V (Suzu et al., 1992a, 1992b, 1997). PG-M-CSF also binds bFGF through its carboxyl terminus. The binding sites for PG-M-CSF and heparan sulfate on bFGF are located close together. Interestingly, PG-M-CSF reduces the mitogenic action of bFGF (Suzu et al., 1994). Osteoblastic cells produce both the secreted 85-kDa M-CSF as well as the proteoglycan form (Ohtsuki et al., 1995). The PG-M-CSF is produced by bone lining cells and binds bone-derived collagens allowing its extraction from human bone. This suggests that the bone surface itself is a site for osteoclast formation.

Gene regulation Regulatory sequences Monopoiesis is induced by Maf-B, c-Jun, or Egr-1 and is dependent upon the transcription factors PU.1, Sp1, and ICSBP. c-Jun:PU.1, ICSBP:PU.1, and Maf:Jun complexes induce monocytic genes. High levels of PU.1 are required for monopoiesis. Cell cycle arrest is important for terminal myelopoiesis, possibly due to the expression of p53 or hypo-phosphorylated Rb (Friedman, 2002). Dexamethasone interestingly increases the expression of membrane M-CSF on a large number of transduced tumor cells expressing M-CSF suggesting nuclear hormone sensitive regulatory sites upstream of the M-CSF gene (Dan et al.,

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2001). Metallothionine expression mediates zinc regulation of M-CSF gene expression at the transcriptional level as well (Kanekiyo et al., 2002).

PROTEIN STRUCTURE The structure of the recombinant human M-CSF dimer, determined by X-ray crystallography, contains two bundles of four alpha helices laid end-to-end, with an interchain disulfide bond (Pandit et al., 1992). Residues in or near helix A and helix C are involved in receptor binding. Truncated, non-glycosylated Escherichia coli-produced forms of recombinant MCSF (rM-CSF) are functionally equivalent to the glycosylated rM-CSF form secreted from mammalian cells. The N-terminal domain of 149 amino acids, produced by all splice variants, is bioactive. Heterodimeric rMCSFs from different splice variants are fully active, but are not observed in vivo (Taylor et al., 1994; Koths, 1997). A soluble form of the M-CSF receptor, c-Fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. Each dimeric M-CSF molecule binds two soluble receptor molecules, supporting the notion that M-CSF signaling is linked to receptor dimerization.

CELLULAR SOURCES OF M-CSF This cytokine is produced by several normal and malignant cell types including bone marrow stromal cells (Pogue-Geile et al., 1995), osteoblasts and bone lining cells (Ohtsuki et al., 1994, Takahashi et al., 1988), astrocytes (Thery et al., 1990), stromal cell lines (Ohtsuki et al., 1994), and T-cell lines (Takahashi et al., 1988; Zisman et al., 1993) The supportive cells within bone marrow stroma, promoting osteoclastogenesis, are restricted to a specialized population expressing both M-CSF and RANKL (with relatively less of the inhibitor, osteoprotegerin) and sensitive to Vitamin D [1alpha, 25(OH)(2)D(3)] presence (Takeshita et al., 2001). Several tumor cells including myeloma cells (Nakamura et al., 1989), leukemias (Li et al., 2002), and pancreatic cancer cell lines (Ralph et al., 1986) express M-CSF. Expression of CSF-1 and CSF-1R

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correlate with tumor cell invasiveness and poor clinical prognosis (Kascinski, 2002).

BIOCHEMICAL RESPONSES TO CSF-1 CSF-1 is a homodimeric glycoprotein that binds to high-affinity receptors expressed primarily on cells of the monocyte–macrophage lineage (Sherr, 1991). Homozygous disruption of CSF-1 coding sequences in osteopetrotic (op/op) mice severely impairs production of monocyte-derived cells, emphasizing the importance of CSF-1 for their development (WiktorJedrzejczak et al., 1990; Yoshida et al., 1990). CSF-1 exerts pleiotropic effects (e.g., survival, proliferation, differentiation and activation on mononuclear phagocytes) (Stanley, 1985; Vairo and Hamilton, 1991; Stanley et al., 1994). A number of morphological, biological and biochemical changes are evident soon after the addition of CSF-1. For macrophages to enter S phase, CSF-1 must be present throughout the G1 phase of the cell cycle until they pass a late G1 restriction point, after which they become committed to DNA synthesis (Tushinski and Stanley, 1985; Matsushime et al., 1991). The rate-limiting steps that control this transition and lead to DNA synthesis are regulated, at least in part, by G1cyclins and their associated catalytic subunits, the cyclin-dependent kinases (cdks) (Sherr, 1993). A critical substrate of the cyclin D-dependent kinases is likely to be the retinoblastoma (RB) protein whose phosphorylation late in G1 reverses its growth suppressive function (Nevins, 1992). Addition of CSF-1 to quiescent macrophages results in synthesis of cyclin D1 (Matsushime et al., 1991; Cocks et al., 1992; Vadiveloo et al., 1996) and cdk4 (Matsushime et al., 1992; Vadiveloo et al., 1996), as well as phosphorylation of the retinoblastoma protein (Vadiveloo et al., 1996). The CSF-1 receptor (CSF-1R) is encoded by the c-fms proto-oncogene, the cellular counterpart of the v-fms oncogene encoded by the genomes of both Susan McDonough and Hardy-Zuckerman’s five strains of feline sarcoma virus, and consists of an extracellular ligand-binding domain joined through a single membrane-membrane-spanning helix to a cytoplasmic protein tyrosine kinase (PTK) domain (Coussens et al., 1986; Sherr, 1988). The CSF-1R is

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more closely related structurally to the  and

receptors for platelet-derived growth factor (PDGF), the c-kit proto-oncogene product (the receptor for stem cell factor) and flt3/flk2 than to other PTK receptor subfamilies. The extracellular domains of these receptors consist of the five immunoglobulin-like loops (Wang et al., 1993) and their PTK domains are each interrupted by kinase insert (KI) sequences of variable lengths (Sherr, 1991) (see below). Many of the downstream signaling cascades that have been claimed to be modulated following c-Fms activation are discussed below.

The role of Ras Ras activation The Ras genes encode small GTP-binding proteins whose functions are important for both cell growth and differentiation. Ras proteins have low instrinsic GTPase activity that can be stimulated by a GTPaseactivating protein (GAP) by converting active p21 Ras (GTP bound) into the inactive form (GDP bound). CSF-1 activates Ras in NIH3T3 cells expressing human c-Fms (Gibbs et al., 1990) and in BAC1.2F5 cells (Xu et al., 1993, 1995; Büscher et al., 1995). Transgenic mice expressing dominant suppressors of Ras signaling display altered macrophage differentiation, whereas mature peritoneal macrophages exhibited changed responses to CSF-1, including increased dependence on CSF-1 for survival, changes in the morphological responses and suppression of the increased mRNA expression of a CSF-1-responsive gene (Jin et al., 1995). CSF-1 rapidly and reproducibly induces tyrosine phosphorylation of GAP in both NIH3T3 fibroblasts and in the murine myeloid cell line 32D, when transfected with human c-fms (Heidaran et al., 1992). These findings are fairly consistent with those obtained in the NIH3T3 cells transfected with the murine c-fms (Reedijk et al., 1990), although there are disagreements about the role of the KI domain in the two studies. In contrast to these results showing tyrosine phosphorylation of GAP, no such phosphorylation was observed in Rat-2 cells expressing murine c-Fms or in BAC1.2F5 cells (Reedijk et al., 1990). The growth of fibroblasts transformed by the constitutively active product of the v-fms oncogene can

be suppressed by microinjection of antibodies to p21Ras (Smith et al., 1986) or by overexpression of the catalytic domain of GAP (Bortner et al., 1991). Similarly, the enforced expression of the GAP catalytic domain in NIH3T3 cells expressing human c-Fms inhibits the activation of genes controlled by ‘Rasresponsive elements’ and suppresses CSF-1-induced mitogenesis (Bortner et al., 1991). Using NIH3T3 cells transfected with human c-Fms and expression of an Ets–LacZ fusion protein, it was concluded that the Ets family of transcription factors plays a central role in integrating both c-Fms and Ras-induced mitogenic signals and in modulating the c-myc response to CSF-1 stimulation (Langer et al., 1992). The working model developed from these experiments was that CSF-1 stimulation of c-Fms results in a flow of information from a cytoplasmic effector that interacts with the receptor in the region surrounding tyrosine 809 (Y809) through a GTP exchange factor to p21 Ras, through Ets transcription factors, and ultimately through c-Myc (Figure 40.1). However, it was concluded that the proposed Ras-Ets-Myc pathway was probably not sufficient but rather necessary for the proliferative response to CSF-1 (Langer et al., 1992). More recent data with NIH3T3 cells containing the human c-Fms Y809F mutant indicated that the defective mitogenic signaling and concomitant c-Myc expression could be rescued by D-type cyclins, leading to the proposal that c-Myc and cyclin D1 must either act in parallel cross-concomitant pathways or, alternatively, that Myc function may be required both upstream and downstream of cyclin D1 for cells to proliferate continuously (Roussel et al., 1991). Again using NIH3T3 cells expressing human c-Fms and microinjection techniques, Barone and Courtneidge (1995) appear to have drawn different conclusions in that they suggested that, in response to CSF-1, Src family kinases activate a pathway (most probably Ras independent) which culminates in the transcription of c-myc and which is required for DNA synthesis (Figure 40.1). They also concluded that an important downstream component of the Ras pathway is the induction of c-Fos expression and not that of c-Myc (Figure 40.1). Neither the Src pathway nor the Ras pathway alone is sufficient to induce DNA synthesis. In the same cells, CSF-1 activated three Src family kinases, Src, Fyn and Yes, as well as their concomitant association with the ligand-activated c-Fms

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FIGURE 40.1 The role(s) of Ras in CSF-1R signaling? There is debate about whether c-Myc or c-Fos expression lies downstream of Ras activation (pathways (a) and (b), respectively) (Langer et al., 1992; Barone and Courtneidge, 1995); whether or not MAP (Erk) activity is involved (Hipskind et al., 1994; Treisman, 1995) is unknown. Alternatively, c-Myc expression has been proposed to lie downstream of Src activation (pathway (c)) (Barone and Courtneidge, 1995). The classical Ras–Raf-1–MEK–MAPK (Erk) pathway may not be of major significance as there is evidence for Ras independence for MAPK and Raf-1 activation (Büscher et al., 1995; Xu et al., 1995); Raf-1 may not be the MEK kinase in CSF-1-activated macrophages. (Courtneidge et al., 1993); the Y809F mutation reduced both their binding and enzymatic activation. It should be noted from the discussion later that the binding site for the Src family kinases on human cFms has been mapped to Y561 and surrounding amino acids (Alonso et al., 1995).

Ras and MAP kinase A variety of mitogens lead to the formation of membrane-associated signaling complexes that deliver an essential signal via Ras to a protein kinase network involving the Raf-1 kinase, the mitogenactivated protein kinase (MAPK) activator, namely MEK and Erks (Blenis, 1993). The Raf-1 kinase interacts directly with the p21Ras proto-oncogene product while activated Raf-1 in turn phosphorylates and activates MEK in vivo and in vitro. It has therefore been proposed that these kinases are arranged in a linear cascade, in which Raf-1 phosphorylates and activates MEK, which in turn phosphorylates and activates Erk. The signal is then carried by Erk to the nucleus where

they phosphorylate and activate transcription factors capable of mediating immediate–early gene induction (e.g. c-fos). The general scheme of the Ras-Raf-lMEK-Erk pathway is assumed to be well conserved in higher eukaryotes (Egan and Weinberg, 1993). It has been shown that CSF-1 treatment of murine macrophages leads to activation of Raf-l, MEK and Erk (Baccarini et al., 1990; Choudhury et al., 1990; Reimann et al., 1994). The existence of two separate signaling pathways for the modification of distinct ternary complex factors (TCFs) has been reported: one dependent on Ras and Erk and converging on TCF/Elk-l, and the other targeting TCF/SAP-I independently of Ras and MAPK (Hipskind et al., 1994). Transient expression of a dominant-negative Ras mutant in the CSF-1-dependent macrophage cell line, BAC1.2F5, reduced, but did not abolish, CSF-1mediated stimulation of MEK and Erk, indicating Ras-dependent and -independent pathways leading to MAPK activation in this cellular system (Figure 40.1). Also in this connection, increased intracellular cAMP activates Erk activity in a MEK-dependent

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manner in murine bone marrow-derived macrophages (BMMs) while blocking Ras activation (Wilson et al., 1998). In contrast, activation of Raf-1 was Ras independent (Büscher et al., 1995). In a separate study with BAC1.2F5 cells treated with CSF-1, such a Rasindependent independent mechanism for Raf-1 activation was claimed (Xu et al., 1995). More recent findings in CSF-1-treated murine macrophages indicate that the protein tyrosine phosphatase SHP-1 is involved in Ras-mediated activation of the Erk pathway but not of Raf-1 kinase (Krautwald et al., 1996). All of the above data are consistent with the hypothesis that Raf-1 may not be the CSF-1-induced MEK kinase in macrophages (Figure 40.1). It is likely that Erk activation by CSF-1 will be involved in c-Fos and/or c-Myc expression (Hipskind et al., 1994; Treisman, 1995). As regards cell proliferation, oncogenically active Raf-1 protein (v-Raf ) conferred CSF-1-independent growth on the BAC1.2F5 cells and led to immediate early gene expression without Erk activation (Büscher et al., 1995). This v-Raf expression suppressed CSF-1stimulated Erk activity, which appeared to be due to a feedback mechanism whereby v-Raf induced a Erkspecific phosphatase, MKP-l (Krautwald et al., 1995). The implication is that Erk activation may not be necessary for CSF-1-induced proliferation, a conclusion that is consistent with our studies of Erk-l activation in cycling and poorly cycling murine macrophages (Jaworowski et al., 1996). However, others have concluded that Erk activity is essential in macrophages (Valledor et al., 2000) and in NIH3T3 cells (Aziz et al., 1999). Similarly, using N17Ras and a MEK inhibitor, a Ras-dependent pathway involving Erk and c-myc has been claimed to be critical for CSF-1-induced mitogenesis (Cheng et al., 1999). Others have found a prolonged Erk activation by CSF-1 with a principal downstream target being Ets-2 (Fowles et al., 1998). In CSF-1-treated BAC1.2F5 cells expression of ets-2 and Bcl-xL with similar kinetics of induction (Sevilla et al., 1999); the results suggested a novel role for Ets-2 in affecting apoptosis through regulation of Bcl-xL transcription. In murine macrophages evidence was obtained for two kinetically distinct pathways of Erk-l activation by CSF-1 with peak activation at 5 and 15 min and which could be distinguished from the action of lipopolysaccharide (LPS), which activated Erk-1 with a single peak corresponding to the later peak of activation by

CSF-1 (Jaworowski et al., 1996). For the activation of the other MAPK family members, p38 and JNK-l, CSF-1, unlike LPS, was a poor stimulator (Jaworowski et al., 1999). As the p38 MAPK is implicated in the control of cytokine translation in LPS-stimulated monocytes-macrophages (Lee et al., 1994), the weak stimulation by CSF-1 is consistent with the observation that CSF-1 is a poor inducer of cytokine secretion in monocyte–macrophages (Strassmann et al., 1991; Hamilton, 1993; Hamilton et al., 1993). However, others have claimed that CSF-1 stimulates p38 activity in FD-Mycobacterium avium complex (MAC) II cells (Foltz et al., 1997).

Phospholipase and protein kinase C activation In CSF-1-treated BMMs and human monocytes, no evidence for phospholipase C-mediated hydrolysis of phosphoinsositides (i.e. phospholipase C activity) could be found (Whetton et al., 1986; Hamilton et al., 1989; Imamura et al., 1990), in contrast to what has been found in response to PDGF; it has been proposed that v-Fms could be coupled to a guanine nucleotidedependent polyphoinositide-specific phospholipase C generating diacylglycerol and inositol trisphosphate (Jackowski et al., 1986). In NIH3T3 cells transfected with human c-fms, and in BAC1.2F5 cells, no tyrosine phosphorylation of phospholipase C could be detected (Downing et al., 1989). Phospholipase C2 has been shown to be activated in CSF-1-treated FDCP1 cells transfected with murine CSF-1R (Bourette et al., 1997). In prefusion osteoclasts phospholipase C is a common downstream mediator for adhesion and CSF-1 signals (Nakamura et al., 2001). Several reports have shown that CSF-1 stimulates diacylglycerol formation in a variety of cell systems (Imamura et al., 1990; Choudhury et al., 1991a; Veis and Hamilton, 1991; Xu et al., 1993; Xu et al., 1995); however, further experiments with murine BMMs in our laboratory could not confirm this (Jaworowski et al., 1994). In BAC1.2F5 cells the stimulation of diglyceride production via phospholipase C hydrolysis of phosphatidylcholine was proposed to be an early event in the mitogenic action of CSF-1 (Xu et al., 1993). Cell extracts from CSF-1-treated receptorbearing cells did not show evidence for activation of a phosphatidylcholine-specific phospholipase C

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(Choudhury et al., 1991b). We could find no evidence for phospholipase D or protein kinase C (PKC) activation in murine BMMs in response to CSF-1 (Jaworowski et al., 1994); in human monocytes, however, PKC is activated by CSF-1 (Imamura et al., 1990). When PKC activity (particularly PKC activity) is monitored in BAC1.2F5 cells, CSF-1 has been claimed to be able to activate it in one report (Xu et al., 1995) but not in others (Büscher et al., 1993; Xu et al., 1993; Reimann et al., 1994; Büscher et al., 1995). CSF-1 has been found to induce expression of mitogenactivated protein kinase phosphatase-1 through a protein kinase C-dependent pathway (Valledor et al., 1999). In human monocytes, CSF-1 triggered the phosphorylation and activation of cytosolic phospholipase A2 that appears to correlate with arachidonic acid release and prostaglandin E2 (PGE2) formation (Nakamura et al., 1992). However, we could not confirm PGE2 formation in this system (Hamilton et al., 1993). In murine macrophages phosphorylation of cytosolic phospholipase A2 by CSF-1 was not accompanied by arachidonic acid release and PGE2 formation (Jackowski et al., 1990; Puri et al., 1991; Strassmann et al., 1991; Xu et al., 1994). CSF-1 activated PLA2 in macrophages in a MEK-dependent manner (Qui et al., 1998).

S6 kinase Phosphorylation of the S6 protein of the 40S ribosomal subunit is a highly conserved response of animal cells to treatment with a wide range of stimuli, including growth factors. p70s6K is the physiological S6 kinase activity in mammalian cells. Rapamycin, which can prevent activation of p70s6K in many cells via its ultimate target mTOR/RAFT/FRAP, inhibits CSF-1stimulated DNA synthesis in BAC1.2F5 cells (Kato et al., 1994) and cell proliferation in murine BMMs (Cooper et al., 1994). However, for CSF-1-stimulated BMM DNA synthesis, we found that rapamycin had a relatively incomplete inhibitory effect (Hamilton et al., 1998). In this same study data were obtained that were consistent with phosphatidylinositol-3 (PI-3) kinase lying upstream of p70s6K activity.

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G proteins G proteins are known signal transducers for many hormones, neurotransmitters and chemotactic substances, but not for cytokines. Pertussis toxin can inhibit the activity of some G proteins by ADP ribosylation of the active  subunit of the heterotrimeric Gprotein complex. CSF-1 increased GTP binding to and raised GTPase activity in human monocyte membrane preparations and a variety of responses of these cells to CSF-1 are pertussis toxin sensitive (Imamura and Kufe, 1988; Imamura et al., 1990). A similar conclusion was reached based on studies of signal transduction in hamster fibroblasts expressing human c-Fms (Hartmann et al., 1990) and in studies with murine BMMs but not with murine macrophage cell lines (Zie et al., 1993). In some cases the actions of CSF-1 on murine hemopoietic cells, including macrophages, appear to be less sensitive to the effects of pertussis toxin (He et al., 1988; Hamilton et al., 1991). Inhibition of Gi2 function inhibits CSF-1mediated signal transduction in BAC 1.2F5 cells, leading to the conclusion that the metabolic pathways regulated by Gi2 proteins and c-Fms converge on a common effector necessary for the regulation of macrophage survival and proliferation (Corre and Hermouet, 1995). In BAC1.2F5 cells CSF-1 initially activates Cdc42 and Rac in parallel to stimulate the formation of filopodia and lamellipodia/membrane ruffles, although Cdc42 can also act upstream of Rac. Subsequently, Rac acts upstream of Rho to induce actin cable formation (Allen et al., 1997).

Na/H antiport activity We have shown that CSF-1 induces a rapid increase in Na/H exchange activity in murine BMMs, the accompanying Na influx subsequently increasing Na /H-ATPase (Na pump) activity (Vairo and Hamilton, 1988; Vairo et al., 1990a); persistent stimulation of this ‘Na cycle’ occurred for most of the BMM cycle. Inhibition of Na/ H exchange activity with dimethylamiloride completely abolished CSF-1induced BMM proliferation even when added up to 8 h after the growth factor, suggesting that Na/H  antiport activity late in the G1 phase of the BMM cell cycle is required for S-phase entry (Vairo et al., 1990a).

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It was subsequently found that dimethylamiloride and other amiloride analogues suppressed the induction of mRNA expression for M1 and M2 subunits of ribonucleotide reductase but not for proliferating cell nuclear antigen or cyclin D1, indicating selective suppression of ‘cell cycle’ genes by Na/H antiport inhibitors in this system (Vairo et al., 1992).

Tyrosine phosphorylation Several proteins are tyrosine phosphorylated and/or modulated after c-Fms activation and many are beginning to be identified (Sengupta et al., 1988; Yeung et al., 1992; Lioubin et al., 1994). Several of these are now discussed.

PI-3 kinase activation and function Function. There are reports in various cell populations that CSF-1 can activate PI-3 kinase activity including in antiphosphotyrosine immunoprecipitates (Varticovski et al., 1989; Reedijk et al., 1990; Shurtleff et al., 1990; Choudhury et al., 1991c; van der Geer and Hunter, 1991; Reedijk et al., 1992; Yusoff et al., 1994). We have found that the PI-3 kinase p85 subunit is tyrosine phosphorylated on CSF-1 stimulation in primary cultures of murine macrophages Kanagasundaram et al., 1996), agreeing with a recent report on human monocytes (Saleem et al., 1995). These findings seem to differ from observations made in fibroblasts engineered to express human c-Fms where there was little if any tyrosine phosphorylation of PI-3 kinase following receptor activation (Shurtleff et al., 1990). A stable association of PI-3 kinase with c-Fms, c-Cbl and several other tyrosine phosphorylated proteins has recently been reported for CSF-1stimulated murine macrophages (Kangasundaram et al., 1996). These last observations are interesting given the view that ‘the tyrosine phosphorylated CSF1R does not appear to physically associate with many other intracellular signaling molecules’ (Stanley et al., 1994). The evidence for this notion comes from the observations that activated c-Fms does not associate stably with phospholipase C (Downing et al., 1989), Raf-1 (Baccarini et al., 1990), ras GTPase activating protein (GAP) (Reedijk et al., 1990), SHP-1 (Yeung et al., 1992) or other proteins that can be labeled with 35 S-methionine or 32P-phosphate (Stanley et al., 1994).

In the same study, where the stable association of activated c-Fms with several tyrosine-phosphorylated proteins was observed (Kanagasundaram et al., 1996), we found that the only detectable 35S-labeled protein found in anti-c-Fms immunoprecipitates whose levels increased consistently upon CSF-1 stimulation appeared to be the PI-3 kinase p85 subunit. As regards the involvement of PI-3 kinase in the proliferative response, early reports using either the murine c-Fms Y721F mutant in Rat-2 fibroblasts (Reedijk et al., 1990; van der Geer and Hunter, 1993) or the KI insert mutant of human c-Fms in NIH3T3 cells (Shurtleff et al., 1990; Choudhury et al., 1991c) suggested a relationship based on concordant reduction in kinase activity and cell proliferation (Y721 in the KI domain is believed to be the binding site for the PI-3 kinase p85 regulatory subunit (Reedijk et al., 1992) (see below). However, data with both human c-Fms Y809F in NIH3T3 cells (Roussel et al., 1990) and with murine c-Fms Y807F in Rat-2 fibroblasts (van der Geer and Hunter, 1991) show that the reduction in the proliferative response (see later) was not paralleled by a change in PI-3 kinase activity. More recent reports using microinjection of antibodies to the p110 PI-3 kinase catalytic subunit into NIH3T3 cells expressing human c-Fms (Roche et al., 1994) or using the PI-3 kinase inhibitor, wortmannin, with CSF-1-treated BMMs (Hamilton et al., 1998) failed to find a major role for PI-3 kinase in the control of DNA synthesis; in the latter study, and by the same approach, it was concluded that PI-3 kinase activity does not have a role in the control of c-fos mRNA expression and activation of Erk-1 activity, in contrast to other growth factor cellular responses. More recent studies with CSF-1-treated BAC1.2F5 cells confirmed the findings in the BMMs but found a role for PI-3 kinase in CSF-1dependent cell survival (Murray et al., 2000a); consistent with these latter findings in human monoctyes it has been proposed that CSF-1 promotes cell survival through Akt in a PI-3 kinase dependent manner (Kelley et al., 1999). In myeloid 32D cells, PI-3 kinase activation appears to be on a parallel one to that involving ERK activation (Lee and States, 2000). In BAC1.2F5 cells the CSF-1-stimulated maintenance and translocation of vesicles is PI-3 kinase-dependent (Murray et al., 2000b). PI-3 kinase has been shown to play a a role in the endocytic process for growth factor receptors, such as

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PDGF-R (Joly et al., 1995). However, wortmannin does not significantly affect c-Fms degradation in CSF-1treated murine macrophages, suggesting a lack of requirement for PI-3 kinase activity in this case (Kanagasundaram et al., 1998).

c-Cbl and ubiquitination Among the cellular proteins that are tyrosine phosphorylated in response to CSF-1 in murine macrophages is the proto-oncogene product c-Cbl (Tanaka et al., 1995; Kanagasundaram et al., 1996; Wang et al., 1996). Upon tyrosine phosphorylation, c-Cbl associates with tyrosine-phosphorylated Shc and an unknown phosphorylated protein, pp80, and is transiently ubiquitinated and translocated to the plasma membrane (Wang et al., 1996). Under these conditions, c-Fms is also ubiquitinated (Mori et al., 1995; Wang et al., 1996). As noted earlier, a stable association of c-Cbl with c-Fms, PI-3 kinase and several other tyrosine phosphorylated proteins has been observed in CSF-1-stimulated murine macrophages (Kanagasundaram et al., 1996; 1999). c-Cbl negatively regulates the macrophage proliferative response to CSF-1 (Lee et al., 1999); this negative regulation is associated with targeting of CSF-1R ubiquitination by c-Cbl and a subsequent increase in the CSF-1R endocytic rate.

Shc, Grb2 and other adaptor proteins For the positive regulation of Ras activity in many cells, the guanine nucleotide exchange factors, for example, Sos and other Ras guanine nucleotidereleasing factors, act by increasing the rate of exchange of GDP for GTP. Two adaptor molecules, Grb2 and Shc, seem to be involved in coupling Ras regulators, such as Sos, to PTK receptors. After CSF-1 stimulation of murine myeloid cell lines expressing the murine c-Fms, p52shc and p46shc are tyrosine phosphorylated and associated with Grb2 and c-Fms (Lioubin et al., 1994; Welham et al., 1994), although for some reason this association was not noted in CSF-1treated BAC1.2F5 cells (Xu et al., 1995). The conclusion from one of these publications (Lioubin et al., 1994) was that CSF-1 stimulation of myeloid cells could activate Ras through Sosl by Grb2 binding to Fms, Shc or p150 (subsequently identified as p150Ship;

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see below) and that Fms signal transduction in myeloid cells differs from that in fibroblasts. With CSF-1-treated human monocytes, Kufe’s laboratory has evidence for at least three Grb2 pools with independent association of Grb2 with c-Fms and Shc as well as separate Grb2 complexes with pp125 focal adhesion kinase (FAK) and dynamin, and with PI-3 kinase (Kharbanda et al., 1995; Saleem et al., 1995). The last complex was proposed as providing a potential mechanism for CSF-1-induced interaction of PI-3 kinase and Ras (Saleem et al., 1995). If this were so, and if Ras is controlling Fos/Jun expression as discussed above (Barone and Courtneidge, 1995), then it would be predicted that PI-3 kinase inhibition would give rise to a concomitant inhibition of Fos expression; however, wortmannin failed to suppress CSF-1stimulated c-fos mRNA expression in murine macrophages (Hamilton et al., 1998). Recent evidence has implicated tyrosine phosphorylation of Shc in the control of CSF-1R-driven macrophage differentiation in M1 myeloid cells (Csar et al., 2001). It has been shown (Chen et al., 1996) that Grb2 becomes associated with the tyrosine-phosphorylated phosphatase SHP-1 and other tyrosine-phosphorylated proteins in CSF-1-treated murine macrophages (see below), the purpose of which may be to recruit to SHP-1 substrates that bind to the Grb2 Src homology region 3 (SH3) domains as a potential new function for Grb2 (Chen et al., 1996). The relationships of the various Grb2-containing complexes listed above in CSF-1-treated cells are unclear; as mentioned, Y697 in murine c-Fms would appear to be the Grb2 association site based on experiments with Rat-2 fibroblasts. Another adaptor, Gab2, has been suggested to provide a mechanism for the association of Shc and SHP2 in myeloid FDC-P1 cells and for Erk-dependent differentiation (Lui et al., 2001). The adaptor protein CrkII has been reported to associate with a multimeric CSF-1R-containing complex containing the p85 PI-3 kinase, c-Cbl and Grb2 in CHO cells (Husson et al., 1997).

Phosphatases SHP-1. SHP-1 (PTP1C, HCP, SHPTP1) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hemopoietic cells. It has been reported that SHP-1 becomes tyrosine phosphorylated

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following CSF-1 stimulation of BAC1.2F5 cells, although for some reason it was a major substrate in one study but was weakly phosphorylated in the other (Yeung et al., 1992; Yi et al., 1993); in contrast to c-Kit, association between SHP-1 and c-Fms was not observed (Yi et al., 1993). Upon CSF-1 stimulation of murine macrophages, SHP-1 has been found to be associated with a 130-kDa tyrosine phosphoprotein (Chen et al., 1996) shown subsequently to comprise PIR-B and the SHPS-1-related protein, BIT (Timms et al., 1998; Berg et al., 1999). Genetic evidence suggests that SHP-1 is a negative regulator of hemopoietic cell signal transduction. Homozygous mutations in the SHP-1 gene give rise to motheaten (me/me) and motheaten viable (mev/me) mice (Shultz et al., 1993). Because of an early frameshift mutation, me/me mice are essentially SHP-1 null; the mev/mev mutation results in SHP-1 species with markedly decreased phosphatase activity. Moth-eaten mice display multiple defects in hemopoietic cells, which result in immunodeficiency, systemic autoimmunity and premature death. One study claimed that splenic macrophages from me/me mice proliferate in the absence of CSF-1 (McCoy et al., 1984), while it was found recently that their BMMs retain an absolute requirement for CSF-J for both viability and proliferation and are hyper-responsive to CSF-1 (Chen et al., 1996). BMMs from me/me mice were used to show that Grb2 is associated, via its SH3 domain, with several tyrosine-phosphorylated proteins, leading to the concept (see above) that Grb2 may recruit substrates for SHP-1 which may therefore be a critical negative regulator of CSF-1 signaling in vivo (Chen et al., 1996). RASGAP-associated p62DOK constitutively associates with SHP-1 in BMMs and is a major SHP-1 substrate whose tyrosine phosphorylation correlates with CSF-1-independent survival in macrophages (Berg et al., 1999). By using macrophages expressing dominant-negative Ras and the mev /meV mouse mutant, it has recently been shown that SHP-1 is activated by CSF-1 in a Ras-dependent manner and that its activity is necessary for the Rasdependent activation of the MAPK pathway but not that of Raf-1 kinase (Krautwald et al., 1996). p150Ship. The SH2 domain containing inositol 5-phosphatase (p150ship) is a 145-kDa protein that was initially observed to be tyrosine phosphorylated in

CSF-1-treated FDC-P1 myeloid cells (but not in fibroblasts expressing CSF-1R) (Lioubin et al., 1994, 1996). S1. Mutations in c-Fms that abolish its interactions with Grb2/Sos (Y697F) and PI-3 kinase (Y721F) (see below) do not abrogate p150Ship phosphorylation and its association with Grb2 and Shc. Likewise the Y706F and Y807F mutations do not affect these parameters but the triple mutation, Y697–706–807F, does. Data have been presented for a role for p150Ship as a negative regulator of cell growth (Lioubin et al., 1996). PP2A. The serine-threonine protein phosphatase 2A (PP2A) is rapidly activated in CSF-1-treated BAC1.2F5 cells and can be associated with Raf-l (Abraham et al., 2000). Others. The protein tyrosine phosphatase phi mediates CSF-1-induced morphological changes in macrophages by dephosphorylating paxillin (Pixley et al., 2001). Overexpression of the low molecular weight tyrosine phosphatase reduces the mitogenic response to CSF-1 and tyrosine phosphorylation of CSF-1R (Rovida et al., 1998). The expression of DEP-1, a receptor protein tyrosine phosphatase, is regulated by CSF-1 (Osborne et al., 1998).

JAK-STAT pathways CSF-1 causes the tyrosine phosphorylation of TYK2, a protein kinase belonging to the family of Janus kinase (JAK) kinases, in murine BMMs as well as in NIH3T3 cells expressing human c-Fms (Novak et al., 1995); in the latter cell type, but not in the former, JAK1 was also phosphorylated. In addition, CSF-1 activated the signal transducers and activators of transcription, STAT1, STAT3, STAT5A and STAT5B (Novak et al., 1995; 1996). It has been reported that the suppressor of cytokine signaling (Socs1) interacts with CSF-1R and negatively regulates its proliferation signal (Bourette et al., 2001).

Cytoskeleton CSF-1 induces upregulation of avb5 integrindependent phosphorylation of paxillin in human macrophages via a PKC-dependent mechanism (DeNichilo and Burns, 1993). It also tyrosine phosphorylates the adhesion focal tyrosine kinase Pyk2 in

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human monocytes/macrophages presumably an important step in adhesion or migration (Hatch et al., 1998); along the same lines it also tyrosine phosphorylates MAYP, the major F-actin-associated protein (Yeung et al., 1998). CSF-1-induced osteoclast spreading is accompanied by rapid reorganization of the actin cytoskeleton and phosphorylation of c-Src (Insogna et al., 1997) and a functional association between PI-3 kinase and c-Src (Grey et al., 2000).

Vav The proto-oncogene product, Vav, associates with receptors by SH2 domains and to be tyrosine phosphorylated (Margolis et al., 1992). However, CSF-1 treatment of BAC1.2F5 cells did not lead to increased tyrosine phosphorylation of the constitutively expressed protein (Margolis et al., 1992; Xu et al., 1995).

Effects of cAMP and other inhibitors of CSF-1-stimulated cell cycle progression A number of agents have been found to suppress the proliferative response of macrophages to CSF-1 and used as tools to understand CSF-1 action (Vairo and Hamilton, 1988; Vairo et al., 1990a; Vairo et al., 1990b, 1991, 1992a, 1992b, 1995; Cocks et al., 1992; Rock et al., 1992; Vairo et al., 1992a; Vairo et al. 1992b; Kato et al., 1994; Vairo et al., 1995; Vadiveloo et al., 1996; Sester et al., 2000). The studies have indicated that different antiproliferative agents (8BrcAMP, LPS, interferon- (IFN-), dimethylamiloride) arrest CSF-1treated murine macrophages at distinct checkpoints in the G1 phase of the cell cycle and that their effects on cell cycle machinery are linked to the position at which they arrest cells in G1 (Hamilton et al., 1992; Vadiveloo et al., 1996). We have evidence that increased cAMP activity, which results in increased protein kinase A activity, reduces cyclin D1 and cdk4 expression in CSF-1treated murine macrophages (Cocks et al., 1992; Vadiveloo et al., 1996), in contrast to a study where no such reduction occurred but induction of p27Kip1 was given as the explanation for the cAMP-induced G1 phase arrest (Kato et al., 1994). 8BrcAMP also resulted in down-modulation of c-myc mRNA expression. Overexpression of c-Myc was not able to abrogate the

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antiproliferative effect of 8BrcAMP. However, it did reverse the proliferation inhibition caused by the addition of IFN- , indicating that, while both agents shared an ability to lower c-Myc expression, they also acted via distinct mechanisms to bring about growth arrest (Vairo et al., 1995). It was also observed that endogenous type 1 interferon in murine macrophage cultures could suppress drastically the degree of proliferation found in cultures of CSF-1-treated murine macrophages, thereby masking the proliferative response (Hamilton et al., 1996); the varying interferon levels might explain the variability in the proliferative response in such cultures. We have found that tumour necrosis factor- (TNF) suppresses CSF-1-induced murine macrophage proliferation (Vairo et al., 1991), whereas others have reported that it, in fact, can enhance this proliferation (Branch et al., 1989; Shieh et al., 1989). In part this may be due to changes in phenotype and function of cells depending on their origin.

CSF-1 RECEPTOR c-Fms dimerization, internalization and activating mutations Dimerization Upon addition of CSF-1 to the murine macrophage cell line BAC1.2F5, cell surface c-Fms undergoes the following sequence of changes: non-covalent dimerization → autophosphorylation and signaling → covalent dimerization via disulphide bonds → modification (homodimer to heterodimer) → tyrosine dephosphorylation and internalization (Li and Stanley, 1991). It was proposed that ligand-induced non-covalent dimerization activated c- Fms kinase activity, whereas the covalent dimerization led to the subsequent changes. In contrast, for the myeloid FDC-P1 cells engineered to express murine c-Fms, dimers could be detected only using cross-linkers (Carlberg and Rohrschneider, 1994).

Internalization In the absence of CSF-1, c-Fms has little or no tyrosine kinase activity and remains, apparently

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unaltered, on the cell surface for several hours (Rettenmier et al., 1987; Carlberg et al., 1991). Internalization of CSF-1-bound receptors occurs rapidly; this internalization is via clathrin-coated pits and vesicles (Manger et al., 1984) and targeted to lysosomes, where both the receptor and ligand are degraded (Guilbert and Stanley, 1986). A mutant c-Fms protein lacking tyrosine kinase activity was rapidly internalized after CSF-1 binding but not degraded (Carlberg et al., 1991). Another mutant c-Fms molecule that lacked most of the KI region was similarly internalized after CSF-1 binding and was also not degraded. These data indicate that the signal for internalization is separate from that directing degradation of the receptor. It was also claimed (Carlberg et al., 1991) that CSF-1-induced degradation of c-Fms may require tyrosine phosphorylation of a protein other than that of c-Fms itself and that the kinase insert region may be necessary for recognition of this substrate. An amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine-569 in murine c-Fms would appear to be important for internalization because its mutation to alanine eliminates ligand-induced rapid endocytosis of receptor molecules (Myles et al., 1994). The mutant Fms Y569A also lacks tyrosine kinase activity. Thus, tyrosine-569 within the juxtamembrane region of murine c-Fms is essential for kinase activity and CSF-1-dependent internalization. However, receptor tyrosine kinase activity was shown not to be essential for its endocytosis as the kinase-inactive receptor c-Fms K614A underwent endocytosis, albeit at a reduced rate.

et al., 1992). Mutations at amino acids 301 and 374 in the fourth immunoglobulin-like loop of the extracellular domain of murine c-Fms are necessary, but not sufficient, for receptor dimerization in the absence of CSF-1 (Carlberg and Rohrschneider, 1994). Only receptors with a truncated C-terminus as well as the extracellular domain mutations, dimerized efficiently in the absence of CSF-1, suggesting that the C-terminus of c-Fms also regulated receptor oligomerization. Receptors with mutations in both regions underwent ligand-independent aggregation, transphosphorylation, and phosphorylation of cellular proteins, followed by rapid internalization and degradation. These results suggest that CSF-1 binding to c-Fms initiates activation by inducing conformational changes in both the cytoplasmic C-terminal domain and the fourth immunoglobulin-like loop of the extracellular domain, leading to the formation of stable receptor dimers (Carlberg and Rohrschneider, 1994). There are data to indicate that the cell type in which a mutated c-Fms is expressed can also determine the biological outcome of the mutation (see also below). In the myeloid FDC-P1 cells, c-Fms D802V is a very potent ‘transforming’ mutation (Glover et al., 1995; Morley et al., 1999). However, this mutation in c-Fms could not induce Rat-2 fibroblast foci, even in the presence of CSF-1; in this respect c-Fms D802V has the characteristics of an inactivating mutation in Rat-2 fibroblasts (Glover et al., 1995), despite the fact that it was selected as an activating mutation in FDC-P1 cells. Mutation at Y969 conferred IL-3-independent stimulation of FDC-P1 cell growth (McGlynn et al., 1988).

Activating mutations The transforming mutations in v-fms, which are missing in c-fms, lead to activation even in the absence of CSF-1. These transforming or activating mutations were identified by assaying the ability of c-fms and v-fms chimeric genes to induce foci formation of Rat2 or NIH3T3 fibroblasts (Roussel et al., 1988; Woolford et al., 1988). In this way, it was shown that C-terminal deletion in v-Fms had a transforming effect. In addition, v-Fms has transforming point mutations in the extracellular domain. Many additional single point mutations, which alter the encoded extracellular domain, can also activate c-Fms (van Daalen Wetters

c-Fms tyrosine phosphorylation Upon CSF-1 binding and dimerization the tyrosine kinase domain of c-Fms is activated, resulting in transphosphorylation of specific tyrosine residues within the cytoplasmic domain (Ohtsuka et al., 1990) with the potential to create high-affinity binding sites for Src homology region 2 (SH2) domains, PTB domains, etc., contained within cytoplasmic signaling proteins. Several tyrosine ‘autophosphorylation’ sites have been identified to date in the Fms protein, either directly by tryptic phosphopeptide analysis or inferred by other approaches. For the

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CSF - 1 RECEPTOR

murine receptor these are: Y697, Y706, Y721 (each located in the KI region), Y807 and Y973 in the tyrosine kinase domain, and Y559 in the juxtamembrane region (Tapley et al., 1990; van der Geer and Hunter, 1990; Reedijk et al., 1992; Joos et al., 1996) (Figure 40.2); the equivalent tyrosines in human Fms are at positions 699, 708, 723, 809, 969 and 561, respectively (Sherr, 1991). However, it should be noted that it is unlikely that all of the tyrosine phosphorylation sites in activated c-Fms have been discovered (van der Geer and Hunter, 1990; Downing et al., 1991); this consideration is given further credibility by studies with v-Fms where an additional ‘major autophosphorylation site’ has been claimed (Joos et al., 1996) (see below). One of the difficulties in assigning all of the sites is that the c-Fms tyrosine phosphorylation resulting from CSF-1 action is transient; also, it is still possible that some of the assigned sites may not actually result from an autophosphorylation reaction but be due to an indirect effect of another tyrosine kinase. It would seem that further studies should be undertaken. The roles of the identified tyrosine ‘autophosphorylation’ sites in cellular function will be discussed in turn, as will the binding of specific proteins.

Y809 (human) and Y807 (murine) Ligand-activated human c-Fms can induce continuous proliferation when ectopically expressed in murine NIH3T3 fibroblasts, replacing their requirement for PDGF and insulin-like growth factor-1 (Roussel and Sherr, 1989). In contrast, a receptor mutant containing a phenylalanine for tyrosine substitution at codon 809 (here designated c-Fms Y809F) was mitogenically inactive (Roussel et al., 1990) and ligand-stimulated cells remained arrested in early G1 phase (Roussel et al., 1994). Although the cells expressing c-Fms Y809F exhibited normal receptor tyrosine kinase (R TK) activity in vitro and in vivo, and unimpaired c-fos and c-jun expression (Roussel et al., 1990), c-myc mRNA was poorly induced and its levels were unsustained. Enforced expression of cMyc in these cells overcame the G1 arrest and restored the proliferative response, suggesting that cMyc is required for CSF-1-induced mitogenesis in this cellular system (Roussel et al., 1991); also, in this system constitutively active MEK led to c-myc

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expression and S-phase entry (Cheng et al., 1999) while a truncated PI-4-phosphate-5-kinase complemented the mitogenic defect (Davis et al., 1997). In contrast, the equivalent murine c-Fms Y807F mutant had decreased autophosphorylation in vivo and PTK activity in vitro, and induced lower c-fos and c-jun mRNA expression, but was able to induce an increase in growth rate (albeit reduced) in Rat-2 fibroblasts in response to CSF-1 (van der Geer and Hunter, 1991). In agreement with this last study with the murine c-Fms Y807 mutant, we showed in NIH3T3 fibroblasts expressing human c-Fms that the Y809F c-Fms was significantly less phosphorylated than the wild-type receptor in response to CSF-1, showing only a small increase in tyrosine phosphorylation (Novak et al., 1995). Any loss in receptor kinase activity due to a mutation clouds any assignment of downstream pathways to a dependence on the residue being mutated (e.g. Y807/809) as the pathways might be differentially sensitive to quantitative change in the kinase activity (Stanley et al., 1994). Findings with the murine c-Fms Y807F expressed in FDC-P1 cells differed again (Bourette et al., 1995). In this mutant system, CSF-1-dependent proliferation over a few days actually increased relative to cells expressing the wild-type receptor, the in vivo tyrosine kinase activity of the receptor being comparable, but c-myc mRNA expression being reduced by 50%; one suggestion resulting from the mutation studies in the FDC-P1 cells was that c-Myc induction is not absolutely required for growth (Bourette et al., 1995). Interestingly, in the FDC-P1 cells and in M1 cells (Csar et al., 2001) the mutation in the Y807 site totally abrogated CSF-1-induced differentiation (instead of proliferation) indicating, in these cells at least, that Y807 is required for initiation of a differentiation signal and not a proliferation one. The different conclusions regarding the effects of the Y807F (or Y809F) mutation on the proliferative response to CSF-1 are depicted in Figure 40.2. A model has been proposed in which tyrosine phosphorylation of Y807 controls the conformation of the receptor and its differential interaction with signaling molecules (Bourette et al., 1997; Bourette et al., 2000). More specifically, a change in the cytoplasmic domain occurs such that p85-PI-3 kinase is released in favor of phospholipase Cc2 binding to the same phosphorylated Y721 site.

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Tyrosines in the c-Fms KI domain In NIH3T3 cells genetically engineered to express human c-Fms mutants, deletion of 67 amino acids from the KI domain, including Y699, Y708 and Y723, resulted in a partially impaired ligand-dependent proliferative response with an intact in vivo and in vitro tyrosine kinase activity (Shurtleff et al., 1990; Choudhury et al., 1991c). However, in Rat-2 fibroblasts transfected with mutated murine c-fms, CSF-1dependent growth signaling was abrogated in cells containing the Y697F mutant, the Y721F mutant or the mutant with Y697, Y706 and Y721 all changed to phenylalanine (Reedijk et al., 1990; van der Geer and Hunter, 1993); for the last mutant there was a reduction in autophosphorylation activity (van der Geer and Hunter, 1993). Again, in contrast to the above studies with transfected fibroblasts, for FDC-P1 cells transfected with the receptor with Y697, Y706 and Y721 all mutated to phenylalanine, CSF-1-dependent proliferation was slightly increased with in vivo

tyrosine kinase activity intact (Bourette et al., 1995). This uncertainty over the role of the tyrosine phosphorylation sites in the K1 domain in the proliferative response to CSF-1 in the various cell systems is indicated in Figure 40.2.

Y973 It has been implied that Y973 in the C-terminal region of murine c-Fms (Y969 in human c-Fms) is tyrosine phosphorylated (Joos et al., 1996) and has been implicated ill the lineage switching of pre-B cells to macrophages (i.e. differentiation) (Borzillo et al., 1990) and in the negative regulation of growth in CSF-1-treated NIH3T3 cells expressing human c-Fms (Roussel et al., 1987) (Figure 40.2). Y973 has been claimed to be the binding site for c-Cbl (Mancini et al., 2002; Wilhelmsen et al., 2002). Point mutations at codon 969 of the FMS gene have been identified in patients with acute myeloid leukemia and myelodysplasia (Ridge et al., 1990; Tobal et al., 1990).

FIGURE 40.2 Cytoplasmic regions of c-FMS and downstream pathways? The activated murine c-Fms protein is depicted with the TK and KI domains shown. It would appear that phosphorylated Y559, 697, 706 and 721 are the binding sites for the Src family, Grb2, STAT1 and PI-3 K p85 subunit, respectively. A role for Y807/809 in the CSF-1 proliferative response has been proposed (Bourette et al., 1995; for v-FMS there is association with tyrosinephosphorylated p120 Ras GAP Trouliaris et al., 1995), but there are no reports for any protein to be associated with Y807/809 in c-Fms. The roles of the KI domain and PI-3 kinase (PI3-K) in the proliferative response are debated. There is the suggestion that Y973 is phosphorylated and involved in differentiation signaling (Roussel et al., 1987; Burzillo et al., 1990; Joos et al., 1996) and is the c-cbl binding site (Mancini et al., 2002; Wilhelmsen et al., 2002). Numerous complexes (nine or more) containing Grb2 have been described; their relationships are unclear. The equivalent tyrosine to Y544 in v-Fms has been shown to be a ‘major autophosphorylation site’ (Joos et al., 1996) but not reported in c-Fms. THE CYTOKINES AND CHEMOKINES

CSF - 1 RECEPTOR

Substrate binding to tyrosine phosphorylated c-Fms Following tyrosine phosphorylation after activation, RTKs acquire the ability to bind to proteins containing SH2 domains, PTB domains, etc. The specificity of the interactions is determined both by these SH2 domains, for example, and by the sequence surrounding the phosphorylated tyrosine (Reedijk et al., 1992). The evidence for the binding of SH2 domaincontaining proteins to activated c-Fms is now listed and the conclusions are summarized schematically in Figure 40.2. A novel adaptor protein called ‘Mona’ for monocytic adaptor has been identified which contains one SH2 domain and two SH3 domains closely related to the Grb2 adaptor. Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Mona is express in the spleen and peripheral blood mononuclear cells (Bourette et al., 1998; Bourgin et al., 2002). Grb2. In Rat-2 fibroblasts transfected with murine c-fms, the adaptor protein Grb2 associates with the receptor upon CSF-1 treatment and Y697 had been identified as its binding site (van der Geer and Hunter, 1993). As discussed earlier, at least nine Grb2containing complexes have been claimed (Lioubin et al., 1994; Kharbanda et al., 1995; Saleem et al., 1995; Chen et al., 1996). PI-3 kinase. As mentioned above, there is evidence in various cell populations that CSF-1 can activate PI-3 kinase activity (Varticovski et al., 1989; Reedijk et al., 1990, 1992; Shurtleff et al., 1990; Choudhury et al., 1991c; van der Geer and Hunter, 1991; Yusoff et al., 1994); it has been reported that Y721 regulates this activity in Rat-2 fibroblasts containing mutant murine c-Fms and is the binding site for the p85 regulatory subunit, most likely through SH2 domains (Reedijk et al., 1992). In CSF-1-treated murine macrophages, the p85 subunit of PI-3 kinase is the major [35]S-methionine-labeled protein associated with the receptor (Kanagasundaram et al., 1996). STAT1. In a study with FDC-P1 cells transfected with c-fms mutants, it has been reported that Y706 is required for efficient activation by CSF-1 of the transcription factor STAT1, but not of STAT3 (Novak et al., 1996); also, addition of phosphopeptides spanning

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Y708 of human c-Fms (Y706 in murine c-Fms) to electrophoretic mobility shift assays led to competition of STAT1-containing complexes with the DNA probe, suggesting that this tyrosine is the binding site for STAT1. The Y807F mutation in murine c-Fms expressed in FDC-P1 cells led to a block in the activation of STAT1, STAT3, STAT5A and STAT5B in response to CSF-1, but Y807 does not seem to be a STAT binding site. Src. It has been proposed that Src family members may interact with the phosphorylated Y561 residue in the juxtamembrane region of activated human Fms (Y559 in murine Fms) (Alonso et al., 1995). Support for this has been obtained using the receptor with this mutation expressed in M1 myeloid cells CSF-1dependent Src activation and macrophage-like differentiation are both abrogated (Marks et al., 1999). Src-dependent Erk activation and cyclin D expression in CSF-1-dependent systems have been reported (Corre et al., 1999; Dey et al., 2000; McMahon et al., 2001).

v-Fms tyrosine phosphorylation and regulation of CSF-1 signaling The v-fms oncogene encodes a modified PTK which differs in only seven amino acid positions and in the C-terminal sequence from c-Fms (Sherr, 1988). The entire cytoplasmic domain of v-Fms contains 18 tyrosine residues that are conserved in c-Fms. All c-Fms proteins, however, contain an additional tyrosine residue at the C-terminus. Y-543 has been shown to be a ‘major autophosphorylation site’ in v-Fms (Joos et al., 1996). Strangely, there has been no claim for autophosphorylation of the equivalent tyrosine in cFms (Y544 in the mouse, Y546 in the human) after activation (Figure 40.2). Interestingly, in the same study Y558 (equivalent to Y559 in murine c-Fms and to Y561 in human c-Fms) was not considered to be a major autophosphorylation site, even though it has been inferred for c-Fms (Alonso et al., 1995). No association of any protein with Y807 has been reported for c-Fms (Figure 40.2), but for v-Fms there is association with p120 Ras GAP, which is tyrosine phosphorylated (Trouliaris et al., 1995). In v-Fms, it has been published that Y543 associates with a tyrosinephosphorylated p55 protein (Joos et al., 1996).

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CSF-1/CSF-1R SIGNALING – FUTURE DIRECTIONS From the above discussion it can be seen that there are serious disagreements regarding key issues in CSF-1R signaling, for example the role of Y807 and the degree of kinase activity retained upon its mutation, pathways governing c-Myc expression, the degree of significance of the ‘classical’ Ras→Raf-1→MEK→MAP kinase (Erk) pathway in CSF-1-treated macrophages, the role of the K1 region in the proliferative response, the nature of the Grb2/Shc-containing complexes (e.g. whether p150Ship is present), the effects of the c-Fms D802V, the degree of tyrosine phosphorylation of PI-3 kinase, etc. For certain types of experiment, particularly those involving mutational analysis, it has been necessary to insert c-Fms into cell types in which the receptor is not usually found. An important caveat of this type of study is that the ability of the receptor to induce a response in such cells (e.g. proliferation) may involve the recruitment of second messenger systems that are not normally engaged in cells in which the receptor is normally expressed (Sherr, 1991; Stanley, 1994); perhaps differences in the actual contents of signal transducers in various cell types may be a significant contributing factor. It could be argued that non-monocyte/macrophage myeloid cells, such as FDC-P1 cells, although not ideal, may more closely reflect the normal situation than fibroblast-type cells. Findings from these heterologous systems will have to be checked where possible to determine their relevance for CSF-1-dependent monocyte/macrophage biology. While the discordant findings obtained from different cell types, which were outlined above, could result from the presence of c-Fms in vastly different cell types, there are other inconsistent findings using the same or similar cellular systems to analyse the CSF-1 response which are more difficult to explain: for example, the relationship of Ras activity to subsequent c-fos or c-myc expression, the role of c-Myc and PI-3 kinase in proliferation, the degree of tyrosine phosphorylation of SHP-1 and GAP, whether PKC and phospholipase C are activated, whether PGE2 is produced, the differing actions of 8BrcAMP on cyclin D1 expression, etc. Obviously further experimentation is needed before these issues are clarified. The same or very similar molecules are activated by

different mechanisms and function in different pathways depending on the cellular background and on the stimulus used. One example of this, which is referred to above, is the activation of the Erk pathway where, in macrophages, it could be that the paradigm Ras–Raf-1–MEK–Erk pathway may not be that significant (Büscher et al., 1995; Krautwald et al., 1996) even though each of the components appears able to be activated. In other words, to have any significance, the findings will have to explain ultimately the in vivo role of CSF-1 in the development and function of cells of the monocyte/macrophage lineage and of the other cell types in which c-Fms is expressed, for example trophoblasts (Stanley, 1994). Data from c-src and c-fos knockout mice (Soriano et al., 1991; Grigoriadis et al., 1994) suggest that the products of these genes are at least dispensable for macrophage development. The recent availability of CSF1-R gene-deficient mice (Dai et al., 2002) should provide answers to some of these questions. The advent of microarray and mass spectrometry-based proteomics technologies also offers potential.

CLINICAL DETECTION OF CSF-1 Presence in normal sera and in inflammation Normal human blood serum or plasma contains on average 118±9 U/ml of M-CSF, which is biologically active (Shadle et al., 1989). M-CSF has been found at increased amounts in chronic inflammatory states including multiple sclerosis (Werner et al., 2002), chronic hepatitis, correlating with the amount of sTNFR p75 (Itoh et al., 1999a), and following hepatic damage induced by transcatheter embolization (Itoh et al., 1999b) correlating (p0.001) with increases in serum alanine aminotransferase and type IV collagen-7S. Advanced glycation end products (AGEs) are found in the serum of patients with long-term hemodialysis, correlating with increases in M-CSF and IL-18 suggesting a state of macrophage activation in these patients (Uchimura et al., 2002). Similarly, M-CSF levels in dialysis patients are about double of that of normal individuals and positively correlated with measures of atherosclerosis (Nitta et al., 2001) including estimates of aortic calcification evaluated

THE CYTOKINES AND CHEMOKINES

ROLE OF M - CSF ON MONOCYTES , TISSUE DERIVED MACROPHAGES AND DENDRITIC CELLS

by CT scan (r0.596, p0.01). Kawasaki disease (KD) in children is associated with profound activation of macrophages. The serum M-CSF levels measured during the first week of illness is higher than than observed in subsequent weeks (1710 compared with 867–1121U/ml, p0.001) and are noted to be particularly elevated in patients with valvular insufficiency and persistent coronary dilatation (Igarashi et al., 2001). Low cholesterol and triglyceride levels in children with aplastic anemia are associated with elevated M-CSF levels and a poor prognostic factor (Yokoyama et al., 2000). In apparently normal pregnancies with good outcome, M-CSF levels rise throughout pregnancy and preeclampsia did not occur if maternal serum M-CSF levels increased more than 100% throughout the course of the pregnancy (Keith et al., 2000).

Presence in sera of patients with cancer M-CSF has been noted to be increased in the serum of patients with several tumor types. These include ovarian cancer (Moradi et al., 1993), chronic lymphocytic leukemia (Jewell et al., 1994), adult T-cell leukemia (Yamada et al., 1996), malignant histiocytosis (Ozaki et al., 1995), breast cancer (Scholl et al., 1994, 1996), and lung cancer (Katsumata et al., 1996). In general, higher levels are associated with poor prognosis. Levels in women with ovarian cancer diminished with successful resection of tumors (p0.0001), but were not otherwise related to stage, histology, grade of differentiation, age, or residual disease (Gadducci et al., 1998). M-CSF levels were also higher in the serum of patients with clinically evident disease than in women with no clinical evidence of disease (p0.0001) on follow-up. Increased levels in the serum of women followed with primary breast cancer (Scholl et al., 1994) also negatively correlated with survival, predicting a shorter disease free interval (p0.03). In a multivariate analysis, including tumor size, clinical node status, histological grade, and hormone receptor status, levels remained significantly associated with poor outcome (relative risk of relapse: RR: 3.3 [1.3–8.5]), tumor size (RR: 2.8[1–8.2]) and clinically positive nodes (RR: 4.1[2.1–8]). Conversely, increases in M-CSF in the serum of patients treated with alpha interferon with CLL correlated with improvement (Jewell et al., 1994).

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ROLE OF M-CSF ON MONOCYTES, TISSUE DERIVED MACROPHAGES AND DENDRITIC CELLS The SV40-immortalized mouse macrophage cell line, BAC1.2F5, is dependent on CSF-1 for survival in culture. Introduction of human CSF-1 cDNA into these cells eliminated their growth factor dependence but these cells were not transformed and unable to grow as tumors (Roussel et al., 1988). Peripheral blood monocytes incubated in human M-CSF enhanced ADCC activity (Munn and Cheung, 1989; Qi et al., 1995) using antibodies to human melanoma and neuroblastoma cell lines with modest antibodyindependent cytotoxicity. Thus M-CSF serves to enhance monocyte/macrophage survival and some effector functions. Apoptotic uptake inhibits proliferation of bone marrow derived macrophages stimulated to proliferate by M-CSF (Reddy et al., 2002). These cells are capable of maturing into phenotypic DCs following inductive influence of LPS or IL-1 and CD40L. In the interstitium, they survive by coordinate expression of TRANCE and RANK (Wong et al., 1997; Josien et al., 1999; Cremer et al., 2002). Macrophages generated from CD34() human progenitor cells using M-CSF die with the addition of soluble RANK, blocking autocrine stimulation of survival by cell surface RANK. When matured with LPS and CD154, they now also become dependent on exogenous TRANCE for survival. TRANCE (RANKL) induces mucosal secondary lymphoid (Peyer’s patch) DC to produce IL-10, and peripheral nodal DC to produce IL-12, thereby regulating tolerance and activation to environmental antigens (Williamson et al., 2002). Following tissue activation and subsequent migration through the lymphatics, DC become dependent on provision of exogenous sources of TRANCE and other survival factors in lymph nodes. Nodes draining sites of epithelial cancer have significantly less DCs than normal lymph nodes and this might be ascribed to the lack of such survival signals at those sites (Azizi et al., 1998; Laguens et al., 2002). The lack of survival factors may represent another mechanism by which tumors evade immune recognition. When exogenously derived DC are injected intravenously they home to splenic sites and when injected intralymphatically, they home to

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nodal sites, persiting for 1 to 7 days (Mackensen et al., 1999) in patients with melanoma. This suggests that the lymph nodes themselves in such cancer patients at least do not lack the survival factors necessary for DCs. The presence of IL-12, induced by tissue macrophage derived IL-1 and CD40 ligation, may provide these survival factors (Luft et al., 2002) or they may reside in the serum (Loudovaris et al., 2001).

ROLE OF M-CSF ON OSTEOCLASTS Mature osteoclasts can be indentified as cells that express tartrate resistant acid phosphatase, carbonic anhydrase II, cathepsin K, vitronectin, calcitonin receptors, vacuolar-type H()-ATPase, and promote lacunar resorption and are supported critically by M-CSF (a product of osteoblasts). They are also supported by IL-1, TNF, and IL-17 (Kermanac et al., 2002) but not by other cytokines including GM-CSF, IL-3, IL-6 or IFN gamma (Hattersley et al., 1988). M-CSF is necessary for bone marrow derived progenitors to proliferate and enter the osteoclast pathway (Fan et al., 1997) but inhibits osteoclastogenesis, promoting multinucleated macrophage-like cells, at higher doses (Shiotani et al., 2002). Fetal bone conditioned media containing M-CSF also induce the multinuclearity of mononuclear progenitors (van der Wijngaert et al., 1989) and inhibits osteoclast generation. Notch ligands expressed on bone marrow stromal cells critically regulate cell fate determination, promoting DC maturation (Weijzen et al., 2002) and inhibiting osteoclast generation (Yamada et al., 2002).

Factors modifying M-CSF activity on osteoclasts TRANCE (osteoprotegerin ligand, osteoclast differentiation factor or RANKL) and TNF (Kudo et al., 2002a), both as cell surface and soluble molecules, enhance RANK expressing osteoclast survival, and along with M-CSF, promote bone resorption. Calcitonin, conversely, directly inhibits the motility and resorptive activity of osteoclasts (Kudo et al., 2002b). The recently defined osteoclast associated receptor, OSCAR, a member of the leucocyte receptor family, engages a counter-receptor on osteoblasts and promotes bone resorption. This is inhibited by a soluble

form of the molecule (Kim et al., 2002). Conversely, GM-CSF and IL-3 promote common progenitors of DCs and osteoclasts to enter the DC pathway, coincident with downregulation of c-fos and Fra-1 (Miyamoto et al., 2001). When osteoclasts form in the absence of osteoblastic cells, markedly higher levels of TRANCE mRNA are expressed. When osteoclast progenitors are stimulated in the presence of peripheral blood lymphocytes with M-CSF and TRANCE, osteoclast generation is limited unless leptin, IL-4, IL-10, IL-13, or TGF is added (Massey et al., 2001; Scopes et al., 2001; Holloway et al., 2002; Kermanac et al., 2002; Quinn et al., 2002a). Direct promotion of osteoclast generation from bone marrow progenitors is also suggested in some studies with TGF in the absence of apparent lymphoid cells (Quinn et al., 2002b). In aggregate, these studies suggest that immune cells modify osteoclast activity and that the context for osteoclast generation is critically dependent on M-CSF presence. Environmental factors including inflammation play a major defining role on the biologic activity of these cells.

Survival and regulation of osteoclasts by M-CSF Ingestion of particulates in bone by resident macrophages results in differentiation into osteoclasts (Haynes et al., 2001), capable of osteolysis. The factors responsible for osteoclast survival include M-CSF as well as IL-1, osteoclast differentiation factor and TRANCE. These factors also downregulate a novel apoptosis inducing kinase, DRAK (death-associated protein [DAP] kinase-related apoptosis-inducing protein kinase 1) which, when transfected into osteoclasts, induces apoptosis of these cells (Kojima et al., 2001). Although osteoclasts are found in reduced numbers associated with reduced bone resorption in op/op mice, they are still identifiable. FLT3L appears to be the factor responsible for supporting some level of osteoclast survival in the absence of M-CSF (Lean et al., 2001). Although TRANCE induces interferon

from progenitors, which in turn would inhibit osteoclastogenesis, it also induces the suppressors of cytokine signaling, SOCS1 and 3, which limit interferon signaling (Hayashi et al., 2002). Increased osteolysis is noted by increased functionality of osteoclasts in patients with rheumatoid arthritis and this is inhbited by corticosteroids (Hirayama et al., 2002a).

THE CYTOKINES AND CHEMOKINES

PRECLINICAL AND CLINICAL MODELS OF CSF - 1 DELIVERY

However, steroids also enhance the proliferation and differentiation of osteoclast progenitors (Hirayama et al., 2002b). Parathyroid hormone (PTH) regulates osteoclast formation and activation through its reciprocal up and down-regulation of RANKL and osteoprotegerin (OPG), respectively (Kondo et al., 2002) by bone stromal cells. Parathormone increases RANKL and macrophage colony-stimulating factor (M-CSF) expression, while decreasing that of OPG, through cAMP and protein kinase A signaling.

ROLE OF M-CSF ON MICROGLIA Microglia are highly specialized cells residing within the brain that belong to the mononuclear phagocyte system. Microglia are derived from a common bone marrow progenitor giving rise also to macrophages, osteoclasts, and dendritic cells promoted by the hematopoietic stem cell factor, Flt3-ligand (FL). In response to M-CSF and glial cell-conditioned medium (GCCM), they commit to becoming microglia (Servet-Delprat et al., 2002). It appears that the parenchymal microglia are uncomitted progenitors that remain in an undifferentiated state with empty Class II MHC molecules and a pattern of cathepsins consistent with lack of antigen presenting function (Santambrogio et al., 2001). Although these cells can indeed serve as functional antigen presenting cells, with culture in GM-CSF they progressively lose this capability unless cocultured with astroglia or conditioned media from these cells (Fischer and Bielinsky, 1999). The factor made by astrocytes, supporting the c-fms positive microglia appears to be M-CSF and the supportive activity of astrocytes is blocked with antibodies to it. Gene expression analysis reveals that GM-CSF increases antigen presentation associated gene products whereas M-CSF does not (Re et al., 2002).

Microglia and inflammation During quiescence, microglia appear to induce T-cell apoptosis, preventing unnecessary inflammation in the brain Perry, 1998). Inflammation including T. gondii infection allows functional upregulation and maturation of the APC and IL-12 producing activity of

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these cells (Fischer and Reichmann, 2001). In the setting of multiple sclerosis, there are markedly increased numbers of microglia within the plaques but they fail to express M-CSFR or M-CSF (Werner et al., 2002). Microglia are also increased in Alzheimer’s disease with marked increase in nitric oxide production and inflammatory cyotkines including iL-1, MIP1, but in vitro they do not appear to induce neuronal death (Vincent et al., 2002). Beta amyloid promotes survival of macrophage and increases their response to M-CSF (Hamilton et al., 2002). In the setting of HIV infection within the CNS, viruses persist in macrophages and microglia. Treatment of infected cells with either GM-CSF or M-CSF increased antiviral

chemokines including RANTES with induction of MIP1, MIP1 as well as M-CSF but not GM-CSF (Si et al., 2002). GM-CSF appears to be produced by locally activated astrocytes.

PRECLINICAL AND CLINICAL MODELS OF CSF-1 DELIVERY The half-life of truncated M-CSF forms injected IV into rats increased with the MW of 10 kD polyethylene glycol linked to forms of rM-CSF (Koths, 1990). M-CSF transfection of tumors causes substantial infiltration with macrophages (Dorsch et al., 1993; Hock et al., 1993). Tumor growth was only slightly affected by MCSF and in spite of transfection cells grew as tumor in vivo. Conversely, M-CSF antisense treatment suppresses human tumor xenograft growth in murine models (Aharinejad et al., 2002). Administration to cynomolgus monkeys by continuous intravenous infusion or subcutaneous injection, at a dose of 50 to 100 micrograms/kg/day causes a progressive 7-fold increase in the number of large, granular, and extensively vacuolated circulating monocytes (Munn et al., 1996). These cells expressed HLA-DR, LFA3, CD11b, and CD14, and CD16 but with little cytotoxicity against melanoma cell lines. M-CSF augments the antifungal activity of macrophages against conidia/hyphae of Aspergillus fumigatus by enhancing oxidation-dependent destruction in in vitro studies (Roilides et al., 1995). Not surprisingly, M-CSF enhances human macrophage HIV-1 infection in vitro (Gruber et al., 1995). HIV-infected cells also produced M-CSF at levels

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5- to 24-fold higher than uninfected cells, dependent on viral entry and active replication of HIV-1. Exogenous M-CSF delivery increased the frequency of macrophage infection, the amount of HIV mRNA, and proviral DNA expressed per infected culture. In contrast, infection with measles or respiratory syncytial virus, does not induce M-CSF but rather MIP-1alpha. Addition of anti-M-CSF antibodies or soluble M-CSF receptors inhibits HIV replication and reduced the production of MIP-1alpha, suggesting a possible strategy for therapy (Kutza et al., 2000, 2002).

M-CSF IN ANIMAL MODELS OF INFECTION M-CSF is increased in the serum and organs (brain, kidney, liver and lung) of mice following infection with C. albicans (Cenci et al., 1991). It remains elevated for 2 weeks until a T cell response is induced. Although early studies suggested that administration of M-CSF enhanced infection (Hume and Denkins, 1992), in subsequent studies, it increased survival times and decreased recovery of C. albicans from the organs of treated mice alone and in combination with amphotericin (Kuhara et al., 2000). Daily injection in combination with fluconazole decreases the median titer of C. albicans and improves the median survival time in a rat model (Vitt et al., 1994). Similar studies with IL-1 alone and in combination with fluconazole lowered the number of C. albicans organisms in neutropenic mice but not normal mice (Kullberg et al., 1992). Treatment with recombinant G-CSF or murine interferon-gamma alone is ineffective in SCID mice infected with C. albicans, and combinations with a suboptimal dose of fluconazole is not synergistic. These studies suggest that the cytokines made during the initiation of the adaptive immune response are not themselves helpful in eradicating this organism but this would need to be tested now in combination with M-CSF and in the setting of an intact immune system.

INFLAMMATORY ANIMAL MODELS INVOLVING M-CSF Lipopolysaccharide (LPS) stimulates macrophage activation but inhibits response to M-CSF prolifera-

tive signals, including expression of cyclin D1 (Vadiveloo et al., 1998). Macrophages proliferate in response to M-CSF, expressing cyclins D1 and D2 but not D3. LPS blocks M-CSF-induced proliferation and cyclin D1 expression in bone marrow derived macrophages but stimulates increases in the level of unstimulated and M-CSF-stimulated cyclin D2 mRNA and protein. Fas deficiency in MRL mice is associated with autoimmunity, macrophage accumulation and proliferation as well as altered macrophage (Hamilton et al., 1998). DNA synthesis responses of peritoneal macrophages and those derived from bone marrow precursors do not require the addition of M-CSF to enter the cell cycle and respond vigorously to GM-CSF (Hamilton et al., 1998). In the setting of atheroma, oxidized LDL has been predicted to stimulate macrophages to promote arterial lesions. Indeed in murine macrophages they can induce macrophage survival, DNA synthesis, and enhance proliferative response to M-CSF and GM-CSF (Hamilton et al., 1999, 2001). Delivery of either M-CSF or GM-CSF into the knee of mice injected with bovine serum albumin or in the setting of collagen induced arthiritis, induces enhanced inflammatory responses (Bischoff et al., 1996; Campbell et al., 2000). M-CSF deficient op/op mice are resistant to collagen-induced arthritis. Many particulate adjuvants including aluminum hydroxide, oil-in-water emulsions, calcium phosphate, and silica, also enhance murine bone marrow-derived macrophage survival, proliferation synergizing with exogenous GM-CSF or M-CSF (Hamilton et al., 2000). They enhance survival and the number of cells important for antigen presentation and cytokine production.

CLINICAL TRIALS WITH M-CSF M-CSF enhances antibody dependent phagocytosis and killing of murine tumors (Sanda et al., 1992a). We applied M-CSF in a dose rising schedule as well as following continuous intravenous administration in patients with cancer with a single apparent response in a patient with pulmonary metastases from renal cell cancer (Sanda et al., 1992b). This may have been mistaken pulmonary candidiasis rather than malignant disease. M-CSF was given by

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IV bolus infusion every 8 hours for 7 days. Dose escalation from 10 to 100 000 micrograms/m2/day was administered in 23 patients. Interestingly, five patients developed inflammation with transient signs of ocular/periorbital reddening and iridocyclitis. The absolute monocyte count increased with modesty decrease in platelets during therapy. The half-life varied in individual patients from 1 to 6 hours. In a subsequent study (Cole et al., 1994), twelve patients received two 14-day cycles of rHuMCSF by continuous infusion, with an interval of 2-weeks. Dose levels of 50, 100, and 150 micrograms/kg were given over 24 hours. Severe thrombocytopenia at the highest dose was observed in two patients. Peak serum levels occurred on Day 1. Conjunctival reddening, episcleritis and asymptomatic perilimbal and retinal hemorrhages were observed. No objective responses were observed in these patients with cancer. More frequent administration of M-CSF with infusion every 8 hours 5 days and repeated after a 9-day rest at dose levels of 20-1, 100 micrograms/m2 revealed a similar serum halflife of 25–84 min, consistent with saturable clearance (Redman et al., 1992). With this regimen, monocyte number increased fourfold when compared with baseline. Antitumor activity was observed in two patients with hepatic metastatic leiomyosarcoma metastatic to the liver. rM-CSF has also been administered subcutaneously for 10 to 14 days of treatment with cycles repeated every 28 days at dose levels from 0.1 to 25.6 mg/m2/day. Toxicity included thrombocytopenia and iritis. Dose-dependent monocytosis occurred at doses 3.2 mg/m2/day. Increases in secretion of TNF-alpha and IL-1b by monocytes after stimulation with LPS was noted. Continuous infusion of M-CSF was associated with decreased cholesterol (and the other findings observed with other means of administration) with an anecdotal response in multiple lesions in a single patient (Jakubowski et al., 1996). Other studies have been done in the setting of concurrent antimelanoma antibody administration (Schmid et al., 1995). rhM-CSF increased cell size, relative percentages of CD14, HLA-DQ, CD11b, and CD16 monocytes, and enhanced cell-surface expressions of HLA-DR and CD11b, promoting enhanced ADCC. Similarly, patients with metastatic melanoma receiving continuous infusion of 80

micrograms/kg/day with the R24 antibody with 3/19 patients attaining tumor regression in breast, liver, and lymph node metastases with prolonged survival noted in these and some of the other treated patients (Minasian et al., 1995). In a Phase II study in patients with metastatic gastrointestinal cancer, the mAb D612 in combination with rhM-CSF at 80 micrograms/kg/day 1–14, peripheral monocytosis without apparent clinical antitumor responses (Saleh et al., 1995). Patients with fungal infection following bone marrow transplantation received M-CSF in addition to conventional antifungal regimens (Nemunaitis et al., 1991). Infection resolved in six of the treated patients, twelve were inevaluable and six had no response. Long-term follow-up of bone marrow transplant patients with severe fungal infections who received M-CSF as adjunctive therapy treatment (Nemunaitis, 1998) revealed enhanced survival of M-CSF treated patients when compared with an historical control group (27% versus 5%). Additional studies will need to be done to demonstrate the utility of M-CSF in fungal infections (Hubel et al., 2002).

ACKNOWLEDGEMENTS The authors thank Jacqueline Reed for her support during the preparation of this revised chapter and colleagues at the University of Pittsburgh for their encouragement in returning to the fold of academic clinicians. The National Health and Medical Research Council is acknowledged for a Senior Principal Research Fellowship to John A. Hamilton.

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41 EGF family ligands David C. Lee, C. Leann Hinkle, Leslie F. Jackson, Shunqiang Li and Susan Wohler Sunnarborg University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

There’s something happening here, and what it is ain’t exactly clear. Stephen Stills, from ‘For What It’s Worth (Stop, Hey, What’s That Sound)’ 1967

OVERVIEW The mammalian ErbB signaling network has vital roles in development and homeostasis, and its components are frequent and important targets for deregulation leading to cancer and other diseases (reviewed in Alroy and Yarden, 1997). The network comprises multiple polypeptide ligands belonging to the epidermal growth factor (EGF) superfamily, and four related type I receptor tyrosine kinases, ErbB1–ErbB4, which initiate signal transduction.

EGF family proteins The EGF superfamily includes members of the EGF and Neuregulin (also known as Heregulins/Neu differentiation factor) subfamilies. The EGF group includes six independent gene products: EGF, transforming growth factor-alpha (TGFa), amphiregulin (AR), heparin-binding EGF (HB-EGF), betacellulin (BTC), and epiregulin (EREG). An apparent seventh The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

member, epigen, was recently described (Strachan et al., 2001). The Neuregulins are multiple polypeptides derived from four distinct genes by alternative splicing (Wen et al., 1992; Carraway et al., 1997; Zhang et al., 1997; Harari et al., 1999). The hallmark of this superfamily is a structural motif (the EGF-like sequence) corresponding to a sequence of 36–40 amino acids that includes six conserved Cys residues (Figure 41.1). Disulfide bonds formed between specific Cys pairs (C1–C3, C2–C4, C5–C6) give rise to a three-loop structure that must be intact for high affinity receptor binding, and thus appreciable biological activity. Comparing EGF subfamily members, the consensus sequence is XnCX7CX4–5CX10CXCX3YXGXRCXn. Preservation of this motif allows other EGF subfamily ligands to compete with EGF for binding to the EGF receptor and to mimic the biological actions of EGF, even though overall sequence conservation across the family is only ~20–25%. A large number of transmembrane or secreted proteins of diverse function, including EGF receptor, Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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EGF TGFa: Epigen: HB-EGF: BTC: EPR: AR:

1 3 53 38 30 1 38

NSYPG-CPSSYDGYCLNGGVCMHIESLDSYTCNCVIGYSGDRCQTRDLRWWELR SHFNK-CPDSHTQYCFHG-TCRFLVQEEKPACVCHSGYVGVRCEHADLLA LKFSHPCLEDHNSYCING-ACAFHHELKQAICRCFTGYTGQRCEHLTLTSYA GLGKKRDPCLRKYKDYCIHG-ECRYLQEFRTPSCKCLPGYHGHRCHGLTL KVKTHFSRCPKQYKHYCIHG-RCRFVVDEQTPSCICEKGYFGARCERVDLFY VQITK-CSSDMDGYCLHG-QCIYLVDMREKFCRCEVGYTGLRCEHFFL RNKKKKNPCTAKFQNFCIHG-ECRYIENLEVVTCNCHQDYFGERCGEKSM

FIGURE 41.1 Alignment of the EGF motifs of the seven mouse EGF family members. The position of the first amino acid depicted relative to the N-terminus of the mature forms is indicated. Residues conserved throughout the family are indicated, and arrows denote residues that are conserved in all family members except AR.

contain related – often repeated – EGF-like motifs. However, the number and spacing of the Cys residues is less well conserved, and the Gly and Arg residues are often absent. Hence, related motifs do not bind to EGF receptor and instead probably function in protein–protein interaction and/or Ca2 binding.

Precursor processing An initially surprising feature of EGF family growth factors is that the mature ligands are invariably derived from the extracellular regions of type I transmembrane precursor proteins by proteolytic cleavage. Whether these transmembrane precursors have distinct biological activities, ensure proper folding of the EGF peptide, or prevent inappropriate intracellular binding to receptor (Wiley et al., 1998) is a subject of considerable interest. The precursors share a similar architecture which includes (from the N-terminus) a signal peptide, an N-terminal proregion of variable length, the mature peptide sequence containing the EGF motif, a single transmembrane domain, and a relatively short cytoplasmic tail. Invariably, the mature EGF-like peptide is located close to the membrane, separated only by a short juxtamembrane stalk. Except for the EGF motif, the various precursor proteins display no significant homology. Among EGF family members, mature forms of AR and HB-EGF are distinguished by the ability to bind heparin; this is due to an N-terminal extension from the EGF motif that is rich in basic residues. However, the significance of heparin binding in EGF family actions is not fully understood. Conversion of EGF family precursors to soluble growth factors is normally slow in cell culture, but is

rapidly stimulated by phorbol esters, elevated cytosolic calcium levels, and serum factors (Pandiella and Massague, 1991a, 1991b). This stimulation occurs as part of a global process (Arribas and Massague, 1995) termed ectodomain shedding that additionally regulates other cell surface proteins, including receptors and cell adhesion molecules. Release of soluble EGF family ligands is also transactivated by G-proteincoupled receptors (Prenzel et al., 1999) and autoregulated by EGFR signaling (Baselga et al., 1996). Cleavage occurs at the cell surface, is sensitive to metalloprotease inhibitors (Bosenberg et al., 1993; Arribas et al., 1996), and appears to be important for autocrine signaling (Dong et al., 1999). Variable processing of the precursors, especially at the N-terminus, often gives rise to multiple soluble species that may have different biological potencies. Major challenges are to definitively identify EGF family convertases, and to determine if their action in releasing soluble ligands are regulated, and if so, to decipher the underlying mechanisms. As shown originally for TGFa (Brachmann et al., 1989; Wong et al., 1989) and EGF (Mroczkowski et al., 1989; Dobashi and Stern, 1991), the precursors to soluble EGF family growth factors are bioactive while retained in the membrane – i.e. they can productively engage receptors on opposing cell surfaces, leading to signal transduction (termed ‘juxtacrine’ signaling (Massague and Pandiella, 1993)). The pro-forms may elicit distinctive biological responses compared to corresponding soluble forms (Iwamoto et al., 1999). It has also been suggested that they could serve as cell surface receptors and participate in ‘reverse’ signaling complexes via their cytoplasmic tails. However, neither physiologic roles for EGF family precursors

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nor the efficiency with which they are processed in vivo are firmly established.

EGF TGFα AR

HB-EGF BTC EPR

NRG1 NRG2

NRG3 NRG4

Receptors EGF family growth factors all bind and activate the 170-kDa EGF receptor (EGFR/ErbB1/HER1) with relatively high, albeit varying affinity. Studies with EGF or TGFa primarily have revealed a paradigm in which binding stabilizes a receptor homodimer (or oligomer), leading to conformational changes, activation of the receptor tyrosine kinase, and receptor transphosphorylation. It appears likely that two ligand molecules bind per receptor dimer (Lemmon et al., 1997; Lu et al., 2001). Prominent signaling pathways activated as a result of SH2-based interactions include the Ras/Raf/ MAP kinase cascade, Ca2 responses dependent on phospholipase C-c-generated IP3 and DAG, and PI3 kinase/Akt-dependent pro-survival and growth responses. Other, possibly indirect responses include activation of Stat latent transcription factors, non-receptor tyrosine kinases (e.g. Src), etc. Whether the different ligands activate an EGFR homodimer to produce qualitatively different signals is an unsubstantiated but attractive notion. Furthermore, for affected EGF family members, the consequence of heparin binding for regulation of receptor interactions or responses is not yet understood. [Note added in proof: crystal structures of the EGFR ectodomain bond to EGF or TGFa were recently reported in the September 20, 2002 issue of Cell ]. A subset of EGF family members (HB-EGF, BTC, EPR) can additionally bind and activate ErbB4 (Figure 41.2), though the remaining ligands may also bind ErbB4 with low affinity (Beerli and Hynes, 1996; Riese et al., 1996a; Elenius et al., 1997; Komurasaki et al., 1997). Moreover, family members are able to activate various ErbB receptor heterodimers; this offers a likely explanation for signaling by the orphan ErbB2 (HER2/neu), which functions as the preferred heterodimerization partner (Graus-Porta et al., 1997), and by the tyrosine kinase-inactive ErbB3 (Guy et al., 1994). It also offers a potentially important mechanism for diversifying signaling. The different EGF family members favor specific heterodimer combinations (Riese et al., 1995; Beerli and Hynes, 1996). The physiological importance of receptor heterodimerization for EGF family actions has not been firmly established, however.

EGFR ErbB1 HER1

C-Neu ErbB2 HER2

ErbB3 HER3

ErbB4 HER4

FIGURE 41.2 Specificity of ligand binding to ErbB receptors. Note that within the EGF family, HB-EGF, BTC and EPR are distinguished by the ability to bind ErbB4/HER4 with relatively high affinity. Note that the additional ligands likely bind ErbB4 with low affinity.

Genes The genes encoding bona fide ErbB ligands vary over 10-fold in size (10 to 120 kb), but are conserved with respect to exon number (6) and organization. Thus, exon 1 typically encodes the 5 UTR and signal peptide of the growth factor precursor, exon 2 the N-terminal pro-region, exon 3 the first two loops of the EGF sequence, exon 4 the third loop together with the transmembrane domain, exon 5 the cytoplasmic region, and exon 6 the 3 UTR (Blasband et al., 1990b; Plowman et al., 1990). In contrast, the EGF-related sequences in diverse secreted and membrane proteins are often encoded entirely by single exons. Three of the EGF subfamily genes (AR, BTC, EREG) are located in a tightly linked cluster on mouse chromosome 5/human chromosome 4 (Pathak et al., 1995), suggestive of gene duplication events. The recently described Epiga is also in this cluster. EGF family genes are often induced during neoplastic progression as a result of transcriptional or post-transcriptional mechanisms (Berkowitz et al., 1996a; Knebelmann et al., 1998). Additionally, they are subject to auto- and cross-regulation (Barnard et al., 1994), which could amplify EGFR-mediated autocrine growth loops that underlie neoplastic transformation. Thus, activation of EGF genes is often induced in response to EGFR signaling activated by any member of the EGF family. Although several of the promoters have been characterized, regulatory mechanisms are still poorly understood.

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Activities The effects of EGF family growth factors on cultured cells include altered growth, migration, differentiation and survival. As potent growth regulators, they can also induce transformed growth when present at elevated levels in vitro or in vivo. Despite these overall similarities, distinctions in their bioactivities have been reported. For example, TGFa is frequently more potent in various bioassays compared with EGF. In addition, HB-EGF is a more potent mitogen for smooth muscle cells than EGF or TGFa (Higashiyama et al., 1991), while AR is comparatively poor at stimulating anchorage-independent growth of NRK cells (Shoyab et al., 1989). These distinctions could be due to variations in potency of receptor activation, differential recycling of ligand–receptor complexes (Ebner and Derynck, 1991), or preferential activation of distinct ErbB heterodimers. EGF family ligands are broadly expressed in vivo from early embryos (Rappolee et al., 1988; Johnson et al., 1994) through adulthood, present in milk, saliva and other secretions, and implicated in diverse processes. These include decidualization, placental development, and implantation, development and homeostasis of various tissues, notably skin, mammary gland, lung, kidney, the gastrointestinal tract including pancreas, brain and oral cavity structures. They have been implicated in wound healing, tissue regeneration, cytoprotection and angiogenesis, as well as in certain pathological states: cancer, polycystic kidney disease and psoriasis. Phenotypes of EGFR knockout mice, which include defects in numerous epithelial tissues (e.g. lung, gastrointestinal tract) and embryonic or neonatal lethality (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995), are consistent with broad roles for this signaling system. On the other hand, knockout mice lacking individual EGF family members have either no or limited phenotypes, indicating that the ligands have broad overlapping functions. This speculation is supported by findings that combinatorial ligand knockouts have more exaggerated phenotypes (Luetteke et al., 1999). Additionally, the ligand knockouts have thus far revealed that EGF family actions are predominantly local (autocrine or paracrine),dependentonsolubleligand, and mediated by EGF receptor.

EPIDERMAL GROWTH FACTOR Discovery Epidermal growth factor (EGF), the first bona fide polypeptide growth factor discovered, was identified by Levi-Montalcini and Cohen as an activity distinct from nerve growth factor in extracts of mouse salivary gland. When injected into neonatal mice, it stimulated precocious eyelid opening and incisor eruption, and inhibited hair growth (Cohen, 1960, 1962; Levi-Montalcini, 1960). Originally named ‘toothlid factor’, it was renamed in light of its ability to promote epidermal growth and keratinization (Cohen and Elliot, 1963; Cohen, 1965). Subsequent characterization of the mature, 53-amino-acid EGF protein sequence and disulfide bonding confirmed its identity with urogastrone, a potent inhibitor of gastric acid secretion from the urine of pregnant women (Gregory, 1975). Surprisingly, despite its early discovery, many aspects of EGF regulation and function remain obscure.

Protein An unexpected revelation from early cDNA cloning was that the 53-amino-acid EGF is expressed as a transmembrane precursor protein (preproEGF) of ~1200 amino acids (130 kDa) containing the mature sequence in its extracellular domain (Gray et al., 1983; Scott et al., 1983; Bell et al., 1986). Interestingly, this preproEGF also contains eight other EGF-like motifs N-terminal to the mature EGF sequence, a feature distinguishing it from other EGF family precursors, which contain only one EGF motif. No physiological function is yet ascribed to the additional EGF-like sequences. PreproEGF is processed by cleavage of Arg/His and Arg/Asp dipeptide bonds at N- and Cterminal ends, respectively, to release soluble peptides, including a species similar in size to the intact precursor (Mroczkowski et al., 1988). As with other family members, the transmembrane precursor is bioactive in model systems – it supports cell growth and competes with mature EGF for receptor binding (Mroczkowski et al., 1989). Finally, aberrant production of the soluble 165-kDa precursor has been suggested as the cause of a form of congenital polycystic

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kidney diseases (Lakshmanan and Eysselein, 1993), but this has not been confirmed. Predictions from 2-D NMR data are that mature EGF (and by inference the EGF motif of other family ligands) is composed of two slightly overlapping domains (reviewed in Carpenter and Wahl, 1990). The N-terminal domain includes a major antiparallel b-sheet structure and two of the disulfide bonds, while the C-terminal domain includes a double hairpin structure, a minor antiparallel b-sheet and the third disulfide bond (Cooke et al., 1987; Montelione et al., 1987; Kohda et al., 1988; Hommel et al., 1992; Montelione et al., 1992). A predicted hydrophobic surface, generated by clustered aromatic residues, is believed to act in concert with the three intramolecular disulfide bonds to establish a protein structure that is remarkably stable to extremes of heat and pH (Holladay et al., 1976) – a hallmark of EGF family ligands. In contrast to the highly structured core of mature EGF, the N- and C-terminal tails appear largely unrestrained. An EGF crystal structure confirms these predictions, and indicates that EGF might dimerize (Lu et al., 2001). A similar model has been derived for the structure of mature TGFa, albeit the structure may be more flexible (Montelione et al., 1989). Mutagenesis studies in numerous laboratories have confirmed the importance of the conserved cysteines and glycines, as well as residues Met21, Leu26, Asn32, Tyr13, Ile23, Arg41, and Leu47 of mature EGF in high affinity receptor binding (reviewed in Groenen et al., 1994).

Receptors Available evidence indicates that EGF actions are mediated by EGFR (ErbB1) homodimers, or possibly ErbB1:ErbB2 heterodimers, depending on the relative levels of the two receptors. However, reports now suggest that EGF binds ErbB4 with low affinity, and hence EGF may signal through other ErbB heterodimers (Alimandi et al., 1997; Tzahar et al., 1997; PinkasKramarski et al., 1998).

Gene The EGF gene is located on chromosome 4 in humans and chromosome 3 in mice (Brissenden et al., 1984; Zabel et al., 1985; Morton et al., 1986). A relatively

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large gene, it comprises 24 exons and 23 introns and spans 110 kb (Bell et al., 1986). Individual exons encode the eight EGF-related motifs, whereas exon 20 encodes the first two disulfide loops of mature EGF and exon 21 specifies the third disulfide loop together with the transmembrane domain of the precursor. The mature mRNA of ~4.8 kb is expressed at especially high levels in the granular convoluted tubules of submaxillary glands of male mice, with 10-fold lower levels in the female submaxillary gland (Rall et al., 1985). This sexual dimorphism presumably reflects regulation by androgens (Greslik et al., 1985). Levels of the mRNA are also relatively high in the convoluted portions of the distal tubules of the kidney. Conserved, TATA-containing human, mouse and rat EGF promoters have been characterized (Bell et al., 1986; Pascall et al., 1994; Fenton et al., 1996), and functional binding sites for Sp1 (Pascall and Brown, 1997) and a Yin-Yang-1-like repressor (Fang and Sheffield, 1998) described. The mouse promoter with ~5 kb of flanking sequence directed higher levels of T-antigen expression in male submaxillary gland of transgenic mice compared with the female counterpart (Pascall et al., 1994), consistent with endogenous gene expression. However, whether androgens directly regulate this promoter, influence EGF mRNA levels postranscriptionally (Sheflin et al., 1996), or affect expression indirectly, remain enduring questions.

Activities As the first discovered member of the family, EGF has been found bioactive in a wide array of processes in vitro and in vivo. The list of actions ascribed to EGF (reviewed in Carpenter and Wahl, 1990) is too long to summarize here, but two points are worth noting. First, any or all of these actions could be assigned to other EGF family members given their ability to mimic EGF actions generally. Second, physiological roles of EGF are not well understood. Recently, animal models have been derived to explore EGF action in vivo. Overexpression of a modified EGF precursor lacking the eight EGF-related sequences in transgenic mice was associated with hypospermatogenesis, stunted growth and impaired bone development (Wong et al., 2000). In contrast to TGFa transgenic models derived in other laboratories, tumorigenesis was not observed. Directed expression

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of an EGF transgene in the pancreas caused cell proliferation, and disorganized islet growth (Krakowski et al., 1999). Knockout mice lacking EGF have also been developed. These mice display no overt phenotype (Luetteke et al., 1999), but develop more severe duodenal lesions in response to a low dose of the ulcerogen, cysteamine, compared with wild-type counterparts (Troyer and Lee, 2001). Moreover, triple null mice that additionally lack AR and TGFa develop spontaneous duodenal ulcers, and display stunted and fragile villi in the ileum as neonates (Troyer and Lee, 2001). These observations are consistent with broadly speculated roles for EGF as a developmental and/or cytoprotective factor in the gut. In light of the more severe intestinal defects exhibited by EGFR-null mice, they also indicate roles for other EGF family ligands in this tissue. The AR/EGF/TGFa-deficient triple null mice also exhibit mammary gland defects, including dramatic stunting of epithelial ducts during puberty, and aberrant differentiation of milk producing glands in pregnancy (Luetteke et al., 1999). While these mammary gland defects are principally due to loss of AR (they are not observed in EGF/TGFa double null mice), they are markedly aggravated by loss of these additional family members. Collectively, these gastrointestinal and mammary gland defects combined with loss of these growth factors from maternal milk, as well as the presumed poor quality of the milk, may all account for the gut defects noted above, as well as the transiently runted growth of triple null neonates.

TRANSFORMING GROWTH FACTOR ALPHA Discovery Transforming growth factor alpha (TGFa) is perhaps the best studied member of the EGF family. It was initially identified in the conditioned media of sarcoma cells through its ability to promote a transformation of normal cultured cells. Cells exposed to TGFa lost contact inhibition in monolayer and formed large colonies in soft agar (de Larco and Todaro, 1978). Originally termed ‘sarcoma growth factor’, it was renamed ‘transforming growth factor (TGF)’ when similar

activities were extracted from chemically transformed cells and tumors (Roberts et al., 1980; Todaro et al., 1980). Further purification revealed the presence of two independent activities, a mitogenic activity (TGFa) and an unrelated protein (TGFb) that collaboratively induced anchorage-independent growth (Anzano et al., 1982; Roberts et al., 1982). TGFa was found to bind and activate EGFR with EGFlike potency (Pike et al., 1982; Massague, 1983). Upon purification and sequencing, it was found to be homologous to EGF (Marquardt et al., 1984). Thus, 16 of the 53 EGF residues, including the six cysteines, were preserved in TGFa. Although initially suspected as a tumor-specific product, TGFa was subsequently shown to be expressed in embryos (Twardzik et al., 1982a, 1982b), and in normal decidua (Han et al., 1987) and skin (Coffey et al., 1987). Its overexpression in a striking number of tumors and transformed cell types remains a conspicuous feature, however.

Protein The mature, 50-amino-acid TGFa is derived by limited proteolysis from a transmembrane precursor (proTGFa) of 159 or 160 amino acids that is encoded by alternately spliced transcripts (Derynck et al., 1984; Lee et al., 1985; Bringman et al., 1987; Gentry et al., 1987). This precursor is variably modified by N- and O-glycosylation of the extracellular domain (Bringman et al., 1987; Teixido et al., 1987; Luetteke et al., 1988), and palmitoylation of the cytoplasmic domain (Bringman et al., 1987). It contains dominant signals for basolateral localization in polarized MDCK cells (Dempsey and Coffey, 1994), and its two C-terminal valine residues specify efficient routing to the cell surface (Briley et al., 1997; Urena et al., 1999), possibly as a result of interactions with PDZ domaincontaining proteins (Fernandez-Larrea et al., 1999; Kuo et al., 2000). Cell-surface proTGFa is biologically active in the absence of processing (Brachmann et al., 1989; Wong et al., 1999), and it has served as a model for juxtacrine signaling. Activation of EGFR by membrane-anchored TGFa may spatially limit activity and may also lead to prolonged EGFR signaling (Yang et al., 2000), thus promoting transformation (Blasband et al., 1990a). It has been further proposed that proTGFa may simultaneously stimulate cell adhesion and proliferation (Anklesaria et al., 1990).

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Both mature and proTGFa sequences are highly conserved across mammalian species (Derynck et al., 1984; Lee et al., 1985), leading to speculation that the precursor has unique activities. Interestingly, proteins of 86 and 106 kDa are found to associate with the cytoplasmic and transmembrane domains of proTGFa and this complex exhibits serine/threonine and tyrosine kinase activity (Shum et al., 1994, 1996). Additionally, reported association between the extracellular regions of proTGFa and the tetraspanin protein, CD9, apparently leads to inhibition of precursor processing with resulting higher levels of surface proTGFa and juxtacrine signaling involving the precursor in co-culture models (Shi et al., 2000).

Precursor processing ProTGFa has been a model for membrane-anchored growth factor processing. Release of mature TGFa from its precursor requires cleavage of a membrane proximal (C-terminal) Ala-Val bond; additional cleavage of a more distal (N-terminal) Ala-Val bond gives rise to the fully processed, 50-amino-acid growth factor. Processing at the two sites appears unlinked, since the N-terminal site is cleaved more efficiently in CHO cells (Teixido et al., 1990), while higher molecular weight TGFa species resulting from cleavage exclusively at the C-terminal site are prominent in media from transformed cells (Ignotz et al., 1986; Luetteke et al., 1988) and urine from cancer patients (Sherwin et al., 1983). Based on these findings, it has been proposed that different enzymes mediate these two cleavage events (Teixido et al., 1990). Conversion of proTGFa to soluble TGFa is normally slow in cell culture, but is rapidly activated when cells are treated with phorbol esters, calcium ionophores, GTP, serum factors and growth factors (Pandiella and Massague, 1991a, 1991b; Bosenberg et al., 1993; Fan and Derynck, 1999). Signaling via the ERK and p38 MAPK pathways has been implicated in induced and constitutive shedding, respectively (Fan and Derynck, 1999). Conversely, TGFa shedding is inhibited by Zn-dependent metalloproteinase inhibitors (Arribas et al., 1996). These are characteristics common to diverse ectodomain shedding events, and the observation of a general shedding defect in a mutant CHO cell line that also displayed impaired release of TGFa suggests a

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requirement for a common component (Arribas et al., 1997). Domain-swapping experiments involving juxtamembrane sequences from proTGFa, b-amyloid precursor and betaglycan suggest that the ectodomain shedding machinery may recognize general structural determinants rather than a specific sequence. Recently, the tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) was identified as a TGFa convertase. Although TACE-deficient mice frequently die perinatally, survivors are born with open eyes and curly whiskers (Peschon et al., 1998), phenotypes shared by TGFa- as well as EGFR-deficient mice (see below). Moreover, release of TGFa by cells derived from TACE-null mice is greatly reduced compared with wild-type cells, a defect that can be at least partially rescued by restoring TACE. Also, TACE accurately cleaves a soluble proTGFa corresponding to the entire ectodomain at both processing sites in vitro (Sunnarborg et al., 2002). Since TACE is implicated in the shedding of a number of substrates (Black, 2002), its role in TGFa processing is consistent with predictions of common shedding components, but it also raises the important question of how processing is regulated. TACE-deficient cells exhibit residual, metalloprotease-dependent TGFa release (MerlosSuarez et al., 2001; Sunnarborg et al., 2002), indicating the existence of one or more additional proTGFa processing enzymes that may have larger roles in different contexts.

Gene The gene encoding TGFa is located at human chromosome 2p13 (Tricoli et al., 1986) and mouse chromosome 6 (Fowler et al., 1993; Barrow et al., 1994). It comprises six exons, spans 85 kb, and gives rise to a transcript of 4–4.8 kb (Derynck et al., 1985; Blasband et al., 1990b). The promoter lacks canonical CCAAT or TATA motifs (Blasband et al., 1990b; Shin and Kudlow, 1994). Transcription initiates from a single site in the human promoter (Jakobovits et al., 1988), but two clusters of sites in the rat gene (Blasband et al., 1990b). Transcription factors Sp1 and AP-2 are required to efficiently activate the promoter (Shin et al., 1992; Chen et al., 1994; Berkowitz et al., 1997; Wang et al., 1997), with p53 and the TEF1 transcription factor additionally shown to regulate human

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TGFa gene (Shin et al., 1995; Wang and Kudlow, 1999). A region of the promoter has been implicated in autoregulation, a possible autocrine loop mechanism underlying the growth factor independence of colon carcinoma cells (Howell et al., 1998a, 1998b; Jiang et al., 1998) and other tumor types. TGFa gene expression is regulated by various chemical and biological agents, including growth factors, hormones and phorbol esters (reviewed by Lee et al., 1995), but relatively little is known about the mechanisms. In a well-studied model, dramatic induction of TGFa transcripts in transformed rat liver cells results from a combination of increased gene transcription and stabilization of the mRNA (Berkowitz et al., 1996b). However, the underlying mechanisms have not been elucidated. Relevant to these findings, the Von Hippel-Lindau tumor suppressor has been shown to reduce the half-life of TGFa mRNA (Knebelmann et al., 1998). TGFa is widely expressed in mammals, with protein and/or transcript (more reliable) detected in most tissues. Consistent with the phenotypes of mouse models, expression of TGFa is found in skin and hair follicles (Luetteke et al., 1993b), advancing eyelid margins and corneal epithelium (Berkowitz et al., 1996b), developing mammary gland (Luetteke et al., 1999), and all major compartments of the gastrointestinal tract (Troyer et al., 2001). It is also readily detected in brain and nervous system (Lee et al., 1985; Xian and Zhou, 1999; Junier, 2000) and lung (Brown et al., 1990; Liu et al., 1990; Seroogy et al., 1991), and is induced in the decidua around the time of implantation (Han et al., 1987). TGFa overexpression has been associated with renal failure-related parathyroid hyperplasia (Cozzolino et al., 2001; Dusso et al., 2001) and precocious female puberty in rats (Rage et al., 1997), and it has been identified as a possible prognostic marker for Wilms’ tumor, as well as breast and bladder cancer (Umekita et al., 2000; Ghanem et al., 2001; Thogersen et al., 2001). Polymorphisms near the TGFA locus have been associated with cleft lip with or without cleft palate in humans (Hibbert and Field, 1996; Machida et al., 1999), a link that is consistent with the phenotype of EGFR-deficient mice (Miettinen et al., 1999). Finally, a recent study implicates TGFa in circadian regulation. Infusion of TGFa reversibly blocked circadian locomotor activity, and waved-2 mice harboring a hypomorphic EGFR (see below)

were abnormally active during the day compared with wild-type counterparts (Kramer et al., 2001).

Receptor The principal receptor for TGFa is the EGFR (Beerli and Hynes, 1996; Riese et al., 1996b) based on the phenotypic similarities of the respective knockout mice. However, recent studies have shown that TGFa has moderate affinity for ErbB2/ErbB4 heterodimers, and to a lesser extent, ErbB4 (Jones et al., 1999), and that it can stimulate DNA synthesis in cells expressing these receptors (Wang et al., 1998). Structural and domain-swapping studies of TGFa have indicated that determinants throughout the molecule contribute to its three-dimensional structure and thus receptor binding. Important interactions involve the A (first) and C (third) loops, as well as the C-terminal tail, with specific residues providing the majority of binding affinity (McInnes et al., 1996, 1998, 2000). A phage display approach identified variants of TGFa with C-loop substitutions that enhance EGFR binding affinity (Tang et al., 1997). Residues important for receptor binding are in analogous positions to those required in EGF (see above).

Activities As with EGF, a large literature implicates TGFa in a host of normal and disease processes. On the other hand, mice lacking TGFa are viable, generally healthy and fertile, though this may be the result of functional compensation by other family members. TGFa knockout mice derived in two independent laboratories exhibit wavy hair and whiskers, as well as delayed eyelid closure that predisposes the mice to a range of eye anomalies as adults (Luetteke et al., 1993b; Mann et al., 1993). These phenotypes, which are also observed in EGFR-null mice, have thus revealed roles for this EGF family member in eyelid cell migration and in the patterning and function of hair follicles, roles that correlate with localized expression of TGFa in these structures (Luetteke et al., 1993b; Berkowitz et al., 1996b). Similar phenotypes displayed by the spontaneous waved-1 and waved-2 mouse mutants led to the discovery that these mutations impair TGFa expression (Luetteke et al., 1993b; Mann et al., 1993; Berkowitz et al., 1996b) and EGFR tyrosine kinase

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activity, respectively (Luetteke et al., 1994; Fowler et al., 1995). Although the original studies did not find impaired wound healing in TGFa-deficient mice (Luetteke et al., 1993b), subsequent studies have reported delayed wound epithelialization (Kim et al., 2001) and impaired recovery of small intestine from methotrexate challenge (Xian et al., 2002). TGFa was dispensible for liver regeneration, but TGFa-deficient mice had reduced incidence of large, chemically induced hepatocellular carcinoma (Russell et al., 1996). Additional elimination of EGF and AR in triple null mice (see EGF and AR) revealed augmenting or compensatory roles for TGFa in mammary gland development and/or differentiation (Luetteke et al., 1999), as well as in development and homeostasis of the small intestine (Troyer et al., 2001). Importantly, transgenic models have established that overexpression of TGFa can profoundly influence cellular growth and differentiation in numerous tissues (see Lee et al., 1995; Schroeder and Lee, 1997; Humphreys and Hennighausen, 2000; Normanno et al., 2001; Troyer and Lee, 2001, for reviews). Overexpression of TGFa under control of several different inducible or tissue-specific promoters has produced potent stimulation of growth in the gastrointestinal tract (Jhappan et al., 1990; Sandgren et al., 1990), marked inhibition of adipose development (Luetteke et al., 1993a), and precocious induction of alveolar differentiation and inhibition of involution in the mammary gland (Sandgren et al., 1995). It has also been associated with fibrosis and aberrant alveolar development in lung (Korfhagen et al., 1994), pathologies of the eye (Decsi et al., 1994), hyperplasia and adenoma formation in the female pituitary (McAndrew et al., 1995), and protection from motoneuron degeneration following injury (Boillee et al., 2001). The ability of TGFa transgenes to promote multifocal liver tumorigenesis through formation of hyperproliferative, precancerous lesions, as well as to collaborate with c-myc and carcinogens in this process, has received considerable attention (Jhappan et al., 1990; Sandgren et al., 1990; Takagi et al., 1992b; Murakami et al., 1993; Tamano et al., 1994). Enhanced tumorigenesis in TGFa/c-myc bitransgenic mice is associated with elevated levels of TGFa, decreased apoptosis, and excessive DNA damage resulting from overproduction of reactive oxygen species (Factor et al., 1998; Santoni-Rugiu et al., 1998).

Surprisingly, despite the link between TGFa-mediated tumorigenesis and impaired apoptosis, co-expression of a Bcl-2 transgene inhibited tumor incidence and latency (Vail et al., 2001). The mammary gland is also sensitive to transformation by TGFa transgenes (Matsui et al., 1990; Sandgren et al., 1990). In this tissue, tumorigenesis is associated with altered differentiation and especially enhanced proliferation and survival of epithelial cells. It is frequently accompanied by induction of cyclin D1, and is dramatically accelerated by carcinogen, coexpression of a c-myc transgene, or induction of Wnt genes (Coffey et al., 1994; Sandgren et al., 1995; Amundadottir et al., 1996; Schroeder et al., 2000). In the stomach, transgenic expression produces excessive proliferation, altered differentiation leading to dramatic structural and functional lesions, and foveolar hyperplasia. It also produces gastric symptoms reminiscient of human Menetrier’s disease (Dempsey et al., 1992; Takagi et al., 1992a; Sharp et al., 1995) and in the pancreas, fibrosis, redifferentiation and premalignant symptoms (Jhappan et al., 1990; Sandgren et al., 1990; Bockman and Merlino, 1992). When overexpressed from a transgene, TGFa functions as an autonomous tumor promoter in DMBAinitiated transgenic skin (Jhappan et al., 1994), and collaborates with v-fos to produce aberrant skin differentiation and papilloma formation (Wang et al., 1995). Collectively, these studies reveal that aberrant regulation of TGFa profoundly affects cellular proliferation, differentiation and survival, and it thus leads to neoplastic conversion in vivo.

AMPHIREGULIN Discovery Amphiregulin (AR) was the third EGF family growth factor discovered. It was originally identified in media conditioned by MCF-7 cells exposed long-term to PMA (Shoyab et al., 1988). AR was described as a bifunctional regulator of cell growth (hence the name). It inhibited the growth of A431 human epidermoid carcinoma cells, but stimulated proliferation of human fibroblasts. Subsequently, AR was shown to supplant the requirement for EGF or TGFa in murine keratinocyte growth, but to be a much weaker stimulator

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of other cell types (Shoyab et al., 1989). AR was independently discovered as Schwannoma-derived growth factor (SDGF) (Kimura et al., 1990).

Protein Soluble forms of AR are larger than EGF or TGFa, containing in addition to the EGF-like sequence, an N-terminal extension that is rich in basic amino acids, hydrophilic in nature, and contains glycosylation and heparin-binding sites (Plowman et al., 1990; Johnson et al., 1993b). These soluble forms are derived from the extracellular domain of a 252-amino-acid integral membrane precursor (proAR) by proteolytic cleavage at a membrane-proximal site. In cell culture models, variable cleavage at two N-terminal sites six amino acids apart gives rise to two major soluble forms of 78 and 84 amino acids, as well as a larger, 43 kDa protein corresponding to the entire extracellular domain of proAR (Shoyab et al., 1989; Brown et al., 1998). The N-terminal extension present in soluble AR contains two consecutive Lys-Arg-Arg-Arg motifs resembling known nuclear targeting signals (Plowman et al., 1990), and it is frequently associated with the nucleus (Modrell et al., 1992; Martinez-Lacaci et al., 1996). However, the significance of these observations is presently unclear. Interestingly, efficient trafficking of proAR requires the presence of the precursor’s proregion suggesting an interaction between this domain and the N-terminal extension (Thorne and Plowman, 1994). As with proTGFa, proAR is sorted to the basolateral surface in polarized epithelial cells, apparently due to a dominant element in the cytoplasmic domain (Brown et al., 2001).

Precursor processing Cleavage of proAR is stimulated by PMA and pervanadate, inhibited by the metalloproteinase inhibitor, batimastat, and does not require the precursor’s cytoplasmic domain (Brown et al., 1998; Vecchi et al., 1998). Cell transfection experiments indicate that TACE/ADAM17 can stimulate conversion of proAR to soluble forms (Sunnarborg et al., 2002), but a physiological role for this or other metalloproteinases has not been confirmed.

Gene The AR gene is relatively small: it spans ~10 kb of genomic DNA and gives rise to a relatively short transcript of 1.4 kb (Plowman et al., 1990). The gene is composed of six exons that are organized in a manner similar to that of other EGF family genes. The mouse AR gene is located in a tightly linked cluster containing betacellulin and epiregulin on mouse chromosome 5 (Pathak et al., 1995), while the human gene is correspondingly located at 4q13-4q21 near a common breakpoint for acute lymphoblastic leukemia (Plowman et al., 1990). The TATA-containing AR promoter (Plowman et al., 1990) is potently activated by theWilms tumor suppressor,WT1 (Lee et al., 1999), and AR expression in the developing kidney mirrors that of WT1, thus implicating AR in branching morphogenesis in this tissue. However, kidney development occurs normally in the absence of AR (see below). Like other EGF family members, AR is broadly expressed. AR protein and/or mRNA has been identified in normal human ovary, placenta, testis, pancreas, spleen, kidney, lung, breast and colon (Plowman et al., 1990), in keratinocytes (Cook et al., 1992a; Piepkorn et al., 1994) and in cultured mammary epithelial cells (Li et al., 1992). AR is also ubiquitously expressed in mouse tissues, with especially high levels in the epithelium of developing mammary glands (Kenney et al., 1995; Luetteke et al., 1999). Overexpression of AR is observed in human malignancies of the colon (Ciardiello et al., 1991; Johnson et al., 1992; Saeki et al., 1992), stomach (Cook et al., 1992b; Kitadai et al., 1993), pancreas (Ebert et al., 1994), and prostate (Sehgal et al., 1994), as well as in mammary tumors (Normanno et al., 1994; Qi et al., 1994; Ma et al., 1999), including those induced by various oncogenes in transgenic mice (Kenney et al., 1996a). It is believed to function as an autocrine growth factor in many of these contexts. Finally, AR mRNA is also elevated in psoriatic epidermis (Cook et al., 1992a).

Receptors AR binds to EGFR, but with lower affinity than EGF – apparently because it lacks a C-terminal leucine preserved by several family members (Adam et al., 1995). Present evidence suggests that AR action is principally

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mediated by this receptor (Johnson et al., 1993a; Wong et al., 1999), even though AR can induce ErbB heterodimerization in cell culture models. AR stimulation of MCF-10A cell proliferation requires heparin sulfate glycosaminoglycan (Johnson and Wong, 1994).

Activities As with other EGF family ligands, AR has been implicated in diverse physiologic and pathologic processes. These include mammary development and neoplasia (Li et al., 1992; Kenney et al., 1996b), trophoblast growth (Lysiak et al., 1995), lung and kidney branching morphogenesis (Schuger et al., 1996; Lee et al., 1999), and keratinocyte proliferation (Cook et al., 1991). Despite AR’s general ability to mimic EGF or TGFa actions, several studies have pointed to distinctions in biological responses evoked by AR compared with EGF or TGFa in vitro. Thus, in contrast to the latter two ligands, AR only weakly induced anchorageindependent growth of NRK cells in the presence of TGFb, yet was as active as EGF in sustaining proliferation of Balb/MK cells (Shoyab et al., 1989). Pointing to a possible explanation, AR requires the carboxyterminal region of EGFR, including the prominent autophosphorylation sites, to stimulate NR6 fibroblast proliferation, whereas EGF does not (Wong et al., 1999). This may be due to the preferential ability of EGF to promote an EGFR:ErbB2 heterodimer. Transgenic expression of human AR directed by the Keratin-14 promoter resulted in psoriatic-like lesions (Cook et al., 1992b). Knockout mice homozygous for a disrupted AR allele were viable and fertile, but displayed defective mammary gland development (Luetteke et al., 1999). Formation of epithelial ducts was impaired in pubescent AR-deficient females, leading to incompletely arborized fat pads. This defect was significantly exacerbated in triple null mice additionally lacking EGF and TGFa, although both ligands were dispensable in the presence of AR. Thus, ductal elongation was severely stunted in triple null females and their pups displayed runted growth, presumably due to poor milk production. Defective epithelial development was also observed in mammary gland transplants lacking EGFR (Wiesen et al., 1999). Tissue recombination experiments indicated that the critical EGFR population is in the stroma, while AR is selectively expressed in the epithelium

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(Luetteke et al., 1999). Collectively, these results suggest that paracrine stimulation of stromal EGFR by AR constitutes a required epithelial-mesenchymal signal for ductal penetration of the fat pad in the pubescent gland. Lobuloalveolar development in pregnancy was also aberrant in the triple null mice (Luetteke et al., 1999), and was impaired in waved-2 mice harboring a mutant EGFR (Fowler et al., 1995). However, the requirement for EGFR signaling in lobuloalveolar development is controversial (Wiesen et al., 1999).

HEPARIN BINDING-EGF Discovery Heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as a heparin-binding activity in the conditioned medium of human macrophages that was mitogenic for Balb/c 3T3 fibroblasts and smooth muscle cells, but not endothelial cells (Besner et al., 1990; Higashiyama et al., 1991).

Protein Analysis of the human HB-EGF cDNA clone predicts a primary translation product of 208 amino acids (Higashiyama et al., 1991). Like all the ligands in the ErbB network, HB-EGF is synthesized as a transmembrane precursor molecule that is proteolytically cleaved to release soluble, mature growth factor from the cell surface (Goishi et al., 1995; Izumi et al., 1998). The apparent molecular mass of mature HB-EGF isoforms varies from 20 to 22 kDa, owing to N-terminal heterogeneity and O-glycosylation (Higashiyama et al., 1992). The heparin-binding domain of HB-EGF appears to modulate its bioactivity since HB-EGF binding to EGFR and HB-EGF activity are optimized in the presence of heparin-like molecules or cellsurface heparan sulphate proteoglycans (HSPG) and inhibited by heparinase treatment or by a synthetic peptide corresponding to the 21 residues of the heparin-binding domain (reviewed by Raab and Klagsbrun, 1997). Mature soluble HB-EGF is both mitogenic and chemoattractant (Higashiyama et al., 1991, 1993; Hashimoto et al., 1994). However, membraneanchored HB-EGF exhibits several unique biological

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activities (Iwamoto and Mekada, 2000). First, proHBEGF is a high affinity receptor for diptheria toxin (DT) and mediates internalization of the bound DT by endocytosis, an activity that does not require the cytoplasmic domain of HB-EGF. Heparin and heparin-like molecules appear to modulate this activity, since DT binding is maximized in the presence of HSPG. Interestingly, DT binds to the EGF-like domain of human proHB-EGF, but not mouse, due to non-conserved amino acid substitutions (reviewed by Raab and Klagsbrun, 1997). Second, transmembrane HB-EGF may have a role in intercellular communication since it forms a complex with the adhesion molecule integrin a3b1 (Nakamura et al., 1995) localized at cell–cell contact sites in epithelial cells, as well as the tetramembranespanning protein, CD9 (Iwamoto et al., 1991, 1994; Mitamura et al., 1992; Hasuwa et al., 2001). In cell culture, coexpression of CD9 and proHB-EGF increased the number of DT binding sites, as well as DT sensitivity (Mitamura et al., 1992; Iwamoto et al., 1994), and it up-regulated the juxtacrine-like mitogenic activity of proHB-EGF (Higashiyama et al., 1995; Nishida et al., 2000). This up-regulation required the presence of the EGF-like domain, and was found to be due to protein–protein interactions between proHBEGF and CD9, rather than enhanced proHB-EGF transcription (Iwamoto et al., 1994; Higashiyama et al., 1995; Nakamura et al., 2000). More recently, coincident distribution of CD9, HB-EGF, and integrin a3b1 has been demonstrated in normal human tissues by immunohistochemistry (Nakamura et al., 2001). Discovery of complexes of proHB-EGF, CD44 and MMP7/matrilysin in uterine and mammary epithelium suggests a possible role in tissue remodeling (Yu et al., 2002). Finally, proHB-EGF juxtacrine signaling may regulate cell growth and survival. Cells expressing EGFR proliferated in the presence of soluble HB-EGF, but underwent growth inhibition and apoptosis when cocultured with cells expressing proHB-EGF (Iwamoto et al., 1999). Suppressed cell growth was also observed in transfected hepatoma cells stably producing proHB-EGF while cells producing soluble HB-EGF grew rapidly (Miyoshi et al., 1997). It was proposed that formation of a homodimer or homooligomer of proHB-EGF molecules on the cell surface leads to oligomerization and activation of EGFR and recruit-

ment of growth inhibitory and apoptotic signaling molecules (Iwamoto et al., 1999).

Precursor processing Various stimuli induce ectodomain shedding of HBEGF, including TPA, calcium ionophore and G protein ligands (Goishi et al., 1995; Dethlefsen et al., 1998; Izumi et al., 1998; Umata et al., 2001), suggesting that multiple signaling pathways regulate proteolytic processing of this molecule. TPA-induced processing, which implicates protein kinase C (PKC) activation, is blocked by hydroxamate inhibitors, indicating that Zn-dependent metalloproteinases are involved (Lanzrein et al., 1995; Umeda et al., 2001). Evidence for the requirement of the MAP kinase signaling pathway was presented by Gechtman and colleagues (1999), who showed that PMA-induced shedding of HB-EGF was blocked by the MEK inhibitor PD98059. Finally, metalloproteinase-dependent cleavage of proHB-EGF was shown to transactivate EGFR in response to G-protein-coupled receptor (GPCR) signaling (Prenzel et al., 1999). Initially believed to be a ligand-independent mechanism of EGFR-transactivation upon stimulation with GPCR agonists (Daub et al., 1996), this novel pathway has now been shown to be involved in smooth muscle cell migration (Kalmes et al., 2000), keratinocyte proliferation (Marques et al., 2002) and cardiomyocyte hypertrophy (Asakura et al., 2002). Although the identity of the protease responsible for physiological proHB-EGF shedding has not been confirmed, members from two subfamilies of the metzincin metalloproteases have been implicated. The involvement of matrix metalloproteinases (MMPs) is suggested by observations of impaired release of mature HB-EGF upon treatment with synthetic MMP inhibitors or divalent ion chelators. Candidate MMPs include MMP-3 (Suzuki et al., 1997; Bursi et al., 2002) and MMP-7/matrilysin (Yu et al., 2002). Specific members of the membraneanchored metalloprotease-disintegrin glycoproteins (ADAMs) that have been implicated in ectodomain cleavage of proHB-EGF include ADAM17/TACE (Sunnarborg et al., 2002), ADAM10/kuzbanian (Lemjabbar and Basbaum, 2002), and ADAM12 (Asakura et al., 2002). ADAM9/meltrin-c/MDC9 may also cleave proHB-EGF, since over-expression of a

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mutated ADAM9 blocked proHB-EGF shedding (Izumi et al., 1998).

Gene Both the human HB-EGF gene, located on chromosome 5q23, and the mouse HB-EGF gene, located on chromosome 18, are composed of six exons and five introns spanning approximately 14 kb (Fen et al., 1993; Pathak et al., 1995; Harding et al., 1996). A comparison of the HB-EGF cDNAs of the human, monkey, mouse and rat reveals that the sequences are about 80% identical (Abraham et al., 1993). Analysis of promoter sequences in the 5-flanking regions of the mouse and human HB-EGF genes indicates the presence of transcription factor binding sites for AP-1, SP-1, NF-jB and MyoD (Fen et al., 1993; Chen et al., 1995) and identifies an Ets-2-binding, Raf-1 responsive element in the mouse HB-EGF gene (McCarthy et al., 1997), consistent with the rapid induction of the gene by Raf and Ras oncogenes (McCarthy et al., 1995). In cultured cells, rapid and transient upregulation of HB-EGF mRNA, that does not require new protein synthesis, indicates that HB-EGF is an immediate–early gene (Yoshizumi et al., 1992; Dluz et al., 1993; Fen et al., 1993; Barnard et al., 1994). HB-EGF mRNA is widely distributed in mammalian tissues including skin, kidney, lung, heart, brain, skeletal muscle, stomach and mammary gland (Vaughan et al., 1992; Abraham et al., 1993; Murayama et al., 1995; Schroeder and Lee, 1998).

Receptors HB-EGF binds and activates EGFR as well as ERBB4, though activation of the latter receptor does not stimulate cell proliferation, implying that the two receptors mediate different biological processes (reviewed by Raab and Klagsbrun, 1997). Interestingly, HB-EGF was recently found to bind N-arginine dibasic convertase (NRDc) and thus enhance EGFRdependent cell migration. Although NRDc is a metalloendopeptidase, enzymatic activity was not required suggesting it could be a novel and specific receptor for HB-EGF (Nishi et al., 2001).

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Activities To date, there are no published animal models for HBEGF, but the breadth of expression has led to many analyses into possible physiological roles. Several lines of investigation implicate HB-EGF in uterine growth and blastocyst implantation in various mammalian species (Das et al., 1994; Kim et al., 1995; Raab and Klagsbrun, 1997; Leach et al., 1999). In the rodent, HB-EGF expression in the uterus appears to be regulated by estrogen and progesterone suggesting that HB-EGF mediates the actions of steroid hormones on the reproductive system. HB-EGF may promote implantation of the embryo, since its expression is limited spatially and temporally to the luminal epithelium surrounding the mouse blastocyst prior to implantation, and proHB-EGF in the luminal epithelium at the site of blastocyst apposition may be an adhesion factor for blastocysts. HB-EGF may also promote growth of the embryo, for it induced blastocyst EGFR autophosphorylation and also promoted blastocyst growth, zona-hatching and trophoblast outgrowth in culture. Additionally, HB-EGF accelerated mouse trophoblast differentiation in vitro via intracellular Ca2 signaling (Wang et al., 2000) which may involve ERBB4 (Paria et al., 1999). Most recently, HB-EGF was identified in human breast milk and amniotic fluid suggesting wider roles for HB-EGF in pre- and postnatal development (Michalsky et al., 2002). HB-EGF expression appears to be up-regulated at sites of injury, leading many investigators to examine its role in wound healing in various tissues and cell lines (Raab and Klagsbrun, 1997; Tokumaru et al., 2000; Zieske et al., 2000; Ellis et al., 2001; Cribbs et al., 2002). The physiological role of HB-EGF in the central nervous system (CNS) has also been explored since it is expressed during development, and stimulates CNS cell proliferation, motility and survival in culture (Nakagawa et al., 1998; Kornblum et al., 1999; Opanashuk et al., 1999). In addition to its physiological roles, HB-EGF may play a role in pathological processes. HB-EGF expression was documented in tumorigenic cells and cell lines, including the prostate cancer cell lines DU145 and PC3 (Torring et al., 2000), hepatocellular carcinoma cells (Inui et al., 1994), breast carcinomas (Ito et al., 2001b), pancreatic adenocarcinomas (Ito et al.,

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2001a), leukemia cells (Vinante et al., 1999), skin cancers (Downing et al., 1997), lung tumors (Fernandes et al., 1999), and malignant plasma cells (De Vos et al., 2001). Additionally, HB-EGF stimulated anchorageindependent growth of NRK cells (Harding et al., 1999). HB-EGF’s specific role in vascular injury is well studied, for it is expressed in atherosclerotic plaques by aortic SMCs, macrophages and infiltrating T lymphocytes (Temizer et al., 1992; Miyagawa et al., 1995; Peoples et al., 1995; Ouchi et al., 1997). Additionally HB-EGF expression in endothelial cells is inducible by angiogenic factors such as VEGF (Arkonac et al., 1998), and cytokines such as TNFa (Yoshizumi et al., 1992). Immunohistochemical localization of CD9 and proHB-EGF in intimal SMCs of atherosclerotic lesions was recently demonstrated, and coexpression of CD9 and proHB-EGF was shown to enhance SMC growth (Nishida et al., 2000). Finally, HB-EGF’s role in cardiac hypertrophy has received much attention. Increased HB-EGF expression has been demonstrated in cultured cardiomyocytes upon application of hypertophic stimuli (Perrella et al., 1994), and in hypertrophic rodent hearts (Fujino et al., 1998; Schwartzbauer and Robbins, 2001). In addition, exogenous HB-EGF stimulates cellular hypertrophy in vitro (Perrella et al., 1994; Asakura et al., 2002). Most recently, it was shown that HB-EGF shedding and transactivation of EGFR mediated cardiac hypertrophy in vivo via G proteincoupled receptor signaling. The hypertrophic response could be blocked by an ADAM12 inhibitor, suggesting this metalloprotease is responsible for HB-EGF shedding in cardiomyocytes, and that both ADAM12 and HB-EGF are potential therapeutic targets for the treatment of cardiac hypertrophy (Asakura et al., 2002). Preliminary analyses of HBEGF-null mice generated in our laboratory also indicate that HB-EGF plays an important role in heart morphogenesis (unpublished observations).

BETACELLULIN Discovery

ditioned media from a pancreatic b-cell insulinoma cell line that was a potent mitogen for Balb/c 3T3 and smooth muscle cells (Shing et al., 1993). It shares the signature sequence of EGF family members and binds the EGFR.

Protein BTC is synthesized as a 32 kDa, 178-amino-acid primary translation product (proBTC) containing signal peptide and transmembrane domains. The mature sequence, which must be cleaved from the ectodomain of proBTC, contains 80 amino acids and corresponds to the EGF sequence with an N-terminal extension. The EGF-like sequence of human BTC is 50% identical to the corresponding portion of TGFa (Sasada and Igarashi, 1993). Mature BTC migrates with an anomalous molecular weight in SDS-PAGE due to extensive N- and O-glycosylation at two or three sites (Watanabe et al., 1994). However, these modifications have no apparent effect on biological activity (Seno et al., 1996; Dunbar et al., 1999). Cells transfected with a BTC cDNA also produced two smaller forms: one lacking 12 amino acids from the N-terminus of mature BTC, and a 50-amino-acid form corresponding to the EGF motif (Watanabe et al., 1994). The potency of these truncated forms is identical to that of the 80-amino-acid BTC. A comparison of cDNAs from mouse, human and bovine sources reveals that the precursor sequences from these species are 74–88% identical (Sasada et al., 1993; Shing et al., 1993; Dunbar et al., 1999), with the highest homology in the EGF motif and transmembrane and cytoplasmic domains. Features that distinguish BTC from other EGF family ligands include the presence of two additional, N-terminal cysteines in the mature sequence that bond to form a fourth disulfide bridge. This fourth bridge does not appear to affect BTC activity, however (Watanabe et al., 1994). The precursor is also distinguished by the presence of an Arg-Gly-Asp (RGD) peptide in the external juxtamembrane region. This sequence could serve as an integrin binding site, suggesting that proBTC might have a role in cell adhesion. Finally, the cytoplasmic domain contains a hydrophilic, Arg-Lys-rich region of unknown function.

Betacellulin (BTC) (reviewed in Dunbar and Goddard, 2000) was originally identified as an activity in conTHE CYTOKINES AND CHEMOKINES

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Precursor processing

Receptors

Cleavage of the mouse and bovine precursors occurs prominently at Ala-Asp and Tyr-Leu dipeptides at the N- and C-termini of mature BTC, respectively (Shing et al., 1993; Dunbar et al., 1999), though alternative N-terminal processing that produces a smaller, 8-kDa mature protein lacking the N-terminal extension has also been observed (Seno et al., 1996; Tada et al., 1999; Dunbar and Goddard, 2000). The protease(s) responsible for shedding of BTC has not been identified, though ADAM family disintegrin metalloproteinases have been implicated in the release of other EGF-like growth factors. Interestingly, BTC processing is not inducible by PMA (Tada et al., 1999), a fact that distinguishes its processes from that of other EGF family precursors and suggests that distinct proteases may be involved.

BTC binds both EGFR and ErbB4 with high affinity (Watanabe et al., 1994; Riese et al., 1996a; Jones et al., 1999). Several reports suggest that it also binds to ErbB3 in an ErbB2:ErbB3 heterodimer (Alimandi et al., 1997; Pinkas-Kramarski et al., 1998; Wang et al., 1998; Jones et al., 1999). It can thus be considered a pan-ErbB ligand that is capable of activating all commonly observed ErbB heterodimers. Physiological confirmation of this broad role has not been provided, however. Interestingly, a comparison of human T cell responses to BTC and several NRG isoforms revealed that the different ligands evoked varying growth responses, induced different patterns of ErbB4 tyrosine phosphorylation, and recruited different signaling molecules to the receptor (Sweeney et al., 2000). Hence, it appears that despite binding to the same receptor, ErbB ligands may induce quite different responses.

Gene The gene for mouse betacellulin is tightly linked to the AR epiregulin and epigen genes on chromosome 5 (Pathak et al., 1995). The human gene maps to the corresponding locus and 4q13-q21. As with other EGF family ligands, the BTC gene is broadly expressed in tissues, as well as in epithelial and smooth muscle cells. In particular, expression has been documented in pancreas and small intestine, as well as in liver, colon, heart, lung, testis, ovary and mammary glands (Sasada et al., 1993; Seno et al., 1996; Schroeder and Lee, 1998; Dunbar et al., 1999; Troyer et al., 2001). The major BTC transcript is ~3 kb, though smaller alternative transcripts have been variably observed. Enhanced expression in tumors and transformed cells, a hallmark feature of EGF family ligands, is also observed with BTC. For example, significantly elevated BTC expression was observed in pancreatic tumors (Yokoyama, 1995), with evidence of a BTC-mediated autocrine loop in pancreatic cancer cells (Kawaguchi et al., 2000). As with other EGF ligands, BTC expression is autoinduced: increased in response to exposure of cells to BTC or other EGF-like ligands (Barnard et al., 1994; Yokoyama, 1995; Kawaguchi et al., 2000).

Activities BTC is broadly expressed and likely to have redundant, EGF-like actions in many tissues. Considerable attention has been focused on potential roles for BTC in the developing and adult pancreas. Key findings are that it is highly expressed in primitive ducts and islet cell populations in the fetal pancreas (Miyagawa et al., 1999), stimulates proliferation of a fetal pancreatic cell line (Sundaresan et al., 1998), and together with Activin A induces amylase-secreting AR42J pancreatic cells to produce insulin, whereas EGF and TGFa do not (Mashima et al., 1996, 1999). BTC is also permissive for Pdx-1 induction of insulin in a glucagonoma cell line (Watada et al., 1996), mediates proliferation and differentiation of a rat insulinoma cell line (Huotari et al., 1998), and promotes b-cell neogenesis and improved glucose tolerance in a diabetic mouse model in which b-cells are completely destroyed (Yamamoto et al., 2000). Roles for BTC in the gastrointestinal tract are suggested by its presence (along with EGF and TGFa) in bovine milk and colostrum (Dunbar et al., 1999; Bastian et al., 2001), and its expression in the epithelium and glands of the stomach, small intestine and colon (Kallincos et al., 2000; Troyer et al., 2001). BTC is also expressed in the developing mammary gland

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(Schroeder and Lee, 1998), functions as a mitogen for normal and tumorigenic mammary cells (Beerli and Hynes, 1996; Daly et al., 1999), and induces mammary epithelial cells to assume a differentiated phenotype (Pinkas-Kramarski et al., 1998). It is also implicated in fertility and is coexpressed with other EGF family ligands in the uterus adjacent to implantation sites (Das et al., 1997). BTC may also be critical for the development of the gastrointestinal tract. Finally, BTC may have important roles in atherosclerosis, since it is a mitogen for vascular smooth muscle cells (Seno et al., 1996; Dunbar and Goddard, 2000), expressed in the smooth muscle cells of the aortic wall, and has been localized to atherosclerotic lesions, with significantly higher expression in advanced stages (Tamura et al., 2001). Knockout mice lacking BTC derived in our laboratory are viable, healthy and fertile, and do not display an overt phenotype (unpublished observations), indicating overlapping or compensating functions by other EGF family members.

EPIREGULIN Discovery Epiregulin (EPR) is a more recently discovered member of the EGF family, and thus only limited information is available. It was initially purified from the conditioned medium of a tumorigenic clone of NIH3T3 fibroblasts as an activity that inhibited the growth of several epithelial tumor lines, but stimulated the proliferation of Balb/c 3T3 fibroblasts and primary hepatocytes (Toyoda et al., 1995a). The 46-amino-acid protein displayed the EGF signature sequence and was 24–50% identical to other EGF family members, with residues known to be important for EGF action conserved.

Protein EPR is expressed as a putative, 162-amino-acid transmembrane glycoprotein precursor with similar organization to that of other EGF family precursors (Toyoda et al., 1995a). The cytoplasmic region contains a relatively basic, Arg/Lys-rich sequence similar to that found in some other EGF family precursors.

The mouse and human proEPR sequences are ~85% identical (Toyoda et al., 1997). The mature 46-aminoacid, 5.4 kDa protein is derived from the ectodomain of proEPR, and like EGF and TGFa – but unlike AR, HB-EGF and BTC – does not contain an N-terminal extension. It does not include N-glycosylation consensus or heparin-binding sites.

Precursor processing Processing of proEPR has not yet been studied in detail. One study reports that the phorbol ester, TPA, rapidly induces ectodomain shedding of EPR, resulting in the disappearance of the full-length precursor and concomitant accumulation of the cytoplasmic tail (Baba et al., 2000).

Gene The mouse and human EPR genes are located on chromosome 5 and 4, respectively (Toyoda et al., 1995a; Baba et al., 2000) in the BTC/AR/EPR/epigen cluster, and give rise to transcripts of ~4.5–5 kb. An initial study reported that in contrast to the broad expression of other EGF family members, EPR transcripts were not detected in a range of adult mouse tissues, but were present in early mouse embryos (Toyoda et al., 1995a). However, expression was detected by semi-quantitative RT-PCR in the pregnant mammary gland (Schroeder and Lee, 1998) and small intestine (Troyer et al., 2001) of mice. Human EPR transcript was detected in adult peripheral blood macrophages and placenta, as well as in heart tissue. Lower levels of expression were detected in bone marrow, ovary, small intestine, colon, lung and liver (Toyoda et al., 1997). The overall impression is that EPR is expressed at lower levels compared with other EGF family ligands. Expression of the EPR gene is frequently elevated in neoplastic tissues and cells. Relatively high levels of transcript were detected in cell lines derived from carcinomas of the bladder, lung, kidney and colon, as well as epidermoid carcinomas (Toyoda et al., 1997). Expression did not always correlate with levels of TGFa transcript. EPR transcripts (as well as those of TGFa and HB-EGF) were particularly elevated in muscle-invasive bladder cancers and were inversely correlated with patient survival

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(Thogersen et al., 2001). EPR was also up-regulated in Ki-ras-transformed human colon carcinoma cells, and when transfected into a clone demonstrating low EPR expression, promoted tumorigenicity in nude mice (Baba et al., 2000). EPR transcripts were induced five- to 15-fold in vascular smooth muscle cells exposed to the GPCR agonists angiotensin II, endothelin-1, and a-thrombin, potentially reflecting a contribution of this growth factor to vascular remodeling (Taylor et al., 1999).

Receptors EPR binds directly to EGFR and ErbB4, albeit with relatively low affinity, and thus activates both of these tyrosine kinase receptors – a property it shares with BTC and HB-EGF. It has been reported that EPR is a more effective ligand for EGFR than BTC, whereas BTC is a more effective ErbB4 ligand (Riese et al., 1998). However, this apparent preference is controversial (Komurasaki et al., 1997). The situation is complicated by the fact that ErbB2 enhances the sensitivity of ErbB4 to EPR, presumably because ErbB2:ErbB4 heterodimers have a higher affinity for this ligand. Although EPR does not bind to ErbB2 or ErbB3, it nevertheless causes transactivation of these receptors through the phenomenon of ErbB heterodimerization (Komurasaki et al., 1997; Shelly et al., 1998). Thus, EPR can be considered another pan-ErbB ligand, though there are distinctions: BTC, also a panErbB ligand, has a much stronger effect on ErbB3 phosphorylation compared with EPR. This ability to promote heterodimerization presumably explains why EPR is much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts even though it binds to EGFR with much lower affinity than EGF (Toyoda et al., 1995b; Jones et al., 1999). This apparent discrepancy, which is partly explained by differential down-regulation of the various ErbB proteins, underscores the importance of ErbB heterodimerization.

smooth muscle cells (Toyoda et al., 1995b; Sasaki et al., 1997; Taylor et al., 1999). It also appears to be an autocrine growth factor for normal human keratinocytes, and in these cells is subject to both auto- and cross-induction – i.e. it induces both its own expression as well as the expression of other EGF family genes (Hashimoto, 2000; Shirakata et al., 2000). As noted previously, EPR was up-regulated in Ki-raspositive human colon carcinoma cells and contributed to the tumorigenicity of these cells in nude mice (Baba et al., 2000). Thus, it may have a broad role in transformation and tumorigenesis. Finally, EPR-null knockout mice have been derived, but display no overt phenotype (D. Threadgill, personal communication).

EPIGEN Discovery Epigen is the most recently discovered member of the EGF family, and in fact, a single study is the source of all the information cited here. Epigen was identified as an expressed sequence tag in an immature mouse keratinocyte library (Strachan et al., 2001). Its name reflects its activity as a potent epithelial mitogen.

Protein Proepigen is a predicted 152-amino-acid transmembrane glycoprotein with features characteristic of EGF family precursors, including the presence of an EGF-like sequence in the extracellular domain. This EGF-like sequence contains the six conserved cysteines, together with the invariant Gly and Arg residues that are essential for bioactivity. The entire epigen sequence is 24–37% identical to other EGF family precursors, though a 51-amino-acid sequence encompassing the EGF motif is ~40% identical to the corresponding sequences of EGF, TGFa and Epr.

Activities EPR exhibits bifunctional regulatory properties: it inhibits the growth of several human epithelial tumor cells, yet stimulates the proliferation of primary hepatocytes, cultured gastric cells, and vascular

Gene Limited Northern analysis detected a 1.7 kb epigen transcript in adult mouse testis and liver, with reduced expression in heart (Strachan et al., 2001).

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Still lower levels were detected in lung and kidney. No information is currently available regarding the epigen gene.

Receptors Epigen stimulation of HaCaT cell proliferation was blocked with anti-EGFR to a similar extent to that of TGFa-induced proliferation. Moreover, tyrosine phosphorylation of EGFR was increased in response to epigen stimulation. These results indicate that epigen is likely to signal through EGFR, but do not exclude the possibility that it may also bind and activate other ErbB receptors.

Activities Recombinant, refolded epigen induced proliferation of HaCaT epithelial cells, though a maximal response required ~20- or 200-fold more epigen than commercial TGFa or EGF, respectively (Strachan et al., 2001). Stimulation was associated with increased phosphorylation of EGFR and downstream MAP kinase proteins, and an antibody directed against the EGFR ectodomain blocked the proliferative response. Moreover, epigen activated a luciferase reporter gene controlled by a reiterated c-fos serum response element. These various results suggest that epigen may be a bona fide member of the EGF ligand superfamily, albeit a less potent EGFR agonist than EGF or TGFa.

ACKNOWLEDGMENTS This work was supported by NCI grants CA43793, CA61896, CA85410 to D.C.L.

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Riese, D.J., Bermingham, Y., van Raaij, T.M. et al. (1996a). Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-beta. Oncogene 12, 345–353. Riese, D.J., Kim, E.D., Elenius, K. et al. (1996b). The epidermal growth factor receptor couples transforming growth factor-alpha, heparin-binding epidermal growth factorlike factor, and amphiregulin to Neu, ErbB-3, and ErbB-4. J. Biol. Chem. 271, 20047–20052. Riese, D.J. II, Komurasaki, T., Plowman, G.D. and Stern, D.F. (1998). Activation of ErbB4 by the bifunctional epidermal growth factor family hormone epiregulin is regulated by ErbB2. J. Biol. Chem. 273, 11288–11294. Roberts, A.B., Lamb, L.C., Newton, D.L. et al. (1980). Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction. Proc. Natl Acad. Sci. USA 77, 3494–3498. Roberts, A.B., Anzano, M.A., Lamb, L.C. et al. (1982). Isolation from murine sarcoma cells of novel transforming growth factors potentiated by EGF. Nature 295, 417–419. Russell, W.E., Kaufmann, W.K., Sitaric, S. et al. (1996). Liver regeneration and hepatocarcinogenesis in transforming growth factor-alpha-targeted mice. Mol. Carcinog. 15, 183–189. Saeki, T., Stromberg, K., Qi, C.F. et al. (1992). Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumors. Cancer Res. 52, 3467–3473. Sandgren, E.P., Luetteke, N.C., Palmiter, R.D. et al. 1990). Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121–1135. Sandgren, E.P., Schroeder, J.A., Qui, T.H. et al. (1995). Inhibition of mammary gland involution is associated with transforming growth factor alpha but not c-mycinduced tumorigenesis in transgenic mice. Cancer Res. 55, 3915–3927. Santoni-Rugiu, E., Jensen, M.R. and Thorgeirsson, S.S. (1998). Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Cancer Res. 58, 123–134. Sasada, R. and Igarashi, K. (1993). (Betacellulin: a new growth factor for vascular smooth muscle cells). Nippon Rinsho 51, 3308–3317. Sasada, R., Ono, Y., Taniyama, Y. et al. (1993). Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family. Biochem. Biophys. Res. Commun. 190, 1173–1179. Sasaki, E., Arakawa, T., Fujiwara, Y. et al. (1997). Epiregulin stimulates proliferation of rabbit gastric cells in primary culture through autophosphorylation of the epidermal growth factor receptor. Eur. J. Pharmacol. 338, 253–258. Schroeder, J.A. and Lee, D.C. (1997). Transgenic mice reveal roles for TGFalpha and EGF receptor in mammary gland development and neoplasia. J. Mammary Gland Biol. Neoplasia 2, 119–129. Schroeder, J.A. and Lee, D.C. (1998). Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland. Cell Growth Differ. 9, 451–464. Schroeder, J.A., Troyer, K.L. and Lee, D.C. (2000). Cooperative induction of mammary tumorigenesis by TGFalpha and Wnts. Oncogene 19, 3193–3199.

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42 Flt3 ligand Stewart D. Lyman and Hilary J. McKenna Amgen, 51 University St, Seattle, WA, USA

My boss asked me to do a presentation on productivity, but I thought he said ‘reproductivity’, and now I work for a different company altogether. J. Struhs

INTRODUCTION The interleukins and the colony-stimulating factors (CSFs) play a large role in regulating the survival, growth, and differentiation of hematopoietic cells. In the case of two CSFs, CSF-1 and Steel factor, this regulation was shown in 1985 and 1990, respectively, to result from the interaction of these proteins with specific tyrosine kinase receptors on the surface of these cells (Sherr et al., 1985; Williams et al., 1990). In 1991, the flt3 (fms like tyrosine kinase 3) tyrosine kinase receptor was isolated independently by two groups using different strategies (Matthews et al., 1991; Rosnet et al., 1991a, 1991b). One group used low stringency hybridization with a DNA probe from the kinase domain of the CSF-1 receptor (c-fms) to isolate a partial cDNA clone of a related DNA sequence (Rosnet et al., 1991b). The gene fragment was then used to isolate a full length receptor clone (Rosnet et al., 1991a), which was named flt3. A second group used a PCR-based strategy (again taking advantage of The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

the highly conserved sequences in the kinase domain of tyrosine kinase receptors) to isolate a novel receptor fragment from purified mouse fetal liver stem cells (Matthews et al., 1991). This fragment was used to isolate a full-length receptor clone that was named flk-2 (fetal liver kinase 2). A comparison of the published mouse flt3 and flk-2 receptor sequences (Lyman et al., 1993b) revealed that these sequences differ by only two amino acids in their extracellular domain. However, a large number of differences at the amino acid level were noted in a region near their C-termini. The sequence of the mouse flt3 receptor has been independently confirmed by several groups (Lyman et al., 1993b; Rossner et al., 1994; Zeigler et al., 1994), and the human flt3 receptor sequence is homologous to the mouse flt3 sequence, but not the mouse flk-2 sequence (Rosnet et al., 1993a; Small et al., 1994). No independent confirmation of the sequence identified as flk-2 has been reported. While this issue of the different flt3 and flk-2 sequences has never been fully resolved, the Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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group that originally cloned flk-2 has reported that ‘the validity of the sequence reported as flk2 is still unclear’ (Dosil et al., 1993). Therefore, we refer to the receptor as flt3, and to its ligand as flt3 ligand (Flt3-L). The flt3 receptor has also been referred to as Stk-1 (stem cell kinase-1) (Small et al., 1994), but this name is not widely used, perhaps because it has been previously designated to denote a gene regulating stem cell kinetics (Van Zant et al., 1983), as well as a different receptor tyrosine kinase of the met/sea/ron family (Iwama et al., 1994).

STRUCTURE OF THE FLT3 RECEPTOR The flt3 receptor belongs to the same subfamily of tyrosine kinase receptors as c-kit, c-fms, and the platelet-derived growth factor (PDGF) A and B receptors. Each of the receptors in this family has five immunoglobulin-like domains in its extracellular region, as well as an ‘insert region’ of 75–100 amino acids in the middle of its cytoplasmic kinase domain. The flt3 receptor has nine potential sites for N-linked glycosylation in its extracellular domain (Matthews et al., 1991; Rosnet et al., 1991a, 1993a; Small et al., 1994) and is glycosylated at one or more of these sites (Lyman et al., 1993b). Immunoprecipitation of the flt3 receptor shows two major protein bands of 143 and 158 kDa (Lyman et al., 1993b). Pulse chase analysis reveals that the 158 kDa protein arises from the 143 kDa protein. This size change is likely to reflect glycosylational processing of the protein from one containing high mannose carbohydrates to one containing complex carbohydrates. Consistent with this interpretation is the finding that only the 158 kDa species is found on the cell surface (Lyman et al., 1993b). An isoform of the mouse flt3 receptor has been identified recently that is missing the fifth of the five extracellular immunoglobulin-like regions (the one nearest the transmembrane region) as a result of the skipping of two exons during transcription (Lavagna et al., 1995). This isoform is present at much lower levels than the wild-type receptor. The receptor isoform lacking the fifth immunoglobulin-like domain is functional in that it binds ligand and is phosphorylated as a result of this binding. However, the physio-

logical significance of this isoform, if any, is presently unknown. A partial clone of the downstream part of the human flt3 receptor genomic locus has been isolated and compared with the genes encoding the tyrosine kinase receptors c-fms and c-kit (Agnès et al., 1994). All three receptors share structural similarities, including the number of exons, size of exons and exon/intron boundaries. These data support the idea that the flt3, c-fms and c-kit receptors, all of which are involved in hematopoiesis, are ancestrally related and are derived as a result of gene duplication and divergence. Evidence for genetic drift can be seen in the exon structure of the flt3 receptor, which includes three additional exons in its extracellular domain compared with the c-kit receptor (Abu-Duhier et al., 2001a).

CLONING OF THE LIGAND FOR THE FLT3 RECEPTOR Cloning of Flt3-L was achieved by two groups using different strategies. Lyman and coworkers (Lyman et al., 1993a) used a soluble form of the flt3 receptor to screen a variety of cell lines looking for one that was capable of binding the soluble receptor. A mouse T cell line was identified that specifically bound the soluble flt3 receptor. The ligand was then cloned from a cDNA expression library made from mRNA isolated from these cells. An alternative approach employed by Hannum and coworkers (Hannum et al., 1994) was to purify Flt3-L from medium conditioned by a mouse thymic stromal cell line. Purification of the protein was facilitated by coupling the extracellular domain of the flt3 receptor to a matrix to generate an affinity column. N-terminal sequencing of the purified protein generated a short amino acid sequence. This information was used to construct degenerate oligonucleotide probes to amplify via PCR a portion of the Flt3-L gene, which eventually led to the isolation of a full-length mouse cDNA clone. The mouse Flt3-L cDNA was then used in hybridization experiments to isolate cDNAs encoding the human gene (Hannum et al., 1994; Lyman et al., 1994b). The mouse and human Flt3-L proteins are 72% identical at the amino acid level. Both human and mouse Flt3-L cDNAs encode a transmembrane

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protein that is biologically active on the cell surface and can also give rise to a soluble, biologically active form of the ligand; the structure of the proteins is shown in Figure 42.1. Flt3-L is structurally related to CSF-1 and c-kit ligand (see below). At present there does not appear to be any species specificity with regard to Flt3-L binding or biological activity. Both the mouse and human ligand are fully active on cells bearing either the mouse or human receptors (Lyman et al., 1994a), and the human Flt3-L protein is also active on mouse, rat, cat, rabbit and primate cells. This is in contrast to either CSF-1 or c-kit ligand, which show species–specific limitations in their activities (Lyman et al., 1994a). Flt3-L is not the only molecule known that is capable of binding to and activating the flt3 receptor. A mannose-binding plant lectin known as FRIL (Flt3 receptor interacting lectin) has been isolated from several types of beans and can stimulate hemato-

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Mouse 231 AA Signal peptide 1

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FIGURE 42.1 Structure of the mouse and human Flt3-L genomic loci and their encoded proteins. Exons and introns are drawn to the same scale for both the mouse and human loci. The human Flt3-L locus is larger than the mouse locus as a result of having larger introns (Lyman et al., 1995c). The sequence information encoding the transmembrane from of Flt3-L is contained within exons 1 to 5 plus 7 and 8; an alternatively spliced exon 6 is used to generate a soluble form of the protein. The vertical lines in the protein structures representing cysteine residues, those that are conserved between Flt3-L, CSF-1 and c-kit ligand, are indicated by asterisks*. The letter N is used to denote the position of consensus sites for the addition of asparagine-linked carbohydrates; TM denotes the transmembrane domain.

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poiesis through binding to flt3 (Colucci et al., 1999; Mo et al., 1999; Moorea et al., 2000).

MULTIPLE ISOFORMS OF FLT3 LIGAND Multiple isoforms of both the mouse and human Flt3-L have been identified by analysis of multiple cDNA clones and PCR (Hannum et al., 1994; Lyman et al., 1993a, 1994b, 1995a). The biological significance of these multiple isoforms is presently unknown. The predominant isoform of human Flt3-L is the transmembrane protein (described above) which is biologically active on the cell surface (Hannum et al., 1994; Lyman et al., 1993a, 1994b). This isoform is also found in the mouse, although it is not the most abundant isoform in that species (see below). The transmembrane protein can (at least under some conditions) be proteolytically cleaved to generate a soluble form of the protein that is also biologically active (Lyman et al., 1993a). The protease responsible for this cleavage, its cellular distribution, and the exact site in the Flt3-L amino acid sequence where cleavage occurs have not been identified. It is also not known if the protease that cleaves Flt3-L is the same or different from the one that cleaves CSF-1 and c-kit ligand. The most abundant isoform of mouse Flt3-L is an alternative, 220 amino acid form that is membrane bound (Lyman et al., 1995c), but is not a transmembrane protein (Hannum et al., 1994; Lyman et al., 1995a; McClanahan et al., 1996). This form arises as a result of a failure to splice an intron out of the mRNA (Lyman et al., 1995a). This leads to a change in the reading frame, which then terminates in a stretch of hydrophobic amino acids that anchor it in the membrane (Lyman et al., 1994b). This isoform lacks the spacer and tether regions that contain the proteolytic cleavage site seen in the transmembrane isoform. As a result, this isoform is resistant to proteolytic cleavage (Lyman et al., 1995a), although it is biologically active on the cell surface. This isoform has not been identified in human Flt3-L cDNAs. A third Flt3-L isoform that has been identified in mouse (Lyman et al., 1995a) and human (Lyman et al., 1995c) tissues is a soluble form of the ligand that arises as a result of inclusion in the mRNA of an

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alternatively spliced sixth exon. This exon introduces a stop codon near the end of the extracellular domain, and thereby generates a soluble, biologically active protein. This isoform appears to be relatively rare compared with other isoforms (Lyman et al., 1995c). A variant on this isoform (seen only in the mouse) is one in which exon 6 is present, but 16 bp of the preceding exon have been deleted (Lyman et al., 1995a). The resulting protein is a transmembrane protein that is not biologically active, most likely because it is missing the last of the four cysteine residues that are conserved between Flt3-L, c-kit ligand and CSF-1. Another method of generating soluble Flt3-L in the human is to splice out the transmembrane domain (Lyman et al., 1994b), but the relative abundance of this isoform has not been quantitated. It is interesting to note the difference between c-kit ligand and Flt3-L in regard to their alternatively spliced sixth exons. The amino acid sequences of exon 6 in mouse and human c-kit ligand are nearly identical, whereas amino acids in exon 6 of mouse and human Flt3-L have virtually no homology to each other or to exon 6 in mouse or human c-kit ligand (Lyman et al., 1995c). In the case of c-kit ligand, the sixth exon is normally part of the transmembrane protein and contains the proteolytic cleavage site. In the case of Flt3-L, the sixth exon is not normally a part of the transmembrane protein, and inclusion of the sixth exon results in the generation of a soluble protein due to a shift in the reading frame. Thus, two different uses have been made evolutionarily of the sixth exon, each of which may contribute to the generation of a soluble protein. In addition to the mouse and human proteins, the amino acid sequences of the dog (Yang and Sim, 2000), cat (Yang and Sim, 2000), and cow (Mwangi et al., 2000) Flt3-L proteins have recently been identified through cloning of their respective cDNAs. The cytoplasmic domains of all three of these proteins are more similar to each other than they are to the cytoplasmic domains of either the mouse or human protein.

COMPARISON OF THE STRUCTURES OF FLT3-L, CSF-1 AND C-KIT LIGAND Flt3-L, CSF-1 and c-kit ligand are transmembrane proteins that are similar in size and structure, but are not very homologous at the amino acid level (Hannum et al., 1994; Lyman et al., 1993a, 1994b). Despite this lack of primary sequence conservation, these three proteins are ancestrally related as evidenced by their four cysteine residues that are completely conserved in position between all three proteins and their common exon structure (Lyman et al., 1995c). The Flt3-L and CSF-1 proteins share an additional two conserved cysteine residues that are not found in c-kit ligand. All of these cysteine residues are believed to play a critical role in the formation of intramolecular disulfide bonds that serve to stabilize the three-dimensional structure of the protein. c-kit ligand has two intramolecular bonds (Lu et al., 1991), whereas CSF-1 has three (Pandit et al., 1992). It is likely that Flt3-L also has three intramolecular bonds. All three of these proteins undergo alternative splicing of a sixth exon (Lyman et al., 1995a), and mouse and human Flt3-L genomic clones (Figure 42.1) have a similar exon/intron structure to c-kit ligand and CSF-1 (Lyman et al., 1995c). The crystal structure of human Flt3-L has recently been solved (Savvides et al., 2000), showing that the protein is a homodimer of two short alpha-helical bundles. The crystal structure confirms the relatedness of the structures of Flt3-L, CSF-1 and c-kit ligand.

CHROMOSOMAL LOCATION OF FLT3 RECEPTOR AND LIGAND GENES The mouse flt3 receptor gene has been mapped to region G of chromosome 5 (Rosnet et al., 1991b). It is located near the mouse flt1 tyrosine kinase receptor (Rosnet et al., 1993b), but is separated from the clustered c-kit, PDGFRA, and flk-1/KDR receptors that have been localized to region D-E of this chromosome. The human flt3 receptor genomic locus is about 100 kb in size and maps to chromosome 13q12 (Rosnet et al., 1991b), again near the flt1 receptor locus (Shibuya et al., 1989). The human flt3 and flt1

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genes are physically linked (Rosnet et al., 1993b) in a head to tail fashion and are separated by about 150 kb (Imbert et al., 1994). This region of chromosome 13q12 is deleted in some patients with myeloproliferative diseases. However, examination of several leukemic samples with (del)13 have shown no evidence of deletion of the flt3 receptor (Birg et al., 1992). The flt1 receptor has a similar overall structure compared with the flt3 receptor, except that it has seven immunoglobulin-like domains in its extracellular region (Shibuya et al., 1989), compared with five in flt3. Structural alterations in the flt3 receptor are associated with various forms of leukemia, as defined below. The Flt3-L gene has been mapped to the proximal portion of mouse chromosome 7 and to human chromosome 19q13.3 (Lyman et al., 1995a). There are no genetic diseases associated with these loci in either mice or humans that have a phenotype that would be suggestive of a defect in Flt3-L. Interestingly, it has been reported that trisomy 19 is strongly associated with myeloid malignancies (Johansson et al., 1994). Mice transplanted with gene-modified hematopoietic cell precursors expressing Flt3-L develop transplantable hematopoietic malignancies after many months (Hawley et al., 1998). However, whether Flt3-L over-expression in some way plays a role in the development of human leukemia remains to be determined.

EXPRESSION OF FLT3 LIGAND IN MOUSE AND HUMAN TISSUES Flt3-L is widely expressed in both mouse and human tissues (Hannum et al., 1994; Lyman et al., 1994b, 1995a). The highest levels of Flt3-L mRNA on human tissue Northern blots are in peripheral blood mononuclear cells, but the ligand is also expressed in heart, placenta, lung, spleen, thymus, prostate, testis, ovary, intestine, liver, skeletal muscle, kidney and pancreas. Expression of Flt3-L in total brain was reported to be low by one group (Hannum et al., 1994) and absent by another (Lyman et al., 1994b). This apparent lack of expression in human brain may be an artifact, since it was not specified which specific regions of the brain were removed and used to isolate

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the mRNA. Flt3-L would be expected to be found in brain because flt3 receptor has been shown to be expressed widely in that tissue (Ito et al., 1993). Expression of Flt3-L on cell lines mirrors that seen in tissues: it is very widely expressed on a great number of mouse and human cell lines (Brasel et al., 1995; Meierhoff et al., 1995). These include myeloid, monocytic, megakaryocytic, erythroid and B cell lines. Flt3L is widely expressed on mouse stromal cell lines (Wineman et al., 1996), as are a number of other hematopoietic growth factors. Endothelial cells have also been found to express Flt3-L (Solanilla et al., 2000).

FLT3 RECEPTOR EXPRESSION IN MOUSE AND HUMAN TISSUES Several early reports showed apparent widespread expression of the mouse flt3 receptor in various hematopoietic (bone marrow, spleen, thymus, lymph node, fetal liver), as well as non-hematopoietic (brain, testis, kidney, skin, ovary, placenta, liver) tissues (Rosnet et al., 1991a; Maroc et al., 1993; deLapeyriere et al., 1995). However, these results were not consistent in that tissue expression (as determined by Northern blot) seen in liver, testis and ovary was not observed by in situ hybridization or Western blotting. Other investigators reported that flt3 receptor expression was not widespread, and was in fact restricted to mouse brain, bone marrow and a very small subpopulation of early hematopoietic progenitor cells from fetal liver and thymus (Matthews et al., 1991). The more restricted expression of flt3 receptor in mice was confirmed by a third group that showed expression in bone marrow, brain, thymus, fetal liver and spleen, but not kidney, adult liver, or testis (Rossner et al., 1994), or adult thymus or spleen (Rasko et al., 1995). It is possible that a relatively minor subset of cells that are flt3 receptor positive within the given tissues could have given the impression of widespread receptor expression, when in fact it now seems rather restricted. Expression of flt3 receptor in human tissues has been reported in liver, spleen, thymus, bone marrow, and weakly in placenta (Rosnet et al., 1993a). Most mouse hematopoietic cells are negative for flt3 receptor expression. These include promyelocytes, myelocytes, promonocytes, metamyelocytes,

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polymorphic mononuclear cells, eosinophils and nucleated red blood cells (Rasko et al., 1995). Mouse cells reported to express flt3 receptor mRNA are peritoneal macrophages, B cells, T lymphocytes from thymus, spleen and peripheral blood (Rosnet et al., 1991a) and monocytes (Rasko et al., 1995). Within the B cell lineage, flt3 receptor is expressed by pre-proB cells and pro-B cells, but not by pre-B, immature B, or mature B cells (Wasserman et al., 1995). Flt3 receptor expression has been reported in the most immature population of mouse thymocytes (defined as CD4CD8Thy-1lo/IL-2R), but not by the more mature CD4CD8, CD4CD8, or CD4CD8 cells (Matthews et al., 1991). In the stem and progenitor cell compartment, flt3 receptor has been found on LinSca-1AA4.1 cells (Matthews et al., 1991), LinAA4.1 cells (Rasko et al., 1995), and WGA151.1Rh123 bright and dull cells (Visser et al., 1993). In humans, no expression of the flt3 receptor has been seen on mature lymphohematopoietic cells fractionated from human peripheral blood (Small et al., 1994) or on human B cells, T cells, monocytes, or granulocytes (Birg et al., 1992). However, later reports show monocytes (Rasko et al., 1995; Rosnet et al., 1993a) and granulocytes (Rosnet et al., 1993a) to be weakly positive for receptor expression (Rosnet et al., 1993a). The different results in these studies likely reflect either differential sensitivity of measuring techniques, or contamination of cells with a minor flt3 receptor positive cell population. In the stem and progenitor cell compartment, flt3 receptor has been found in the CD34 fraction of human bone marrow cells (Rosnet et al., 1996; Small et al., 1994), but not in CD34 cells (Small et al., 1994).

THE EFFECTS OF FLT3-L ON THE DEVELOPMENT OF HEMATOPOIETIC CELLS Early efforts to characterize the biological effects of Flt3-L utilized in vitro assays and revealed that Flt3-L affects the growth of primitive pluripotent hematopoietic precursor cells (HPC), as well as myeloid and lymphoid committed progenitors. The effects of Flt3-L on the hematopoietic system are summarized in Figure 42.2. Flt3-L alone has weak proliferative activity on HPC, but is able to support the survival of

HPC, which retain their responsiveness to cytokines that induce growth and differentiation (Gabbianelli et al., 1995; Rasko et al., 1995; Jacobsen et al., 1996; Veiby et al., 1996). Flt3-L synergizes with a wide range of CSFs and interleukins (Hudak et al., 1995; Jacobsen et al., 1995; Muench et al., 1995; Rasko et al., 1995), as well as with soluble TPO and G-CSF receptors (Ku et al., 1996) to promote colony growth of both committed and primitive progenitor cells. Both TGF-b and TNF-a inhibit the effects of Flt3-L (Ohishi et al., 1996; Veiby et al., 1996). Ex vivo expansion of progenitors is not supported by Flt3-L alone, although progenitor cell survival is observed (McKenna et al., 1995). Flt3-L synergizes with cytokine combinations to augment the ex vivo expansion of HPC, including G-CSF or IL-11 (Jacobsen et al., 1995), IL-3, IL-6, G-CSF, or c-kit ligand (Hudak et al., 1995), IL-1  IL-3  IL-6  EPO (McKenna et al., 1995), IL-3  GM-CSF  EPO (Koller et al., 1996), and IL-3  IL-6  G-CSF (Shapiro et al., 1996). However, the most primitive hematopoietic progenitors (long-term culture initiating cells), derived from human bone marrow, are induced to expand by Flt3-L alone in vitro (Gabbianelli et al., 1995; Petzer et al., 1996), and a 30-fold expansion of these cells is achieved with a cocktail containing Flt3L  IL-3  c-kit ligand (Petzer et al., 1996). The implication that Flt3-L has effects on the most primitive HPC is supported by data obtained from mice (generated by homologous recombination) that are deficient in flt3 receptor expression (Mackarehtschian et al., 1995). These animals are viable and healthy, but have a defect in primitive stem cells, as measured in long-term competitive repopulation assays. When bone marrow cells from the flt3 receptor deficient mice are transferred to irradiated recipients, their potential to reconstitute the myeloid and lymphoid lineages is reduced compared with wild-type bone marrow cells. The administration of Flt3-L to mice demonstrates the potent effects of this protein on the hematopoietic system in vivo (Brasel et al., 1996a). Soluble Flt3-L was administered to mice daily and hematological parameters were followed over 2 weeks. During the first 5 days, Flt3-L causes a significant expansion of the number of progenitors in the bone marrow, including an increase in CFU-GM and the more primitive CFUGEMM progenitors (approximately five-fold). Flt3-L also induces expansion of HPC in the spleen (approx-

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FIGURE 42.2 The role of Flt3-L in hematopoietic development. A simplified schema for the development of the various blood lineages is presented. The stages of development at which Flt3-L has an effect are indicated by ⊕ and summarize the data discussed in the text. The lineages in which mature cells are expanded in vivo by Flt3-L treatment or are severely reduced in Flt3-L knockout mice are indicated with a question mark. It is unclear whether Flt3-L has a role in the development of the mature cells in these lineages, although it clearly acts on the precursor cells. The development of DC and the relationships of the subsets is unresolved, but for the sake of simplicity they have been divided into myeloid and lymphoid type DC, arising from myeloid or lymphoid committed precursors, respectively. imately 100-fold) (Brasel et al., 1996a). Following HPC expansion in the BM, an increase in the peripheral blood cell counts and a 200- to 500-fold increase in the number of HPC in the circulation occurs which peaks after 10 days of growth factor administration (Brasel et al., 1996a). Transplantation experiments demonstrate that the hematopoietic precursors present in Flt3-L-mobilized blood include primitive HPC; transfer of Flt3-L mobilized mononuclear cells to lethally irradiated mice leads to survival of the animals and both short-term (1 month) and longterm (6 months) multilineage hematopoietic reconstitution (Brasel et al., 1996b). Upon cessation of Flt3-L treatment, all measurable hematopoietic

parameters return to normal levels, indicating that the effects of Flt3-L in vivo are transient. Similar effects on hematopoiesis of soluble Flt3-L alone or in combination with G-CF or GM-CSF has been reported in other species including mice (Brasel et al., 1997; Molineux et al., 1997; Sudo et al., 1997), rabbits (Pless et al., 1999) and primates (Papayannopoulou et al., 1997; Bucur et al., 1998). Although there are now numerous reports demonstrating Flt3-L stimulates myeloid committed precursors, as well as pluripotent precursors, there is little evidence that Flt3-L has a significant effect on erythroid, eosinophil, platelet, or mast cell development (Broxmeyer et al., 1995; Gabbianelli et al., 1995;

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Hirayama et al., 1995; Hudak et al., 1995; McKenna et al., 1995; Hogge et al., 1997; Hjertson et al., 1999). Flt3-L shares a number of biological characteristics with c-kit ligand. However, the absence of an effect of Flt3-L on erythropoiesis and mast cell production, two lineages on which c-kit ligand has potent effects (Galli et al., 1994), indicates that Flt3-L and c-kit ligand do not have entirely overlapping biological functions. In addition to effects on the myeloid lineage, Flt3-L affects lymphoid development. Flt3-L alone, or in combination with IL-7, stimulates the proliferation of day 14 murine fetal thymocytes (Hannum et al., 1994) and primitive progenitor cells within the thymus (CD4low) are responsive to Flt3-L, undergoing proliferation, but not differentiation in vitro in response to Flt3-L plus IL-3, IL-6 and IL-7 (Moore and Zlotnik, 1997). Human T cell development in vitro is augmented by Flt3-L (Freedman et al., 1996). When human bone marrow-derived CD34 cells are cultured on a primary irradiated fetal-thymic stromal layer plus IL-12, development of T cell receptor positive cells is observed. The addition of Flt3-L to the culture augments T cell production without altering the phenotype of the cells produced. However, thymic cellularity is normal in flt3 receptor (Mackarehtschian et al., 1995) and Flt3-L (McKenna et al., 2000) deficient mice, and administration of Flt3-L to mice does not result in T cell expansion, at least over 14 days (Brasel et al., 1996a). Hunte and coworkers have reported that Flt3-L, in combination with IL-7 or c-kit ligand, stimulates proliferation of the most primitive B220low, CD43, CD24 bone marrow-derived B cell precursors, but no effect of Flt3-L is noted on the more differentiated pro- or pre-B cell progenitors (Hunte et al., 1996). Uncommitted progenitors from mouse yolk sac or fetal liver develop into B lineage cells in response to Flt3-L  IL-7  IL-11, and Flt3-L  IL-7 stimulates expansion of pro-B cells (Ray et al., 1996). Purified progenitors (Lin Sca-1) from mouse bone marrow develop into B lineage cells in the presence of Flt3-L  IL-7, although development only proceeds to the pro-B stage (Veiby et al., 1996). Flt3-L effects on human B lymphopoiesis from fetal liver-derived cells have also been reported. Flt3-L synergizes with IL-3  IL-7 to promote pro-B cell growth and differentiation into pre-B cells (Namikawa et al., 1996). The hypothesis

that Flt3-L influences the development of immature B cell precursors is supported by studies on flt3 receptor deficient mice (Mackarehtschian et al., 1995), and Flt3-L deficient mice (McKenna et al., 2000) both of which have reduced numbers of B cell progenitors in the bone marrow. The administration of soluble Flt3L to mice results in the expansion of immature B lineage cells (B220, sIg) in the spleen and bone marrow (Brasel et al., 1996a). Flt3-L has a role in natural killer (NK) cell development since Flt3-L knockout mice are deficient in NK cells (McKenna et al., 2000), and administration of either soluble Flt3-L (Shaw et al., 1998) or DNA encoding Flt3-L (He et al., 2000) results in expansion of functional NK cells in multiple tissues, including the peripheral blood. In summary, Flt3-L has effects on stem cells and progenitor cells for many but not all lineages. Evidence from in vitro assays, in vivo administration of soluble Flt3-L and analysis of mice deficient for either flt3 receptor or Flt3-L reveal that primitive pluripotent stem cells and precursors for the lymphoid lineages (T, B and NK), and the myeloid lineages (monocytes and neutrophils) are affected by Flt3-L. In contrast it appears that Flt3-L has little or no effect on erythroid, eosinophil, platelet, or mast cell development.

FLT3-L SERUM LEVELS IN HEMATOPOIETIC DISORDERS The results from both in vitro and in vivo experimentation with Flt3-L support the idea that Flt3-L is a critical regulator of early events in the development of hematopoietic cells. Further support for this hypothesis has come from studies on the levels of Flt3-L in the blood of patients with a variety of hematopoietic disorders. Serum levels of Flt3-L in normal, healthy individuals average less than 100 pgml1, which is the limit of detection of this protein by ELISA (Lyman et al., 1995b). This is in contrast to the structurally related proteins c-kit ligand and CSF-1, which are present in serum at levels in the range of 2–6 ng ml1 (Langley et al., 1993) and 2–4 ng ml1 (Shadle et al., 1989), respectively. Flt3-L blood levels are not elevated in a variety of anemias that predominantly

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affect only the erythroid lineage, such as acquired pure red cell aplasia, congenital pure red cell aplasia (Diamond–Blackfan anemia), or a-thalassemia (Lyman et al., 1995b). Flt3-L serum levels are also normal in patients with rheumatoid arthritis, systemic lupus erythematosus, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), or HIV (Lyman et al., unpublished observations). In contrast to these findings (and to what has been observed with c-kit ligand), serum levels of Flt3-L are highly elevated in patients with hematopoietic disorders that specifically affect the stem cell compartment, i.e. pancytopenias. Thus, the majority of patients with Fanconi anemia or acquired aplastic anemia have highly elevated serum levels of Flt3-L (up to 10 ng ml1) (Lyman et al., 1995b). Similarly, about half of patients classified as having the refractory anemia form of myelodysplastic syndrome (MDS) display elevated levels of Flt3-L (Zwierzina et al., 1999). Flt3-L serum levels returned to normal in a Fanconi anemia patient following a cord blood transplant that cured the pancytopenia (Lyman et al., 1995b). Similarly, successful treatment of aplastic anemia patients with either a bone marrow or peripheral blood transplant, or with immunosuppressive treatment (anti-lymphocyte globulin  cyclosporin A), results in Flt3-L serum concentrations returning to normal (Wodnar-Filipowicz et al., 1996). These studies were extended to patients undergoing chemotherapy and radiation treatment for various leukemias, and who consequently had marrow aplasia. The level of Flt3-L in their serum appears to be inversely correlated to the peripheral blood white cell count. Finally, radiation-induced marrow aplasia in non-human primates is correlated with an elevation of Flt3-L serum levels (Bertho et al., 2001). It has been hypothesized that in situations where HPC numbers are severely reduced (Fanconi anemia, acquired aplastic anemic, or chemotherapy/irradiation), Flt3-L levels become elevated in an attempt to stimulate the production of hematopoietic stem cells in vivo (Wodnar-Filipowicz et al., 1996). One interpretation of these data is that the loss of functional stem/progenitor cells leads to a loss of a negative regulator of Flt3-L production. The levels of Flt3-L serum concentrations in the blood of patients with hematopoietic disorders (with a suspected stem cell component) can be elevated to a level that is likely to

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be physiologically active. In summary, these data suggest that restoration of stem cells in these patients is associated with a return of Flt3-L serum levels to those measured in normal, healthy individuals, and that Flt3-L serum levels may be a surrogate marker for stem cell content of bone marrow.

EXPRESSION OF THE FLT3 RECEPTOR IN LEUKEMIAS Within the hematopoietic system flt3 receptor expression appears to be restricted to immature precursor cells (Matthews et al., 1991; Small et al., 1994; Zeigler et al., 1994; Rasko et al., 1995; McKenna et al., 1996a). Like the related receptors c-kit and c-fms, flt3 receptor expression has also been described on leukemic blast cells. However, unlike c-kit and c-fms, whose expression appears to be restricted to myeloid leukemic cells (Birg et al., 1994), flt3 receptor expression has been detected on both myeloid and lymphoid primary leukemic blasts (Birg et al., 1992; Carow et al., 1996; McKenna et al., 1996b; Stacchini et al., 1996). In addition, a variety of myeloid and lymphoid leukemic cell lines express flt3 receptor (Da Silva et al., 1994; Brasel et al., 1995; Meierhoff et al., 1995; Turner et al., 1995), although nonhematopoietic tumor cell lines do not (Serve et al., 1999). Flt3 receptor expression is particularly prevalent on pre-B and monocytic/myeloid leukemic cells. A number of growth factor-dependent myeloid leukemia cell lines proliferate in response to Flt3-L, whereas growth factor-independent lines do not (Drexler et al., 1999). In addition, Flt3-L has significant, anti-apoptotic effects on both primary myeloid leukemic cells, as well as some myeloid leukemia cell lines under serum-free conditions (Drexler et al., 1999). A series of reports has defined a role for the flt3 receptor in the progression of human AML (Nakao et al., 1996; Yokota et al., 1997), acute promyelocytic leukemia (APL) (Kiyoi et al., 1997), ALL (Xu et al., 1999), and to a lesser degree MDS (Horiike et al., 1997), but not in chronic myelogenous leukemia (CML) (Iwai et al., 1997). Internal tandem duplications (ITD) of varying sizes (encoding approximately 7 to 54 amino acids) occur in the juxtamembrane region of the receptor. Approximately 20–25% of adult

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AML samples (Nakao et al., 1996) and APL samples (Kiyoi et al., 1997) and 3% of ALL samples (Xu et al., 1999) and MDS samples (Horiike et al., 1997) examined have been found to contain these ITD. These in-frame duplications appear to constitutively activate the flt3 receptor’s kinase activity (Kiyoi et al., 1997; Hayakawa et al., 2000). Flt3 ITD confer ligand-independent transformation of the 32D cell line and cause a leukemia-like disease in syngeneic mice (Mizuki et al., 2000). Interestingly, flt3 ITD are associated with a proliferation defect in the leukemic cells in response to exogenous cytokines or stromal cells (Rombouts et al., 1999), but an enhanced capacity to repopulate bone marrow in NOD/SCID mice (Rombouts et al., 2000). The mechanism responsible for these observations has not been identified. While flt3 ITD do not appear to be causal in the development of AML, they are associated with a worse clinical response and prognosis for the disease (Kiyoi et al., 1999; Kondo et al., 1999; Abu-Duhier et al., 2000; Rombouts et al., 2000). In fact, the presence of the flt3 ITD was the single most significant, independent prognostic factor for poor outcome in pediatric AML patients (Meshinchi et al., 2001). There is a higher incidence of flt3 ITD in adult leukemia compared with childhood leukemia (Iwai et al., 1999; Xu et al., 1999), suggesting that this alteration is secondary to the development of the malignancy. Consistent with this idea is the finding of flt3 ITD in samples obtained from AML patients in relapse where the original leukemia was negative for flt3 ITD (Nakano et al., 1999). Flt3 ITD are not detected in cells taken from AML patients in remission or in normal lymphocytes from AML patients (Meshinchi et al., 2001), suggesting that these alterations are tumor-specific. Similarly, hematopoietic cells from normal individuals show no evidence that they contain ITD (Ishii et al., 1999). ITD are not the only mutations found in the flt3 receptor associated with tumor formation. In several other studies, mutations in amino acid 835 in the ‘activation loop’ of the flt3 receptor cytoplasmic domain have been identified in about 7% of AML samples (Abu-Duhier et al., 2001b; Yamamoto et al., 2001). These mutations are independent of the ITD mutations, are associated with constitutive tyrosine phosphorylation, and are associated with worse disease-free survival. Mutation at the equivalent amino acid in the mouse flt3 receptor is associated

with receptor autophosphorylation and the capacity to induce ligand-independent growth in 32D cells (Fenski et al., 2000). The combination of the ITD mutations and the activating loop point mutations make flt3 the single most mutated gene identified to date in AML (Griffin, 2001) and a good target for a small molecule inhibitor of the kinase activity. Mutation of the equivalent amino acid in the related c-kit tyrosine kinase receptor has been associated with mast cell transformation (Moriyama et al., 1996; Beghini et al., 1998). Given the expression of flt3 on leukemic cells, and the association of mutations in this receptor with kinase activation and cellular transformation, investigations have begun to determine if inhibition of flt3 tyrosine kinase activity might be a useful clinical target. A molecule of the tyrphostin class of receptor tyrosine kinase inhibitors (AG1296) inhibits Flt3-L induced proliferation of flt3 expressing Ba/F3 cells, as well as a constitutively activated TEL-Flt3 fusion protein (Tse et al., 2001). This inhibitor would be predicted to work on flt3 ITD and mutations at amino acid 835 as well, because it acts at the ATP binding site of the receptor. Similarly, the tyrphostin compound AG1295 was selectively cytotoxic (and inhibited receptor phosphorylation) of flt3 ITD containing AML cells compared with AML cells that did not contain this mutation (Levis et al., 2001). In vitro studies showed that the tyrosine kinase inhibitor herbimycin A inhibited the activity of flt3 ITD, but did not inhibit the ligand stimulated phosphorylation of the wildtype flt3 receptor (Zhao et al., 2000). There have been numerous reports of various cytokine receptors expressed on leukemic blast cells, but the in vitro response of these cells to the respective ligands has not been consistent. Expression of a cytokine receptor has not been predictive of a proliferative response, and it has been difficult to determine whether specific cytokine/cytokine receptor interactions are important in the pathogenesis of leukemia (Zola et al., 1994). The published reports of flt3 receptor expression on leukemic cells, and the response of those cells to Flt3-L, are consistent with these observations (Drexler, 1996). Flt3-L alone has been reported to stimulate the proliferation of some leukemic cells, including AML, B-ALL and T-ALL cells (Piacibello et al., 1995; Eder et al., 1996; McKenna et al., 1996b). Synergistic proliferative effects between

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Flt3-L and other cytokines have been noted also, as has been discussed for normal hematopoiesis. More recently, is has been suggested that Flt3-L can prevent apoptosis in AML blast cells (Lisovsky et al., 1996). The prevention of apoptosis, which is augmented with the addition of G-CSF or GM-CSF, is associated with decreased Bax expression in the surviving cells. Flt3-L expression has been detected on the surface of leukemic cell lines (Brasel et al., 1995; Meierhoff et al., 1995), and some examples of concomitant flt3 receptor and ligand expression have been described in these reports. However, there is as yet no evidence of an autocrine loop in these cells, or that the cells are transformed as a result of this concomitant expression. To date there have been no reports of Flt3-L expression on the surface of leukemic blast cells.

FLT3-L IS A KEY REGULATOR OF DENDRITIC CELLS Dendritic cells (DC) are rare, hematopoietic-derived cells that form a cellular network involved in immune surveillance, antigen capture and antigen presentation (Steinman, 1991). DC are predominantly found in the T cell-dependent areas of lymphoid tissue (Metlay et al., 1990; Steinman, 1991), although a subset of DC has also been identified in the B cell-dependent germinal centers (GC) (Grouard et al., 1996). Immature DC acquire antigens in peripheral tissues, undergo maturation and migrate to lymph nodes, and present these antigens as processed peptides to T cells in the context of major histocompatability complex (MHC) antigens (Metlay et al., 1990; Steinman, 1991; Austyn, 1996; Grouard et al., 1996), thus initiating an antigen-specific immune response. In addition to their capacity for processing and presenting foreign or altered self-antigens to induce immunity (Steinman, 1991), certain DC subsets are believed to negatively regulate T cell immunity (Matzinger and Guerder, 1989; Inaba et al., 1991; Mazda et al., 1991; Süss and Shortman, 1996). Furthermore, there is emerging evidence that DC may regulate the cytokine repertoire of T cells during the induction of immunity (Maldonado-Lopez et al., 1999; Rissoan et al., 1999). The cytokines that govern DC development are not well defined, but the best characterized to date is

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GM-CSF which induces DC expansion in vitro (Inaba et al., 1992) and in vivo (Pulendran et al., 1999; Daro et al., 2000). However the physiological role of GMCSF in DC development is unclear as GM-CSF mutant mice have normal numbers of DC, and GM-CSF receptor beta chain mutant mice, in which GM-CSF cannot signal, have nearly normal numbers of DC (Vremec et al., 1997). Administration of Flt3-L to mice expands a population of cells with the phenotype of DC: MHC class II, CD11c which can be subdivided on the basis of CD11b or CD8 homodimer expression (Maraskovsky et al., 1996). While GM-CSF predominantly expands the CD11b CD8  population of DC (Pulendran et al., 1999; Daro et al., 2000), sometimes referred to as the myeloid-related DC, Flt3-L expands both this myeloid-related and the CD11b CD8  population, referred to as the lymphoid-related population (Maraskovsky et al., 1996). DC expansion in multiple tissues is evident after 5–7 days treatment with Flt3-L, and peaks after 9–10 days treatment. Cessation of treatment results in a return to baseline values. The DC expanded in vivo are relatively immature, expressing low levels of CD40 and the costimulatory molecule CD86, and lacking expression of CD80 (Maraskovsky et al., 1996; Pulendran et al., 1997), but are responsive to maturation signals. Stimulation of the DC in vitro results in IL-12 production, particularly from the lymphoid-related DC (Pulendran et al., 1997). The Flt3-L-expanded DC are as efficient as control DC at stimulating the proliferation of allogeneic T cells in a mixed lymphocyte reaction or syngeneic antigen-specific T cells in a keyhole limpet hemocyanin (KLH) presentation assay (Maraskovsky et al., 1996). Furthermore, Flt3-Lgenerated spleen DC are as efficient as control DC at generating antigen-specific T cells in vivo after being pulsed with antigen ex vivo (Maraskovsky et al., 1996). Elevated numbers of DC are detected in both lymphoid- and non-lymphoid tissue in Flt3-L-treated mice, including the bone marrow, gastro-associated lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen and thymus (Maraskovsky et al., 1996; Shurin et al., 1997; Shaw et al., 1998; Viney et al., 1998). The role of Flt3-L in DC generation from progenitors is further highlighted by examination of mice genetically deficient in Flt3-L (McKenna et al., 2000). Flt3-L-deficient mice have severely reduced numbers of DC in the

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peripheral lymphoid tissues. Both myeloid-related and lymphoid-related DC are reduced (McKenna et al., 2000). DC can be generated in vitro from CD34 progenitors cultured with GM-CSF  IL-4  TNFa (Reid et al., 1992; Santiago-Schwarz et al., 1992; Sallusto and Lanzavecchia, 1994). The addition of Flt3-L to cultures can augment DC numbers generated in vitro from hematopoietic progenitors (Bello-Fernandez et al., 1997), and DC progenitors within the CD34 fraction of BM or G-CSF-mobilized peripheral blood are expanded ex vivo by Flt3-L  SCF (Curti et al., 2001). Leukemic AML cells are induced to develop into DC when cultured with GM-CSF  IL-4  TNFa and the addition of Flt3-L to the culture increases DC numbers (Woiciechowsky et al., 2001). In addition Flt3-L augments DC development from thymic precursors in vitro (Saunders et al., 1996) and Langerhans cell development induced by TGFb in vitro (Strobl et al., 1997). Recently a method for generating DC from murine bone marrow in vitro was described that did not require the addition of GMCSF. Culture of murine bone marrow at high density with Flt3-L alone generates cells with the features of DC. Neutralizing antibodies to IL-6, but not GM-CSF, revealed a role for IL-6 in these cultures (Brasel et al., 2000). Administration of Flt3-L to humans induces DC mobilization into the peripheral blood (Maraskovsky et al., 2000; Morse et al., 2000; Pulendran et al., 2000). Daily administration of doses ranging from 10 to 100 μg kg1 results in increased numbers by around day 6, with a peak increase occurring around day 10 of administration. DC identified as CD11c MHC class II are expanded approximately 40–50-fold in healthy volunteers. Based on the cell surface expression of the co-stimulatory molecules CD80, CD86 and CD40, the Flt3-L-expanded DC are relatively immature, but undergo maturation in vitro when cultured with GMCSF  TNFa (Pulendran et al., 1997). CD11c CD123 DC are expanded though to a lesser degree (approximately 10-fold) (Maraskovsky et al., 2000; Pulendran et al., 2000). Upon infection with virus in vitro, the CD11c CD123 DC release large amounts of IFNa as reported previously for this DC population (Siegal et al., 1999). As in mice, DC numbers return to baseline after cessation of treatment. DC are also expanded by Flt3-L treatment in patients bearing

metastatic colon cancer (Morse et al., 2000). An increase in DC in the blood (four- to 25-fold) and in tissues surrounding resected metastases was noted. The mechanism by which Flt3-L influences DC generation in vivo and in vitro is not completely understood. However, it is unlikely that Flt3-L treatment simply results in the mobilization of existing mature DC from other sites into the lymphoid tissue. First, flt3 receptor is not detected on mature DC, as assessed by flow cytometry, and mature DC do not respond when cultured in Flt3-L alone, as assessed either by microphysiometric analysis of cytoplasmic pH changes or by thymidine incorporation of proliferating cells (Maraskovsky, unpublished observation). Second, elevated numbers of DC are detected in multiple organs and tissues in Flt3-L-treated mice, indicating that there is a generalized expansion of DC throughout these animals. Third, the effects of Flt3-L on the expansion of DC from bone marrow progenitors, its described effects on primitive progenitors in vitro, and the potent effects of Flt3-L on hematopoiesis in vivo suggest that Flt3-L targets and expands the progenitor cell pool in the bone marrow, some of which undergo commitment to the DC lineage. In summary, Flt3-L administration induces the transient expansion of DC subsets in multiple tissues and peripheral blood. The Flt3-L-expanded DC are relatively immature, are susceptible to maturation signals and are functional as demonstrated by their ability to prime naive T cells.

ANTI-TUMOR EFFECTS OF FLT3-L Does the expansion of DC in vivo result in a more robust immune response? This question has been addressed in murine tumor models where Flt3-L was administered to tumor-bearing mice with the aim of inducing tumor-specific effector T cells. In a model of fibrosarcoma, tumor-bearing mice treated with Flt3-L exhibit slower tumor growth rates and in some cases reject the tumor (Lynch et al., 1997). Mechanisms of the anti-tumor effects include expansion of NK effector cells and the generation of tumor-specific CD8 cells. Mice that reject the primary tumor challenge resist a second tumor challenge demon-

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strating that a memory response develops (Lynch et al., 1997). Similar results have been obtained in a rodent model of breast cancer. Tumor cells transduced with a cDNA expressing Flt3-L, but not mocktransfected cells, are rejected by mice. Mice that reject the primary tumor reject a secondary challenge of freshly transplanted, non-transduced breast cancer cells demonstrating the generation of a memory response (Chen et al., 1997). Flt3-L has now been tested in numerous rodent tumor models and effects including NK-mediated tumor lysis and the generation of tumor-specific T cells responses and T cell memory have been reported (Esche et al., 1998, Peron et al., 1998, Borges et al., 1999, Braun et al., 1999, Chakravarty et al., 1999, Fernandez et al., 1999, Ciavarra et al., 2000, Favre-Felix et al., 2000, Mach et al., 2000, Wang et al., 2000, Pawlowska et al., 2001). DC can cooperate with NK cells to augment immune responses and it is possible that the anti-tumor responses noted result from both the interactions of the Flt3-L-expanded DC and T cells (Lynch et al., 1997), as well as Flt3-L-expanded DC and Flt3-Lexpanded NK cells (Fernandez et al., 1999).

EFFECTS OF FLT3-L ON INFECTIOUS DISEASES Just as expansion of NK cells and DC in mice bearing tumors has a beneficial effect, mice infected with pathogens have increased protection after Flt3-L treatment. In a murine model of polyoma viral infection, Flt3-L treatment increases the number of viral-specific CD8 cells and the resulting memory pool of the viral-specific CTL (Drake et al., 2000). Similarly, treatment with Flt3-L improves survival in mice infected with HSV-1 (Smith et al., 2001). Flt3L treatment prior to cutaneous challenge with Leishmania major in susceptible mice results in 40% of the mice clearing the infection, compared with 0% of the controls. Flt3-L treatment resulted in increases in IL-12p40 and nitric oxide in the lymph nodes after infection, but surprisingly no skewing toward IFNcproducing T cells, the hallmark of a protective response was noted (Kremer et al., 2001).

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THE ADJUVANT PROPERTIES OF FLT3-L Immune responses are initiated when naive T cells are primed by DC presenting peptide antigens. When mice are treated with Flt3-L to expand DC, then immunized with soluble protein, enhanced antibody responses are noted; in some cases equivalent to those generated with the potent adjuvant, complete Freunds adjuvant (Pulendran et al., 1998). When T cells transgenic for a T cell receptor restricted to a chicken ovalbumin peptide are transferred to mice that are treated with Flt3-L and immunized with soluble ovalbumin protein, T and B cell responses are generated (Pulendran et al., 1998). No responses are generated to ovalbumin in the absence of Flt3-L. The adjuvant properties of Flt3-L-expanded DC have also been demonstrated in a murine measles virus model. Treatment with Flt3-L followed by immunization with sub-optimal doses of measles nucleoprotein via the buccal mucosa increases the number of virus-specific CTL (Etchart et al., 2001). Mice immunized with the intracellular pathogen Listeria monocytogenes, following Flt3-L treatment develop a protective immune response (Gregory et al., 2001). An interesting report in which mice were immunized with a chimeric DNA encoding the HPV antigen E7 and the extracellular domain of Flt3-L demonstrate a potent adjuvant activity of Flt3-L in this setting (Hung et al., 2001). The adjuvant activity, which is CD8 T cell-mediated, occurs in the absence of DC expansion. Rather, the authors postulated that the adjuvant effects result from increased targeting of the E7 antigen to the endoplasmic reticulum (ER), where MHC loading occurs (Hung et al., 2001). The adjuvant properties of Flt3-L and GM-CSF were compared in vivo, since both cytokines expand DC and it has been reported that GM-CSF has adjuvant activity (Warren and Weiner, 2000). Flt3-L, which expands both myeloid- and lymphoid-related DC, induces TH1 T cell development after immunization, whereas GM-CSF, which drives expansion of myeloid-related DC only, skewed T cells toward a TH2 phenotype (Pulendran et al., 1999). In addition to Flt3-L having adjuvant properties for immunization, Flt3-L also has adjuvant properties for the induction of tolerance to a protein. Oral delivery of protein antigen alone leads to tolerance induction. Administration of Flt3-L prior to oral delivery of

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antigen results in tolerance induction in response to much lower levels of antigen (Viney et al., 1998). This increased sensitivity to tolerance induction in Flt3-Ltreated mice is associated with a large increase in DC in the GALT (Viney et al., 1998; Williamson et al., 1999a). The Flt3-L-expanded GALT DC are not restricted to tolerance induction because oral administration of a protein antigen to Flt3-L-treated mice, along with administration of an activating signal like cholera toxin or IL-1a abrogate tolerance induction and leads to immunity to the antigen (Williamson et al., 1999b). A further role for DC in tolerance induction has been postulated in the area of liver transplantation. Long-term survivors of organ transplants, particularly liver transplants, exhibit multilineage donor cell microchimerism in a variety of tissues (Starzl et al., 1992). Examination of host tissue reveals that donorderived DC are commonly present. It has been hypothesized that immature donor-derived DC are involved in the long-term tolerance of the allogeneic organ by the recipients (Starzl et al., 1993; Lu et al., 1995; Thomson et al., 1995). To determine whether augmenting the number of immature donor DC in host tissues might improve transplantation outcome, mice were given an allogeneic transplant of bone marrow cells in a non-cytoreductive setting, and then treated with Flt3-L to determine whether the increased immature donor DC contribute to chimerism. However, chimerism in this setting is augmented only if recipients of the bone marrow are treated with tacrolimus (FK506) plus Flt3-L; Flt3-L treatment alone reduced bone marrow engraftment (Iyengar et al., 1997). The possibility that the increased chimerism noted with tacrolimus  Flt3-L would lead to acceptance of allogeneic solid organ transplants was tested in a model of cardiac transplantation (Antonysamy et al., 1998). Mice received donor bone marrow, tacrolimus  Flt3-L and a donor strain cardiac transplant. Though this regimen leads to improved chimerism, cardiac graft survival was not enhanced, rather it was abrogated. Augmenting the number of immature DC present in a graft with Flt3-L treatment prior to transplantation does not result in increased graft acceptance; livers or bone marrow from Flt3-L-treated mice are more readily rejected than their control counterparts (Steptoe et al., 1997; Morelli et al., 2000). IL-12

appears to have a major role in the rejection of livers from Flt3-L-treated mice; administration of neutralizing antibodies to IL-12 to organ recipients inhibits liver rejection (Li et al., 2001). The effect of Flt3-L treatment on alloreactivity in vivo has also been demonstrated in a model of myeloablative allogeneic bone marrow transplantation (Blazar et al., 2001). Treatment of the recipient mice with Flt3-L to expand their DC results in a stronger alloreactive donor T cell response, which leads to a more severe graft-versushost disease outcome post-transplant. Collectively these results demonstrate that the large number of immature DC expanded by Flt3-L in vivo, either prior to or after bone marrow or solid organ transplantation, are not tolerogenic, but rather stimulate an allogeneic response in vivo.

POTENTIAL USES OF FLT3-L IN THE CLINIC Although still under intensive study, current knowledge of Flt3-L suggests that it has the potential to be used in a number of different areas in clinical medicine. Results from phase I and phase II clinical trials have revealed that there are no overt toxicities associated with the administration of Flt3-L to humans (Maraskovsky et al., 2000; Pulendran et al., 2000; Stiff, 2000).

Ex vivo stem cell expansion Ex vivo expansion of stem cells for transplantation is under investigation and Flt3-L is a likely candidate for inclusion in the culture milieu. Flt3-L as a single factor maintains human bone marrow-derived HPC ex vivo for 3–4 weeks, and when combined with IL-1  IL-3  IL-6  EPO results in a similar level of CFU-GM expansion as seen when c-kit ligand is added to this combination of factors (McKenna et al., 1995). Flt3-L induces expansion of primitive stem cells as measured by expansion of long-term culture initiating cells (Gabbianelli et al., 1995; Petzer et al., 1996). A very promising combination of factors for the ex vivo expansion of stem/progenitor cells from cord blood is the combination of Flt3-L and TPO, which allows for continuous expansion of these cells for a 5-month period (Piacibello et al., 1997). Numerous follow-on

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studies have confirmed the utility of Flt3 ligand in ex vivo stem cell expansion protocols (Rappold et al., 1999; Gilmore et al., 2000; Nielsen et al., 2000), and at least one study has been done where the expanded bone marrow cells have been administered to breast cancer patients following high-dose chemotherapy (Stiff et al., 2000). The majority of patients receiving expanded cells in this study maintained hematopoietic cell production for at least 24 months.

Gene therapy Flt3-L may be of use in the setting of gene therapy. Transduction of stem cells with retroviruses requires that the cells be actively cycling; therefore Flt3-L may be used to stimulate the proliferation of candidate stem cell populations to facilitate this process. A number of reports have now been published showing that Flt3-L increases the capacity of retroviruses to transduce hematopoietic stem cells (Elwood et al., 1996; Dao et al., 1997; Wognum et al., 2000). In terms of clinical protocols, Flt3-L was part of the retroviral transduction cocktail used to successfully treat several boys with severe combined immunodeficiency-X1 syndrome (Cavazzana-Calvo et al., 2000).

Stem cell mobilization Flt3-L has potent effects on the mobilization of HPC to the peripheral blood for transplantation. Data in both mice (Brasel et al., 1996a), rabbits (Pless et al., 1999) and primates (Papayannopoulou et al., 1997; Bucur et al., 1998) indicate that Flt3-L is capable of increasing the number of HPC in peripheral blood. Flt3L-mobilized cells give rise to long-term, multilineage repopulation upon transplantation in mice (Brasel et al., 1997). Flt3-L can synergize with G-CSF or GM-CSF to facilitate the mobilization of stem cells (Brasel et al., 1997, Molineux et al., 1997, Papayannopoulou et al., 1997, Sudo et al., 1997, Pless et al., 1999). In addition, Flt3-L mobilizes facilitating cells (CD8, TCR) which may have a role in aiding engraftment across allogeneic barriers (Neipp et al., 1998). Flt3-L administered to humans mobilizes CD34 progenitor cells, but with relatively slow kinetics; the peak response occurs 10 or more days after the start of daily Flt3-L treatment (Stiff, 2000). The addition of G-CSF or GM-CSF to Flt3-L for stem

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cell mobilization intended for patients undergoing autologous transplant further increases the yield of progenitors in patients heavily pre-treated with chemotherapy (Stiff, 2000).

Radioprotection Hematopoietic failure is a dose-limiting toxicity of radiation which is used for the treatment of oncology patients. A single injection of Flt3-L 20 h prior to a lethal dose of irradiation significantly protects 65% of irradiated mice (Hudak et al., 1998). The treatment is associated with increased myelopoietic activity postirradiation. A longer treatment course of Flt3-L given to rabbits starting just prior to irradiation also has a significant effect on hematopoietic recovery and survival outcome (Gratwohl et al., 1998).

Tissue regeneration A number of recent papers have provided evidence showing that hematopoietic stem cells can give rise to a variety of other tissues (Krause et al., 2001), including heart muscle (Orlic et al., 2001), liver (Theise et al., 2000a, 2000b), and brain (Mezey et al., 2000) via a process referred to as transdifferentiation. Since Flt3-L serves to expand hematopoietic stem cells, it could have a possible role in the future in facilitating tissue repair.

Immunotherapy The use of DC as cellular vectors for either anti-tumor or infectious disease vaccines or as inducers of tolerance are promising immunotherapy strategies. However, the clinical feasibility of using DC as immunotherapy vectors continues to be limited by the extremely small numbers of DC that can be procured from peripheral blood of normal individuals. The potent ability of Flt3-L to expand DC in vivo may alleviate the problem (Maraskovsky et al., 2000; Morse et al., 2000; Pulendran et al., 2000). One can envisage using Flt3-L as a vaccine adjuvant, whereby DC would be expanded in vivo by Flt3-L treatment, and then tumor or infectious disease antigens would be administered. Alternatively, large numbers of circulating DC from Flt3-L-treated individuals could be isolated for ex vivo manipulation followed by reinfusion of these

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DC into the individual. Recent results using this approach have been reported in which patients with colon carcinoma were treated with Flt3-L to expand their circulating DC, the DC were isolated by apheresis and pulsed with altered carcinoembryonic antigen (CEA) peptide. The patients were then immunized with the pulsed autologous DC and CD4 and CD8 T cell responses to CEA were monitored (Fong et al., 2001). A correlation between expansion of CEAspecific CD8 T cells and clinical response was noted in the 12 patients immunized. The use of an altered self-antigen highlights the requirement in tumor immunity to generate an immune response to antigens that are generally seen by the immune system as self-antigens. The ability to generate an immune response using DC as adjuvants may be easier to achieve in the infectious disease setting, where tolerance to the antigen has not been established. Another interesting possibility lies in the use of Flt3-L to enhance tolerance to orally administered antigens (Viney et al., 1998), though the challenge here will be to induce tolerance to antigens to which an immune response has already been established as in autoimmune disease.

ACKNOWLEDGMENTS The authors wish to acknowledge the contributions of our many colleagues at Immunex who have contributed to this body of work.

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43 Stem cell factor Ian K. McNiece1 and Robert A. Briddell2 1

University of Colorado Health Sciences Center, Denver, CO, USA 2

Amgen Inc., Thousand Oaks, CA, USA

How old would you be if you didn’t know how old you were Satchel Paige

INTRODUCTION For many years investigators have studied and characterized hematopoietic stem cells. Many of these efforts have focused on the investigation of regulatory proteins, which are involved in the proliferation and differentiation of stem cells. Mutant mouse strains initially provided evidence for the existence of such proteins. Mutations at the Steel locus (Sl ) resulted in a deficient hematopoietic microenvironment, while mutations at the white spotting locus (W ) caused stem cell abnormalities (Russell, 1979). Investigations by several groups showed that the W locus encoded the protooncogene c-kit, a transmembrane tyrosine kinase receptor, while the product of the Sl locus was the ligand for c-kit (Yarden et al., 1987; Geissler et al., 1988; Chabot et al., 1988). This protein ligand was isolated independently by several groups, and the

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

protein was termed stem cell factor (SCF), mast cell growth factor (MGF) and c-kit ligand (KL) (Zsebo et al., 1990; Anderson et al., 1990; Huang et al., 1990; Williams et al., 1990). SCF, which is produced by stromal cells, occurs both as a soluble protein as well as a trans-membrane protein (Anderson et al., 1990; Toksoz et al., 1992). The membrane-associated version consists of 248 amino acids, and this protein may be cleaved extracellularly, thus providing the soluble version containing 165 amino acids (Toksoz et al., 1992). SCF also occurs as a 220-amino-acid protein, which results from alternative splicing (Zsebo et al., 1990) and lacks the extracellular cleavage site resulting in a membrane-bound form of SCF. While the biological function of the membrane associated form of SCF is poorly understood, the biology of the soluble version has been studied extensively.

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IN VITRO BIOLOGY SCF binds to its receptor, c-kit, which is a tyrosine kinase receptor protein, however, the mechanism of action is not clearly understood. SCF alone has little cellular effect, however, it synergizes with a number of other growth factors to stimulate a range of cell types (McNiece et al., 1991; Williams et al., 1992; Bernstein et al., 1991; Ulich et al., 1991). Some investigators have proposed a sequential model of activation with multiple growth factors. SCF has also been hypothesized to increase receptor expression of other growth factors. Alternatively, there may be a receptor interaction on signaling pathways that may induce unique signaling events not available to either growth factor alone. SCF alone has very limited activity on human bone marrow (BM) or peripheral blood (PB) cell cultures; however, SCF may stimulate the formation of neutrophil colonies from murine BM in agar-based cultures (McNiece et al., 1991). By contrast, mast cells are generated in liquid-based murine BM cultures. When SCF is combined with other growth factors, such as granulocyte–macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and interleukin-3 (IL-3), synergistic effects occur based on the colony number as well as the number of cells contained in each colony (Bernstein et al., 1991; Williams et al., 1992). SCF has minor effects on lineage differentiation, but all lineages of hematopoietic development are influenced by SCF. SCF and EPO stimulate the formation of burst-forming unit erythroid (BFU-E)derived colonies (McNiece et al., 1991), while SCF and thrombopoietin (TPO) enhance the development and number of megakaryocyte colony-forming cell (MKCFC)-derived colonies (Hunt et al., 1994). SCF potentiates the formation of granulocyte–macrophage colony-forming cell (GM-CFC)-derived colonies when combined with IL-3, GM-CSF, granulocyte colonystimulating factor (G-CSF) and/or colony-stimulating factor-1 (CSF-1) (McNiece et al., 1991). In addition, when combined with interleukin-7 (IL-7), SCF stimulates the proliferation of B-cell and T-cell precursors (McNiece et al., 1991; Namen et al., 1991). Since SCF alone possesses minimal stimulatory effect, second messaging pathways may be active. Many models for this observation exist. SCF might induce increased receptor expression for the other growth factor; however, sequential growth factor addition experiments

do not support this hypothesis. Some investigators suggest that the proliferation of growth factor responsive cells are expanded, thus producing the synergistic effect, while others suggest unique signaling pathways not induced by single growth factor stimulation. Overall, most combinations of growth factors, which include SCF, produce optimal expansion of colony-forming cells. Since the discovery and cloning of SCF, there have been numerous reports that show SCF, in combination with other growth factors, expands both primitive and mature progenitor cells, as well as differentiated cells. In murine liquid cultures of purified BM cells (lin/sca-1), SCF has been reported to synergize with IL-6 to generate the stimulation of primitive progenitor cells, or high proliferative potential colony-forming cells (HPP-CFC) (Williams et al., 1992). When SCF was cultured in combination with IL-3 under similar conditions, the stimulation of mature progenitor cells, or GM-CFC, was observed (Williams et al, 1992). Similar reports using human BM, peripheral blood progenitor cells (PBPC) or CD34-purified umbilical cord blood (UBC) show that SCF, when combined with other growth factors, provides optimal conditions for the expansion of both progenitor and differentiated cells (Shieh et al., 1994). During the past decade, many investigators have described the ability of SCF to synergize with other growth factors in in vitro settings. Therefore, there exists the potential of SCF to expand BM or PBPC in cultures for clinical uses such as the subjugation of neutropenia and thrombocytopenia. However, the clinical development of in vitro-manipulated BM or PBPC grafts involves many regulatory issues. Because of this hurdle, much of the investigative work in this area has stalled at the level of bench science. Since 1995, clinical studies have been initiated and performed at limited sites around the world. Reiffers and colleagues (1999) have recently described in Lancet the ability of ex vivo-expanded CD34 PBPC to nearly abrogate post-chemotherapy-induced neutropenia in breast cancer patients. Similar findings have been reported by McNiece and colleagues (2000). These studies used SCF in combination with megakaryocyte growth and development factor (MGDF) and G-CSF to promote the expansion of CD34 PBPC in teflon bags for 10 to 14 days in defined media. A question exists whether some of the expanded cells still possess

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the qualities of stem cells, since most of the expanded cells have differentiated. These differentiated cells are thought to be responsible for short- and mediumterm engraftment. Previous studies have used unmanipulated as well as expanded cells for transplant. In studies performed at the University of Colorado, McNiece and colleagues (2000) have demonstrated that patients who received only expanded cells show no signs of graft failure 3 years post transplant. This finding proposes that there may exist primitive cells within the expanded graft which are still capable of long-term reconstitution as well as the differentiated cells capable of short- and medium-term engraftment. Alternatively, endogenous stem cells that were resistant to high-dose chemotherapy, may provide the long-term blood cell production. In recent years a great deal of interest has focused on the use of umbilical cord blood (UCB) as a source of hematopoietic cells for patients receiving highdose chemotherapy. UCB has been used as alternate graft source for patients who cannot find suitable donors. In addition, UCB are potentially more immunologically naive than adult BM or PBPC. In 1989, Gluckman et al. demonstrated the ability of UCB cells to engraft successfully a child with Fanconi’s anemia. This important work provided the stimulus for many investigations into the potential uses of UCB. A major problem associated with UCB transplantation is the limited number of cells available per UCB unit. For successful transplantation, cellular products need to contain approximately 2 106 CD34 cells per kilogram of body weight. Previously, therefore, most UCB transplants have been performed on younger individuals with lower body weights. The use of cellular expansion protocols for UCB tissue would allow this source of hematopoietic cells to be useful in transplantation settings involving adults. Recently, Shpall and colleagues (2000) have described studies using CD34 UCB in expansion protocols involving adults. These studies have demonstrated successful expansion of CD34 UCB cells in similar conditions as CD34 PBPC. However, the total cellular output from single UCB has been less than optimal resulting in engraftment delays as compared with PBPC expansion protocols. This was most likely due to low seeding densities of the CD34 UCB

in the expansion cultures, and culture periods too short for optimal expansion of short- and mediumterm engrafting cells. Because of this observation, other methods have been employed such as higher seeding densities and smaller expansion volumes. In addition, cultured hematopoietic cells seem to require the in vivo administration of G-CSF for optimal cellular engraftment and recovery of neutrophils. Andrews and colleagues (1999) reported this information from studies involving CD34 PBPC expansion and transplantation experiments in baboons. These studies suggested that optimal engraftment of expanded cells and recovery of neutrophils was facilitated by the in vivo administration of G-CSF post transplant. Many other studies have been initiated and reported by various groups using differing culture conditions as well as cellular sources. To date, there has been no concensus for the optimal culture and differentiation of hematopoietic cells used in clinical transplant settings.

IN VIVO BIOLOGY The biological effects of SCF have been studied in rodents, dogs, primates and humans. In all of the species, SCF was found to stimulate a broad range of biological responses in hematopoietic tissues. Doses of more than 100 lg kg1 day1 of SCF are required to elicit significant biological responses in healthy animals. The use of pegylated SCF, or polyethylene glycol-modified SCF, has been shown to increase the serum half-life in addition to greater biological activity. Acute and repeat-dose i.v. studies of SCF have been conducted in cynomolgus monkeys and baboons. In these studies, severe anaphylactoid reactions occurred when doses of SCF 40 lg kg1 (baboons) or 6 mg kg1 (monkeys) were administered by rapid i.v. injection. No such reactions were observed in primates administered any dose of s.c. SCF in toxicology studies. A subsequent study in baboons indicated that these systemic allergic reactions could be prevented by prophylactic administration of H1 and H2 antihistamines along with either a corticosteroid or an inhaled bronchodilator.

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CLINICAL INDICATIONS SCF is indicated for use in combination with G-CSF, in the setting of autologous PBPC transplantation, for patients at risk of poor PBPC mobilization to increase the number of PBPC collected in the apheresis harvest, thus increasing the proportion of patients reaching a PBPC target for transplantation.

DESCRIPTION SCF was approved for sale by the respective regulatory boards in Australia, Canada and New Zealand in 1999. Ancestim is a human stem cell factor produced by recombinant DNA technology. STEMGEN is the Amgen Inc. trademark for ancestim (recombinant methionyl human stem cell factor, r-metHuSCF). STEMGEN is a 166-amino-acid protein produced by Escherichia coli bacteria into which a gene has been inserted for soluble human stem cell factor. SCF has a monomeric molecular weight of approximately 18 500 daltons and normally exists as a noncovalently associated dimer. The protein has an amino-acid sequence that is identical to the natural sequence predicted from human DNA sequence analysis, except for the addition of an N-terminal methionine retained after expression in E. coli. Because SCF is produced in E. coli, the product is non-glycosylated. SCF is provided as a sterile, white, preservative-free, lyophilized powder for reconstitution and administration as a s.c. injection. Each single-use vial of SCF contains 1.875 mg of ancestim at a specific activity of 0.84 to 1.8 106 U mg1 (as measured by a cell mitogenesis assay). Vials of SCF are reconstituted with 1.2 mL of sterile water for injection to yield a concentration of 1500 lg mL1. Reconstituted SCF is a sterile aqueous solution containing 4.5% mannitol and 0.5% sucrose buffered at pH 6.0 with 5 mM glutamic acid and 10 mM histidine.

CLINICAL EXPERIENCE In phase I/II studies involving 367 patients with breast cancer, non-Hodgkin’s lymphoma and ovarian cancer, SCF administration over a dose range of 5 to 25 lg kg1 day1, in combination with a fixed dose of G-CSF,

resulted in a dose-dependent increase in circulating PBPC, including CD34 cells, GM-CFC and BFU-E, compared with G-CSF alone. SCF alone did not cause substantial PBPC mobilization at the only dose tested (5 lg kg1 day1). For patients receiving the cytokine combination, this increase in circulating PBPC resulted in apheresis yields that were approximately two- to three-fold greater than those of patients receiving G-CSF alone. With discontinuation of SCF plus G-CSF therapy, PBPC levels returned to baseline, in most cases within 4 to 7 days. In a 219-patient phase I/II breast cancer study which utilized three fixed aphereses, 8% of patients treated with 20 lg kg1 day1 SCF plus G-CSF at 10 lg kg1 day1 and 17% of patients treated with G-CSF alone, failed to reach a minimal number of CD34 cells (1 106 kg1) (Moskowitz et al., 1997). In the phase III controlled clinical trial, 205 breast cancer patients were randomized to receive either 20 lg kg1 day1 SCF in combination with 10 lg kg1 day1 G-CSF (n  101) or 10 lg kg1 day1 G-CSF alone (n  104). On day 5 of cytokine administration, apheresis was initiated. Apheresis and cytokine administration were continued daily until the target of 5 106 CD34 cells kg1 (actual body weight) had been collected, up to a maximum of 5 aphereses. This target was based on results from a preceding phase I/II study in a comparable patient population which indicated that infusion of 5 106 CD34 cells/kg was correlated with a high probability of rapid platelet recovery (by day 14) and a low probability of delayed platelet recovery ( 28 days) (Glaspy et al., 1996). Treatment with SCF plus G-CSF caused a sustained mobilization of PBPC resulting in greater CD34 cell collections and a greater proportion of patients reaching the target yield of 5 106 CD34 cells kg1 (63%) compared with treatment with G-CSF alone (47%). This improved CD34 cell mobilization provided a clinically and statistically significant reduction (P  0.038) in the number of aphereses required to collect the target number of PBPC. Patients receiving SCF plus G-CSF reached the target number of PBPC in a median of four apheresis procedures, while patients receiving G-CSF alone required a median of greater than or equal to six aphereses (i.e. less than 50% of G-CSF-treated patients reached the collection target in five aphereses) (Glaspy et al., 1997). As expected for patients apheresed to the same

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target cell yield, the treatment groups showed similar hematopoietic recovery following high-dose chemotherapy and infusion of the collected PBPC with G-CSF support (10 lg kg1 day1). The proportion of patients that failed to reach a minimal number of CD34 cells (1 106 kg1) to allow high-dose chemotherapy with PBPC support (mobilization failures) or experienced a delayed rise in platelets to 20 109 L1 ( 28 days) (engraftment failures), was lower for patients receiving SCF plus G-CSF, compared with those receiving G-CSF alone (3.5% versus 7.4%) (Glaspy et al., 1997). Several additional randomized, controlled studies also support the efficacy of SCF. An analysis across all PBPC studies, including cytokine alone and chemotherapy plus cytokine mobilization, showed that there was a highly significant improvement (P  0.0001) in each of the efficacy parameters, i.e. (1) apheresis yields of CD34 cells, (2) numbers of patients with apheresis harvests 5 106 CD34 cells/kg and (3) number of aphereses required to collect 5 106 CD34 cells/kg for patients receiving SCF plus G-CSF compared with patients receiving G-CSF alone (Glaspy et al., 1997).

SUMMARY SCF has been shown to be an essential growth factor in development and maintenance of normal blood cell production. It synergizes with other hematopoietic growth factors to stimulate stem cells, primitive progenitor cells and committed progenitor cells. Its clinical utility has been minimized by dose-limiting anaphylactoid reactions; however, clinical studies have demonstrated that in combination with G-CSF, SCF can stimulate mobilization of increased numbers of CD34 cells in PBPC products. If predictors of poor mobilization can be developed, SCF may be useful for these patients to improve PBPC harvests. SCF has been shown to be an essential growth factor for ex vivo expansion of hematopoietic cells. There is a need for more studies to determine the optimal culture conditions and patients who can benefit from ex vivo-expanded cells. However, with the recent demonstration of plasticity of hematopoietic stem cells, generating cells of various tissues including, cardiac, neural, liver, lung and endothelial cells, SCF may

be a key growth factor for ex vivo manipulation of hematopoietic stem cells to produce regenerative tissues for various diseases.

REFERENCES Anderson, D.M., Lyman, S.D., Baird, A. et al. (1990). Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63, 225–233. Andrews, R.G., Briddell, R.A., Hill, R. et al. (1999). Engraftment of primates with G-CSF mobilized peripheral blood CD34 progenitor cells expanded in G-CSF, SCF and MGDF decreases the duration and severity of neutropenia. Stem Cells 17, 210–218. Bernstein, I.D., Andrews, R.G. and Zsebo, K.M. (1991). Recombinant human stem cell factor enhances the formation of colonies by CD34 lin cells, and the generation of colony forming cell progeny from CD34 lin cells cultured with interleukin-3, granulocyte-colony stimulating factor, or granulocyte–macrophage colony stimulating factor. Blood 77, 2316–2321. Chabot, B., Stephenson, D.A., Chapman, V.M. et al. (1988). The protooncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the W locus. Nature 335, 88. Geissler, E.N., Ryan, M.A. and Housman, D.E. (1988). The dominant-white spotting (W ) locus of the mouse encodes the c-kit protooncogene. Cell 55, 185–196. Glaspy, J., Davis, M.W., Parker, W.R. et al. (1996). Biology and clinical potential of stem cell factor. Cancer Chemother. Pharmacol. 38 Suppl, 553–557. Glaspy, J.A., Shpall, E.J., LeMaistre, C.F. et al. (1997). Peripheral blood progenitor cell mobilization using stem cell factor in combination with filgrastim in breast cancer patients. Blood 90, 2939–2951. Gluckman, E., Broxmeyer, H.A., Auerbach, A.D. et al. (1989). Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood transplant from an HLA-identical sibling. N. Engl J. Med. 321, 1174–1178. Huang, E., Nocka, K., Beier, D.R. et al. (1990). The hematopoietic growth factor KL is encoded by the SI locus and is the ligand for the c-kit receptor, the product of the W locus. Cell 63, 226–234. Hunt, P., Bartley, T., Li, Y-S. et al. (1994). Purification and cloning of a megakaryocyte growth and development factor: a novel cytokine found in aplastic plasma. Exp. Hematol. 22, 838. McNiece, I.K., Langley, K.E. and Zsebo, K.M. (1991a). Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3, and Epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp. Hematol. 19, 226–231. McNiece, I.K., Langley, K.E. and Zsebo, K.M. (1991b). The role of recombinant stem cell factor in early B cell development: synergistic interaction with IL-7. J. Immunol. 146, 3785–3790. McNiece, I., Jones, R., Bearman, S.I. et al. (2000). Ex vivo expanded peripheral blood progenitor cells provide rapid

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neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 96, 3001–3007. Moskowitz, C.H., Stiff, P., Gordon, M.S. et al. (1997). Recombinant methionyl human stem cell factor and filgrastim for peripheral blood progenitor cell mobilization and transplantation in non-Hodgkin’s lymphoma patients of a phase I/II trial. Blood 89, 3136–3147. Namen, A.E., Widmar, M.B., Voice, R. et al. (1991). A ligand for the c-kit proto-oncogene (MGF) stimulates lymphoid progenitor cells in vitro. Exp. Hematol. 19, 497. Reiffers, J., Cailliot, C., Dazey, B. et al. (1999). Abrogation of post-myeloablative chemotherapy neutropenia by exvivo expanded autologous CD34-positive cells. Lancet 354, 1092. Russell, E.S. (1979). Hereditary anemias of the mouse: a review for geneticists. Adv. Genet. 20, 357–364. Sheih, J.-H., Chen, Y.-F., Briddell, R. et al. (1994). High purity of blast cells in CD34 selected populations are essential for optimal ex vivo expansion of human GM-CFC. Exp. Hematol. 22, 756. Shpall, E.J., Quinones, R., Jones, R. et al. (2000). Transplantation of adult and pediatric cancer patients with cord blood progenitors expanded ex vivo. Blood 96, 207a.

Toksoz, D., Zsebo, K.M., Smith, K.A. et al. (1992). Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane bound and secreted forms of the human homolog of the steel gene product, stem cell factor. Proc. Natl Acad. Sci. USA 89, 7350–7354. Ulich, T.R., Delcastillo, J., Yi, E.S. et al. (1991). Hematologic effects of stem cell factor in vivo and in vitro in rodents. Blood 78, 1954–1962. Williams, D.E., Eisenman, J., Baird, A. et al. (1990). Identification of a ligand for the c-kit proto-oncogene. Cell 63, 213–224. Williams, N., Bertoncello, I., Kavnoudias, H. et al. (1992). Recombinant rat stem cell factor stimulates the amplification and differentiation of fractionated mouse stem cell populations Blood 79, 58–64. Yarden, Y., Kuang, W. and Yeng-Feng, T. (1987). Human protooncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 6, 3341. Zsebo, K.M., Williams, D.A., Geissler, E.N. et al. (1990). Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213.

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C

H

A

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T

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44 Vascular endothelial growth factor Dmitry I. Gabrilovich1 and Mikhail M. Dikov 2 1

University of South Florida, Tampa, FL, USA 2

Vanderbilt University, Nashville, TN, USA

It is queer how it is always one’s virtues and not one’s vices that precipitate one into disaster. Rebecca West, There is no conversation

INTRODUCTION Organ development and cell growth is dependent on adequate vascular supply. Adequate angiogenesis is a fundamental requirement for normal functioning of the organism. On the other side of the same coin, angiogenesis plays a critical role in many pathological conditions. Tumor cells need vascular supply to support their growth. These cells use angiogenesis to eventually kill organisms. Pathological angiogenesis is one of the important manifestations of diabetes, psoriasis, rheumatoid arthritis and other pathological conditions (Folkman, 1995). Angiogenesis is regulated by a complex network of different cytokines and growth factors. Vascular endothelial growth factor (VEGF) plays a central role in this network. VEGF was discovered in the late 1980s through the efforts of two investigators: Dvorak from Harvard University and Ferrara from Genentech (South San Francisco, CA, USA). Dvorak was interested in the mechanisms of

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

increased vascular permeability observed in tumorbearing hosts. He suggested that tumor produced vascular permeability factor (VPF) and set up an in vivo experimental system to investigate this putative factor. Ferrara was interested in tumor-derived factors able to stimulate proliferation of endothelial cells. He used an in vitro experimental system and was able to identify and characterize the protein named VEGF (Leung et al., 1989b). When later Dvorak and his team sequenced their protein it was found to be identical to VEGF (Senger et al., 1990). For several years in the early 1990s, this protein was called in scientific literature VEGF/VPF. Later, the single name ‘VEGF’ was adopted for this growth factor. After 10 years of intensive studiesVEGF firmly occupies a prominent place as one of the most important angiogenic factors. Its significance is extended well beyond the vascular system. In this chapter we will review recent data on the structure and function of this growth factor, as well as possible clinical applications of VEGF and its antagonists.

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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VEGF STRUCTURE The family of VEGF-related molecules contains presently five mammalian members: VEGF-A (VEGF), VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PlGF). The viral homologues, collectively called VEGF-E, are encoded by different strains of the Orf virus (Veikkola and Alitalo, 1999). Within the superfamily, VEGF shares 20% amino acid sequence identity with platelet-derived growth factor (PDGF) and belongs to the PDGF family, in which the monomers are held in a side-by-side orientation, the two b-sheets lying perpendicular to the two-fold symmetry axis. By their protein structure all VEGF family members belong to the dimeric cystein-knot growth factor superfamily and possess a homology domain containing eight conserved cysteine residues. X-ray crystallography of VEGF fragments (aa 8–109) showed that each monomer is characterized by an intrachain disulphide bonded knot motif at one end of a fourstranded b-sheet (McDonald and Hendrickson, 1993; Murray-Rust et al., 1993; Sun and Davies, 1995; Muller et al., 1997). The four VEGF-related genes generate glycosylated dimers, which are secreted after cleavage of their signal peptides. They encode eight cysteine residues that are highly conservative within the VEGF and PDGF family (Plate 44.1) (see Plate section).

VEGF-A (VEGF) The human VEGF gene resides on chromosome 6p21.3 (Vincenti et al., 1996). The coding region spans about 14 kb and contains eight exons (Houck et al., 1991; Tischer et al., 1991). Alternative splicing of human VEGF mRNA from a single gene gives rise to at least six different isoforms of 121, 145, 165, 183, 189 and 206 amino acid residues (Neufeld et al., 1996; Poltorak et al., 1997; Robinson and Stringer, 2001). Exon 6 is absent in VEGF121 and VEGF165 and exon 7 is absent from VEGF121 and VEGF145. VEGF121, VEGF145 and VEGF165 are secreted and form dimeric proteins, whereas VEGF189 and VEGF206 are thought to remain sequestered in the pericellular matrix upon their secretion. VEGF189 was reported to undergo proteolytic cleavage by urokinase to generate a smaller biologically active isoform, which could bind to VEGF receptor 2 (VEGF-R2) and stimulate proliferation of endothelial cells (Plouet et al., 1997). All isoforms,

except VEGF121, bind heparin to various degrees via a region rich in charged basic amino acid residues encoded by the exon 6 and exon 7-encoded domain (Neufeld et al., 1996). Human VEGF165 is glycosylated at Asn 75 and is typically expressed as a 46-kDa homodimer of 23-kDa monomers. It is the most abundant and most biologically active form of VEGF (Neufeld et al., 1996, 1999). All isoforms of VEGF are secreted as a covalently linked homodimers. Monomers associate initially through hydrophobic interactions and are then stabilized by disulfide bonding between Cys 51 of one chain and Cys 61 of the other (Keyt et al., 1996). The signal peptide (exon 1 and four residues of exon 2), which includes amphipathic a-helix (aa 12–19) essential for dimerization, is cleaved off during secretion (Keck et al., 1989; Leung et al., 1989a; Siemeister et al., 1998). A potential glycosylation site exists at Asn 74 that appears to have no effect on VEGF function, but is required for efficient secretion (Peretz et al., 1992; Claffey et al., 1995). Site-directed mutagenesis identified three acidic residues (Asp 63, Glu 64 and Glu 67) in exon 3 and three basic residues (Arg 82, Lys 84 and His 86) in exon 4 essential for binding to VEGF receptor 1 (VEGF-R1) and VEGF-R2, respectively. Three highly flexible loops are clustered at each pole of VEGF at the dimer interface. Loop II contains VEGFR1 binding determinants and lies close to loop III of the opposing monomer, which binds to VEGF-R2 (Keyt et al., 1996). The positioning of these receptorbinding interfaces at each pole of VEGF seems to facilitate receptor dimerization, which is essential for transphosphorylation and signaling (Siemeister et al., 1998). The structure of the VEGF165 heparin-binding region (aa 111–165) has been resolved separately by NMR and represents a novel type of heparin-binding domain (Fairbrother et al., 1998). VEGF165 binds VEGFR1 and VEGF-R2 with Kd values of 10–20 and 75–125 pM, respectively (de Vries et al., 1992; Terman et al., 1992a). However, other studies reported a rather broad range of binding constants for both receptors (see Table 44.2). It also binds with high affinity to neuropilin-1 (Soker et al., 1998).

VEGF-B Two mRNA splice variants are generated from the VEGF-B gene, which is located on human chromo-

THE CYTOKINES AND CHEMOKINES

VEGF STRUCTURE

some 11q13 (Grimmond et al., 1996; Paavonen et al., 1996; Olofsson et al., 1996). The gene contains seven exons. The coding sequence of the first five exons is incorporated into both splice forms. Alternative splicing results in use of different, but overlapping reading frames in exon 6. Consequently the two isoforms of the polypeptide share the same 115 amino-terminal amino acid residues but have distinct carboxyl termini (Olofsson et al., 1996). After the 21 amino acid signal sequence has been cleaved off, the two polypeptides have 167 (VEGF-B167) and 186 (VEGF-B186) amino acids in length (Olofsson et al., 1996; Grimmond et al., 1996; Townson et al., 1996). The apparent molecular masses of the secreted homodimers of VEGF-B167 and VEGF-B186 are 42 kDa and 60 kDa, respectively. The amino acid sequences of VEGF-B167 and VEGF165 are ~44% identical and their intermolecular disulfide bridging patterns are similar. Exon 6B of VEGF-B167 is homologous to exon 7 of VEGF165; both encode protein sequences rich in basic amino acid residues, which after secretion bind the growth factor to cellsurface heparan sulfate proteoglycans. In contrast, the carboxy-terminal domain of VEGF-B186 is hydrophobic and contains many serine, threonine and proline residues. VEGF-B167 and VEGF-B186 also differ in their glycosylation pattern. VEGF-B167 is not glycosylated, whereas VEGF-B186 contains O-linked glycans. Furthermore, VEGF-B186 is proteolytically processed at Arg 127, giving rise to a 34 kDa dimer (Olofsson et al., 1996). The role of VEGF-B in angiogenesis is not clear. VEGF-B knockout mice are healthy and fertile, but have smaller hearts and exhibited vascular dysfunction after coronary occlusion and impaired recovery following experimental cardiac ischaemia (Olofsson et al., 1999; Bellomo et al., 2000). VEGF-B has been reported to induce expression and activity of urokinase type plasminogen activator (Olofsson et al., 1998).

VEGF-C and VEGF-D The human gene for VEGF-C spans over 40 kb of DNA and consist of seven exons. It has been localized to chromosome 4q34 (Paavonen et al., 1996; Olofsson et al., 1996; Chilov et al., 1997). The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Within the VEGF family of growth factors, VEGF-C and its closest relative, VEGF-D, constitute a subgroup,

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which is characterized by the presence of unique amino- and carboxyl-terminal extensions flanking the common VEGF-homology domain (Joukov et al., 1996; Lee et al., 1996; Achen et al., 1998). The central core (the VEGF homology domain) of VEGF-C exhibits ~30% identity to VEGF-A (Joukov et al., 1996). The VEGF homology domains of VEGF-C and VEGF-D are 61% identical. VEGF-C is synthesized as a precursor protein, which undergoes subsequent proteolytic processing similar to that of PDGF-A and-B chain processing, suggesting an evolutionary relationship (Tischer et al., 1991; Heldin et al., 1993; Joukov et al., 1996, 1997). The carboxy-terminal domain is cleaved upon secretion, but remains bound to the aminoterminal domain by disulfide bonds giving rise to a disulfide-linked tetramer composed of 29 and 31 kDa polypeptides. Proteolytic processing of the aminoterminal pro-peptide releases the mature form, which consists of two 21 kDa polypeptide chains corresponding to the VEGF homology domain (Joukov et al., 1997). Transfection assays suggested that VEGF-C forms disulfide-linked dimers and can activate both the VEGF-R3/FLT4 and VEGF-R2/KDR/FLK1 receptor tyrosine kinases (Joukov et al., 1996). Lee et al. (1996) used competitive binding of purified components to show that VEGF-C and VEGF-R3 bind with a high affinity, suggesting that VEGF-C is a biologically relevant ligand of VEGF-R3 (Lee et al., 1996). VEGF-D is structurally related to VEGF-C. The human VEGF-D gene was mapped to human chromosome Xp22.31. VEGF-D is expressed as a 2.2-kb transcript with highest expression in lung, heart, small intestine and fetal lung, and lower levels in skeletal muscle, colon and pancreas. The protein also contains eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. The human cDNA encodes a protein of 354 amino acid residues while the mouse cDNA encodes 358 amino acid residues. Similar to VEGF-C, VEGF-D is synthesized as a pro-protein requiring proteolytic processing in both the N- and C-terminal regions for activity, and the fully processed growth factor is a non-covalent dimer (Li and Eriksson, 2001). Analysis of VEGF-D receptor specificity revealed that VEGF-D is a ligand for VEGF receptors VEGF-R2 and VEGF-R3, but not VEGF-R1.

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At this time the role of VEGF-C and VEGF-D is not well understood. The major function of VEGF-C may be as a growth factor for lymphatic vessels (Kukk et al., 1996; Jeltsch et al., 1997). Transgenic mice overexpressing VEGF-C in keratinocytes develop enlarged lymphatic vessels in their skin, but mice overexpressing VEGF164 in the same location showed only blood vessel hyperplasia (Jeltsch et al., 1997).

PLGF Three PlGF isoforms are produced by alternative splicing from a single copy gene on chromosome 14 (Maglione et al., 1993). Mattei et al. (1996) used radioactive chromosomal in situ hybridization to map the PlGF gene to 14q24-q31. The protein shares 53% identity with VEGF. A role for PlGF in placental angiogenesis has been proposed (Torry et al., 1999), but PlGF homodimers appear to have little effect on angiogenesis in vivo. However, VEGF/PlGF heterodimer binds to VEGF-R2 and is mitogenic for endothelial cells, and stimulates angiogenesis in vivo (Cao et al., 1996). PlGF-2 differs from the other two PlGF forms as it is the only form containing the 21 basic amino acids encoded by exon 6. These 21 amino acids are responsible for PlGF-2 heparin binding ability (Hauser and Weich, 1993). PlGF-1 and PlGF-2 do not interact with VEGF-R2, but are able to bind to VEGF-R1 (Park et al., 1994). PlGF-2 also binds to neuropilin-1 (NP-1), while

PlGF-1 does not. The binding is strongly affected by heparin and is apparently mediated via a binding site which includes peptide encoded by exon 6 and 7 of PlGF (Migdal et al., 1998).

VEGF-E VEGF-E is a group of proteins with significant homology to VEGF that are encoded by certain strains of orf virus, an epitheliotropic parapoxvirus, which induces proliferative lesions in sheep, goats and rarely humans (Lyttle et al., 1994; Haig and Mercer, 1998; Meyer et al., 1999). All known VEGF-E variants bind VEGF-R2, but not VEGF-R1 or VEGF-R3 (Ogawa et al., 1998; Meyer et al., 1999; Robinson and Stringer, 2001). One variant, NZ2–VEGF-E, was shown to bind neuropilin-1. These viral proteins seem to be as potent as VEGF165 at stimulating endothelial cell proliferation despite lacking heparin-binding ability. Biochemical properties of VEGF are summarized in Table 44.1.

VEGF promoter region The promoter region of VEGF gene contains a single major transcription start site near to a cluster of potential Sp1 factor binding sites. The promoter also contains several AP-1 or AP-2 binding sites (Tischer et al., 1991; Ortega et al., 1998; Robinson and Stringer, 2001). Hypoxia response element (HRE) works

TABLE 44.1 Biochemical properties of VEGF family members

Isotype length, amino acids Gene map locus Homology to VEGF, % Receptor specificity Heterodimers Heparin binding

VEGF

VEGF-B

VEGF-C

VEGF-D

PlGF

VEGF-E

121, 145 165, 183 189, 206 6q12.3 100

167, 186

388

333

131, 152 183

132

11q13 45

4q34 30a

Xp22.31

14q24–31 46

VEGFR1 NP-1

VEGFR2 VEGFR3

VEGFR2 VEGFR3

VEGFR1 NP-1 (PlGF-2)

VEGF165

ND

ND

VEGF-B167 yes VEGF-B186 no

ND

no

VEGF121 VEGF165 no

VEGFR1 VEGFR2 NP-1 (VEGF165) PlGF-1 PlGF-2 VEGF121 yes Others no

a Homology within VEGF-homology region. ND, not determined.

THE CYTOKINES AND CHEMOKINES

VEGFR2 NP-1 (VEGFENZ2

PlGF-1 no PlGF-2 yes

VEGF RECEPTORS

upstream of the VEGF gene as an enhancer (Levy et al., 1995; Liu et al., 1995). This HRE contains a consensus binding site for hypoxia-inducible factor 1 (HIF-1), a heterodimer of the bHLH-type transcription factors HIF-1a and ARNT (Madan and Curtin, 1993; Forsythe et al., 1996). The von Hipple-Lindau tumor suppressor (VHL) negatively regulates hypoxia-inducible genes, including VEGF. The sequence within 3 untranslated region contains several consensus structures known to be involved in the regulation of mRNA stability.

VEGF RECEPTORS The biological effects of VEGFs are mediated via three specific cell surface receptors. All three belong to the class III receptor tyrosine kinases of platelet-derived growth factor subfamily and consist of an extracellular domains comprising seven Ig-like domains, transmembrane domain, followed by kinase domain that is divided in two parts by the insertion of a non-catalytic 100-amino-acid sequence and a C-terminal tail. VEGF-R1 and VEGF-R2 are mainly expressed on endothelial cells, although other cell types including hematopoietic cells can also express these receptors (Shibuya et al., 1999; Petrova et al., 1999). Human VEGF-R1 was first cloned from placental cDNA library (Shibuya et al., 1990); mouse and rat homologues were subsequently identified (Finnerty et al., 1993; Yamane et al., 1994; Choi et al., 1994). VEGF-R2 was isolated from human endothelial cell cDNA library (Terman et al. 1991, 1992a). VEGF-R3 was cloned from human erythroleukemia cells and placental cDNA libraries (Galland et al., 1992; Aprelikova et al., 1992). Human genes encoding VEGF-R1, VEGF-R2 and VEGF-R3 are located in chromosomal regions 13q12-q13, 4q11-q13 and 5q33-q35, respectively (Terman et al., 1992a, 1992b; Galland et al., 1992; Aprelikova et al., 1992; Rosnet et al., 1993b; Spritz et al., 1994). Mouse homologues for VEGF-R1 and VEGF-R2 have been mapped to chromosome 5 (Matthews et al., 1991; Rosnet et al., 1993a) and VEGFR3 to chromosome 11 (Watkins-Chow et al., 1997). All VEGF receptors are structurally similar to the receptors of Fms family: Fms, Kit and Flt3/Flk2. These receptors contain tyrosine kinases with five or seven immunoglobulin (Ig) domains in their extracellular

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region. Fms, Kit and Flt3/Flk2 receptors essential for hematopoiesis have five Ig domains, whereas sevenIg-type receptors, VEGFR-1, -2 and -3, are closely involved in the formation of tube-like structures, blood vessels and lymphatic vessels. Although the overall structure of the exon–intron organization is similar, several clear differences exist between the 5 and 7-Ig-type receptor genes (Kondo et al., 1998). Each of the first and second Ig-like domains in VEGFR1 is split into two exons, whereas these two domains in Fms family genes are fused to two single exons. Additional exons corresponding to the sixth and seventh Ig-like domains are present in VEGFR-1. The localization of the exon encoding the kinase insert region is slightly but definitely different between the VEGFR-1 gene and Fms family genes. Tyr-X-X-Met motif, conserved in essentially all of the Fms family members and important for binding of PI3 kinase subunit and activation of PI3 kinase pathway (Songyang et al., 1993), is not present in any form of VEGF-R1 in mammals (Shibuya, 2001b). Wiesmann et al. (1997) reported the results of domain deletion studies of the extracellular portion of VEGF-R1, as well as the crystal structure to 1.7–angstrom resolution of the complex between the receptor-binding domain of VEGF and VEGF-R1 domain 2. They showed that VEGF-R1 domains 2 and 3 are necessary and sufficient for binding VEGF. Human PDGFRA, c-kit and VEGF-R2 constitute a cluster of genes at 4q12 chromosome encoding closely related type III receptor tyrosine kinases (Spritz et al., 1994). The predicted amino acid sequence contained multiple characteristics (e.g. an ATP binding site, a membrane spanning region, split tyrosine kinase regions) typical of known type III receptor tyrosine kinases (e.g. platelet-derived growth factor receptor, colony-stimulating factor-1 receptor, fibroblast growth factor receptor and Kit). The VEGFR3 tyrosine kinase domain is 79% homologous with VEGF-R1 (Aprelikova et al., 1992). VEGF-R3 expression starts during E8 in developing blood vessels, but subsequently is largely confined to the lymphatic vasculature, consistent with a specific role of this receptor in lymph angiogenesis. Disruption of the VEGF-R3 gene in mice did not prevent vasculogenesis or angiogenesis, but caused defects in normal vascular development, leading to fluid accumulation from leaky vessels by E9.5 (Dumont et al., 1998).

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Neuropilin-1 (NP-1) was recently identified as a new receptor for VEGF (Soker et al., 1998). NP-1 is a non-tyrosine kinase transmembrane receptor with a short cytoplasmic tail and a large extracellular domain (Petrova et al., 1999). It has been earlier identified as a receptor for the semaphorin/collapsin family of polypeptides implicated in axonal guidance (Kitsukawa et al., 1997). Previous work had pointed to a role for NP-1 in cardiovascular development. Overexpression of NP-1 in mice results in diverse vascular abnormalities, including excess capillaries and blood vessels, and in malformation of the heart, while NP-1 knockout mice display impaired neural vascularization, defects in the aorta and other large blood vessels, and aberrant yolk sac vascularization (Kawasaki et al., 1999). In the human fetal heart, NP-1 is expressed in the endocardium, coronary vessels, myocardial capillaries and epicardial blood vessels and is coexpressed with VEGF-R1 and VEGF-R2 in the endocardium and myocardial capillaries, but only with VEGF-R1 in coronary vessels (Partanen et al., 1999). VEGF165, but not VEGF121, was shown to bind to NP-1 with an affinity similar to that for VEGF-R2 (Table 44.2), and coexpression of NP-1 with VEGF-R2 increased binding of VEGF165 four-fold (Soker et al., 1998). NP-1 by itself may not be a functional receptor, but likely plays the role as a co-receptor for VEGF-R2. This may explain why the cardiovascular system fails to develop normally in NP-1/ embryos (Kitsukawa et al., 1997). NP-1 has also been shown to bind VEGF-B and -E and the PlGF-2 splice variant (Migdal et al., 1998; Petrova et al., 1999; Neufeld et al., 1999). The role of NP-1 in the functions of VEGF in vitro and in vivo is not yet known, and the mechanisms by which it acts as a co-receptor is yet to be clarified.

Heparin sulfate proteoglycans may act as an additional low-affinity binding site for VEGF able to modulate binding of VEGF to its receptors. Low concentrations of heparin have been reported to augment the binding of VEGF to human umbilical vein endothelial cell (HUVECs), while higher concentrations reduced binding (Cohen et al., 1995).

VEGF SIGNAL TRANSDUCTION Intracellular signaling of VEGF is mediated via phospholipase C (PLC) activity, change in cytosolic calcium concentration, phosphorylation of several tyrosine kinases and generation of NO and prostacyclin (Plates 44.2 and 44.3). VEGF-R1 and VEGF-R2 have distinct signal transduction properties. In addition, they form heterodimers, which substantially potentiates functions of VEGF-R2 (Huang et al., 2001).

VEGF-R1 (Flt-1) Recent data suggest that VEGF-R1 is able to mediate an array of biological signals when appropriately stimulated. The pattern of responses of PlGFstimulation of VEGF-R1 is distinct from the pattern of responses to VEGF-stimulation of VEGF-R2. Landgren et al. (1998) examined VEGF and PlGF for their ability to stimulate signal transduction in porcine aortic endothelial cells (PAEC) expressing VEGF-R1 or VEGF-R2. VEGF had essentially no effect on VEGF-R1 expressing cells, but induced DNA synthesis and migration of VEGF-R2 expressing cells. PlGF, on the other hand, induced DNA synthesis but not migration

TABLE 44.2 Biochemical properties of VEGF receptors

Alternative name Chromosomal gene locus MW, kDa (glycosylated) Kd for VEGF165, pM Homology to VEGFR1, %

VEGFR1

sVEGFR1

VEGFR2

VEGFR3

Flt-1

sFlt-1

Flt-4

13q12

13q12

KDR, human Flk1, mouse 4q12

5q35.3

10p12

180–185

110

200–230

130–140

10–114 200 for PlGF 100

10–114

100–1000

100

44

195 (chains 120 and 75) 135 for VEGF-C 79 (cDNA)

THE CYTOKINES AND CHEMOKINES

NP-1

400 None

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of the VEGF-R1 expressing PAEC. p38 MAP kinase was activated both by VEGF stimulation of the VEGF-R2 cells and by PlGF stimulation of the VEGF-R1 cells. In contrast, PLC-c was tyrosine phosphorylated only in VEGF-stimulated VEGF-R2 cells, and not in the PlGFstimulated VEGF-R1 cells. Induction of plasminogen activator (PA) was evident in the PlGF-stimulated VEGF-R1 cells, but not in the VEGF-stimulated VEGF-R2 cells. Sato et al. (2000a, 2000b) investigated properties of two VEGF receptors, VEGF-R1 and VEGF-R2, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) by using two newly developed blocking monoclonal antibodies (mAbs). VEGF stimulated the expression of transcription factor Ets-1 as well as matrix metalloproteinase-1 (MMP-1), DNA synthesis and migration of HUVECs. DNA synthesis was mediated by the same signaling system as the expression of Ets-1. In contrast, cell migration was regulated by two distinct signaling systems. The VEGF-R1 homodimer was required for actin reorganization. The VEGF-R2/VEGF-R1 heterodimer and the VEGF-R2 homodimer were required for the assembly of vinculin in focal adhesion plaques by regulating the phosphorylation of focal adhesion kinase and paxillin. Tyrosine 1169 in the VEGF-R1 kinase domain has been reported to provide a binding site for PLC-c (Sawano et al., 1997). VEGF caused activation of PLC-c, but weak activation of extracellular signalregulated kinase (ERK) in VEGF-R1-expressing NIH3T3 cells (Seetharam et al., 1995). VEGF-R1expressing PAEC showed ERK activation, but no effect on PLC-c activity in response to PlGF (Landgren et al., 1998). VEGF-R1 was found to support Ca2 influx in Xenopus laevis oocytes (de Vries et al., 1992) and has been reported to mediate Ca2 mobilization in trophoblast cells (Ahmed et al., 1997), but PlGF and VEGF-R1 did not mediate Ca2 mobilization in HUVECs (Cunningham et al., 1999). VEGF-R1 was also reported to interact with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) in a yeast two-hybrid system (Cunningham et al., 1995). However, so far this has not been associated with a biological activity in VEGF-R1-expressing cells. A recent study suggests that PlGF, but not VEGF, can stimulate ERK activation and mitogenesis, but not migration in VEGF-R1 expressing PAEC (Landgren et al., 1998). VEGF-R1 also mediated migration and increased tissue factor

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expression in monocytes (Clauss et al., 1996b; Barleon et al., 1996) although their biological role in vivo is unclear. Mice expressing only the extracellular domain of VEGF-R1 and lacking its cytoplasmic kinase domain are fertile and develop normally. Suppression of VEGF-induced migration of macrophages in vitro was the only observed phenotype in these mice (Hiratsuka et al., 1998). There is increasing evidence that VEGF-R1 may act as a negative regulator of some VEGF-R2 functions (Shibuya, 2001a; Zachary and Gliki, 2001). A soluble form of the extracellular VEGF-R1 domain, but not soluble VEGF-R2 inhibits VEGF-induced migration and proliferation of human microvascular endothelial cells and HUVECs by forming an inactive complex with VEGF and the full-length VEGF-R2 (Kendall and Thomas, 1993; Roeckl et al., 1998). A study of chimeras made between the extracellular domain of the receptor for colony-stimulating factor 1 (CSF-1) and the kinase domains of either VEGF-R1 or R2, found that activation of the VEGF-R1 kinase domain chimera with CSF-1 suppressed ERK activation and proliferation mediated via the VEGF-R2 kinase chimera (Rahimi et al., 2000). Analysis of domain swapping between VEGF-R1 and VEGF-R2 indicates that a short motif (SSS) unique to the intracellular juxtamembrane VEGF-R1 domain can suppress VEGFR2-mediated signaling and cell migration (Gille et al., 2000).

VEGF-R2 (KDR/Flk-1) VEGF-R2 is activated through ligand-stimulated receptor dimerization and autophosphorylation of tyrosine residues in the cytoplasmic kinase domain. Several major autophosphorylation sites have been identified in human VEGF-R2: Y951, Y996, Y1054, Y1059, Y1175 and Y1214 (Dougher-Vermazen et al., 1994; Takahashi et al., 2001). Like other receptor protein kinases VEGF-R2 has been shown to associate with a number of Src homology SH2 domain proteins, including the adapter proteins Grb2, Nck and Shc and the SH2 domain protein tyrosine phosphatases SHP-1 and SHP-2 (Guo et al., 1995; Kroll and Waltenberger, 1997). VEGF-R2 associate with and is regulated by the protein tyrosine phosphatase HCPTPA (Huang et al., 1999). VEGF, but not the VEGF-R1-specific ligand, PlGF, induced NO and prostacyclin (PGI2) production

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in HUVECs (Wheeler-Jones et al., 1997; Kroll and Waltenberger, 1999). VEGF stimulated NO production, increased constitutive nitric oxide synthase (eNOS) expression, Ca2 mobilization, proliferation and migration of PAEC expressing VEGF-R2, but not in VEGF-R1 PAEC (Waltenberger et al., 1994; Cunningham et al., 1995; Kroll and Waltenberger, 1999; Shen et al., 1999). VEGF also stimulated PLC-c and ERK activity in VEGFR2-expressing NIH3T3 cells and VEGFR2 is strongly implicated in activation of the PI3K/Akt pathway and tyrosine kinase c-Src (Takahashi and Shibuya, 1997; Gerber et al., 1998b; He et al., 1999). When overexpressed in 293T cells, all three VEGF receptors were strong activators of STAT3 and STAT5, suggesting that they activate a specific subset of these signal transducers (Korpelainen et al., 1999). When co-expressed with STAT5, VEGFR1 increased expression of cell cycle inhibitor p21. VEGF induces expression of connective tissue growth factor (CTGF) via VEGF-R1, VEGF-R2 and PI3K-AKT dependent pathways in retinal capillary cells (Suzuma et al., 2000).

VEGF-R3 (Flt4) The vascular endothelial growth factor receptor 3 (VEGFR-3/Flt4) is a receptor tyrosine kinase that regulates angiogenesis and vasculogenesis in response to the binding of the ligands VEGF-C and VEGF-D. VEGF-C treatment of PAEC expressing either VEGF-R3 or VEGF-R2 caused actin reorganization and cell migration (Ahmed et al., 1997). Evidence that VEGFR2 is essential for mediating some biological functions of VEGF-C comes from a study in which a mutant VEGF-C, that can still bind and activate VEGF-R3 but cannot ligate VEGF-R2 or activate ERK, was unable to induce vascular permeability and proliferation of bovine capillary endothelial cells (Joukov et al., 1998; Zachary and Gliki, 2001). Joukov et al. (1996) also demonstrated that conditioned medium from cells expressing VEGF-C could stimulate the growth of endothelial cells in a collagen gel matrix (Joukov et al., 1996).

NP-1 Co-expression of NP-1 with VEGF-R2 increased binding of VEGF165 four-fold and enhanced chemotaxis and mitogenicity of VEGF165. Since NP-1 has a short

cytoplasmic tail with no known signaling function, it is likely that NP-1 by itself is not a functional receptor, but acts as a ‘docking’ co-receptor. A major NP-1 binding site for VEGF165 has been mapped to the domain encoded by exon 7 which is contained in VEGF165 and is absent from VEGF121 (Soker et al., 1998; Migdal et al., 1998).

VEGF EFFECTS ON ANGIOGENESIS VEGF affects angiogenesis on multiple levels. First of all, VEGF is a potent mitogen for vascular endothelial cells. Proliferation of endothelial cells contributes greatly in the formation of new blood vessels. In addition to mitogenic activity, VEGF also works as a survival factor for endothelial cells (Alon et al., 1995; Gerber et al., 1998b). This effect is mediated via several mechanisms. VEGF induces expression of the anti-apoptotic proteins Bcl-2 and A1 in human endothelial cells (Gerber et al., 1998a). Increased survival by VEGF is also mediated via PI3-kinase/Akt signal transduction pathway (Gerber et al., 1998b). Altogether it results in a considerable angiogenic effect of VEGF, which has been observed in a large number of in vivo models (Leung et al., 1989b). VEGF is also known as vascular permeability factor by inducing vascular leakage (Senger et al., 1993; Dvorak et al., 1995). Because of its unique role in physiological angiogenesis VEGF is critically important for embryonic development. Gene targeting studies have shown that both VEGF-R1 and VEGF-R2 are essential for embryogenesis (Fong et al., 1995; Shalaby et al., 1995). Mouse embryos homozygous for a targeted deletion in the VEGF-R1 gene died in utero between days 8.5 and 9.5. Endothelial cells developed in these embryos, but failed to organize in normal vascular channels. It has recently been shown that excessive proliferation of endothelial progenitors is the main factor leading to disorganization (Fong et al., 1999). Deletion of VEGF-R2 gene resulted in severe disruption of hematopoietic precursors and organized blood vessels failed to develop throughout the embryo and yolk sac, resulting in death in utero between days 8.5 and 9.5 (Shalaby et al., 1995). Inactivation of a single VEGF allele in mice also resulted in embryonic lethality

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between days 11 and 12. The VEGF/ embryos exhibited a number of development abnormalities (Carmeliet et al., 1996; Ferrara et al., 1996). To selectively investigate the role of VEGF in postnatal development inducible, Cre-loxP-mediated gene targeting was performed. Alternatively, a soluble VEGF receptor chimeric protein was used to inactivate VEGF in early postnatal life. Partial inhibition of VEGF resulted in increased mortality, stunted body growth and impaired organ development, most notably in the liver (Gerber et al., 1999a). It was reported that PlGF-1 and PlGF-2 cannot induce angiogenesis, endothelial cell proliferation, or blood vessel permeabilization on their own, but that they can potentiate strongly the effect of VEGF on blood vessel permeabilization, and to a lesser extent the mitogenic effect of VEGF (Park et al., 1994). However, other reports indicated that both PlGF-1 and PlGF-2 can induce proliferation of endothelial cells, angiogenesis and permeabilization of blood vessels, and that PlGF-1 may be less active than PlGF-2 (Sawano et al., 1996; Ziche et al., 1997). PlGF-1 can form active heterodimers with VEGF, and these heterodimers are almost as angiogenic as VEGF itself (DiSalvo et al., 1995). Carmeliet et al. (2001) developed PlGF-deficient mice by targeted disruption. Plgf / mice had subtle changes of VEGF-dependent retinal and luteal angiogenesis, but demonstrated impaired angiogenesis and plasma extravasation. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Plgf / mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to FGF or histamine. Ferrara recently reported that VEGF plays an important role in endochondral bone formation. Blocking of VEGF with soluble VEGF receptor resulted in impaired trabecular bone formation. Recruitment and differentiation of chondroclasts was substantially decreased. Resorption of hypertrophic chondrocytes was inhibited, resulting in a three- to six-fold expansion of the chondrocyte zone (Gerber et al., 1999b). These findings indicate that VEGF-dependent blood vessels are essential for bone formation. VEGFmediated angiogenesis is also essential for normal

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function of the ovarian corpus luteum (OCL). Blocking of VEGF with soluble receptor resulted in inhibition of OCL development and release of progesterone. It was associated with failure of maturation of endometrium (Ferrara et al., 1998). VEGF mRNA is rapidly induced by low pO2 in a variety of normal and transformed cultured cell types (Shweiki et al., 1992; Shima et al., 1995) and in organs exposed to hypoxia (Banai et al., 1994; Tuder et al., 1995). Increased level of VEGF was detected during normal pregnancy and especially in pregnancy complicated by pre-eclampsia and eclampsia (Vuorela et al., 1997; Kupferminc et al., 1997). Several cytokines or growth factors including EGF, TGFb, IL-1a, PGE2, and IGF-1 up-regulate VEGF mRNA expression and induce the release of VEGF protein (reviewed in Ferrara and Smyth, 1997). Thus, VEGF is ultimately involved in complex pathways regulating an organism’s response to different physiological and pathological stimuli. In particular, VEGF plays an important role in cancer. It is produced by almost all tumor cells (Ferrara et al., 1992) and is found in the serum of cancer patients (Kondo et al., 1994). VEGF production is closely associated with poor prognosis (Toi et al., 1996; Ellis and Fidler, 1996). High concentrations of VEGF were found in tissues even when serum concentrations were relatively low (Takano et al., 1996). Inhibition of VEGF activity results in suppression of growth of a wide variety of tumor cell lines. Elevated levels of VEGF were found in eye fluids from patients with diabetes proliferative retinopathy (Adamis et al., 1994). This suggests that VEGF may be actively involved in diabetes intraocular neovascularization. Thus, VEGF undisputedly plays a critical role in physiological and pathological angiogenesis.

VEGF EFFECTS ON HEMATOPOIETIC PROGENITOR CELLS Initially, the major VEGF functions were attributed to its effect on angiogenesis. During the last couple of years a new role for VEGF has emerged. VEGF was found to be an important factor for differentiation of hematopoietic cells. All of the different lineages of hematopoietic cells are ultimately derived from a common precursor, the pluripotent hematopoietic

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stem cell (HSC) (Abramson et al., 1977; Mintz et al., 1984; Eglitis et al., 1985; Van Zant et al., 1991). A series of intermediate cells with increasingly limited developmental potential separates the primitive pluripotent cell from the mature cells (Nakahata and Ogawa, 1982; Molinex et al., 1986, Van Zant et al., 1991). Regulation of HSC differentiation is a complex process, which involves multiple signals from cell–cell interactions, soluble factors and the stromal microenvironment (reviewed in Tsukamoto et al., 1995). A direct effect of VEGF on hematopoietic cell differentiation from progenitors might have its roots in embryonic development. Both hematopoietic progenitor cells (HPC) and vascular endothelial progenitor cells are derived from a common embryonic origin, and a significant body of work has been performed in search of a common precursor for both of these lineages, termed the hemangioblast. An essential role for VEGF in hematopoietic development has been established, and both VEGF-R1 and VEGF-R2 have been implicated as important markers of hematopoietic stem cells (Nishikawa et al., 1998; Ziegler et al., 1999). The essential role of VEGF in hematopoietic differentiation is underscored by the fact that heterozygous knockouts of VEGF are embryonic lethal and have defective blood island formation. Several groups reported expression of VEGF receptors and the effects of VEGF on human HPC. Hoehn and colleagues reported the presence of VEGF-R1, but not VEGF-R2-specific mRNA in CD34/Lin/CD38 human umbilical cord blood cells (Hoehn et al., 1996), which is consistent with our findings (Gabrilovich et al., 1996; Oyama et al., 1998a). Katoh et al. reported the presence of mRNA specific for both VEGF-R2 and VEGF-R1 in CD34 cells. They also showed that VEGF suppressed apoptotic cell death of normal hematopoietic stem cells induced by c-irradiation and the lack of mitogenic activity for VEGF (Katoh et al., 1995). VEGF was also found to be able to bind the VEGF-R3 receptor on hematopoietic cells (Wang et al., 1997). In response to VEGF and c-kit ligand, embryonic stem cells give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of hematopoietic precursors (Kennedy et al., 1997). Broxmeyer et al. showed that VEGF enhanced colony formation of mature subsets of granulocyte–macrophage progenitor cells that had been stimulated with a colony-stimulating factor and

inhibited colony formation of progenitor cells not stimulated with such factors (Broxmeyer et al., 1995, 1996). VEGF-R1 is expressed on most leukemic cells and VEGF is considered as an important autocrine and paracrine factor for different types of leukemia (Fiedler et al., 1997; Bellamy et al., 1999, 2001; Dias et al., 2000). Recently a new role of VEGF has been described. In human and mouse systems VEGF was found to have a chemoattractive effect on HPC migration (Young et al., 1999, 2001). It is possible that once in the vicinity of the tumor, these cells can give rise to endothelial cells and contribute to tumor neovascularization. This hypothesis requires further testing. VEGF is chemotactic not only for HPC, but also for endothelial cells, macrophages and several other cell types that can develop from CD34 progenitor cells (Gruber et al., 1995; Clauss et al., 1996a; Isaji et al., 1997). It is important to point out that chemotactic activity of VEGF is mostly associated with VEGF-R1, but not with VEGF-R2 or R3.

EFFECT OF VEGF ON DIFFERENTIATION OF MYELOID CELLS AND IMMUNE RESPONSE IN CANCER Another important function of VEGF is its ability to dramatically affect differentiation of myeloid cells. This growth factor has been implicated in defective differentiation of dendritic cells (DC) in cancer. DCs are the most potent antigen presenting cells critically important for induction of antitumor immunity. Decreased DC generation described in cancer patients and tumor-bearing mice may contribute to the inability of the immune system to control tumor growth (Ishida et al., 1998; Almand et al., 2000). It has been recently reported that elevated levels of VEGF in the vicinity of tumors are closely associated with decreased tumor infiltration by dendritic cells in patients with gastric cancer (Saito et al., 1998). To clarify the in vivo role of VEGF in DC differentiation VEGF was administered into healthy mice using an osmotic pump. The selected dose resulted in serum VEGF concentration comparable with that in cancer patients. Four weeks of continuous VEGF infusions resulted in

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dramatic inhibition of DC production (Gabrilovich et al., 1998). This inhibition was associated with hyperproduction of immature myeloid Gr-1 cells. VEGF significantly increased the number of granulocyte–macrophage colonies during the first week of infusion. This was in agreement with previously reported observations of the stimulatory effect of VEGF in vitro on granulocyte–macrophage colonies (Broxmeyer et al., 1995, 1996). VEGF also blocked the stimulatory effect of Flt3 ligand on the generation of DCs in vivo (Ohm et al., 1999). Detailed in vitro experiments showed that VEGF directly affected growth of myeloid colonies (Gabrilovich et al., 1998). VEGF binds to hematopoietic progenitor cells via one of the VEGF-R1 (Flt-1) receptors (Oyama et al., 1998b). Expression of this receptor decreased with cell differentiation, which correlated with lack of the effects of VEGF-containing tumor cell supernatants on the later stages of DC differentiation. Partial block of VEGF in vivo using neutralizing antibody improved DC production and function in tumor-bearing mice (Ishida et al., 1998; Gabrilovich et al. 1999). Statistically significant correlation was found between elevated levels of VEGF in plasma and decreased presence of DCs in peripheral blood from patients with head and neck cancer (Almand et al., 2000). Taken together these data strongly suggest that VEGF might be one of the important factors responsible for the defective DC differentiation in tumor-bearing hosts. The effect of VEGF was not limited to DCs. VEGF infusion in vivo resulted in a dramatic block in development of T cells and thymic atrophy (Gabrilovich et al., 1998). The mechanism of this effect remains unclear. Cell differentiation is a complex process, which involves multiple genes controlled by multiple transcription factors. One of the transcription factors, NFjB, regulates the transcription of many genes involved in immune responses including those for cytokines and growth factors (reviewed in Verma et al., 1995; Thanos and Maniatis, 1995). NF-jB is present as an inactive complex in the cytoplasm of many cells bound to members of the IjB family of inhibitory proteins. Activation of NF-jB involves serine phosphorylation, dissociation and degradation of IjB by IkB kinase IKK followed by release and nuclear translocation of NF-jB. This activation can be mediated by a variety of stimuli including bacterial

lipopolysaccharide, PMA and TNFa. Authentic NF-jB is composed of 50- and 65-kDa subunits, which bind to a 10-bp motif in the promoter of responsive genes. Several subunits of NF-jB have been identified: p50, p52, p65 (RelA), cRel and RelB. These subunits form both homo- and heterodimeric complexes and differentially regulate gene expression (Liou and Baltimore, 1993). Several studies have recently demonstrated that RelB, a component of NF-jB, is required for the development of DCs (Carrasco et al., 1993; Burkly et al., 1995; Boehmelt et al., 1995; Wu et al., 1998). Block of NF-kB with dominant inhibitor IkBa prevented DC differentiation from progenitor cells in vitro (Oyama et al., 1998b). We suggested that VEGF might affect this transcription factor, which would result in inhibition of DC differentiation. Using electromobility shift assay and reporter gene transcription activation assay we have demonstrated that VEGF inhibited the activation of the transcription factor NF-jB in hematopoietic progenitor cells in vitro (Oyama et al., 1998b) and in vivo (Gabrilovich et al., 1998). This inhibition was seen as early as 2 h after treatment with VEGF and was due to inhibition of IkBa phosphorylation by IKK (Dikov et al., 2001). In experiments in vivo NF-kB activation was blocked in bone marrow cells as early as 7 days after the start of VEGF infusion. This preceded any morphological changes. The same inhibitory effect was detected in tumor-bearing mice (Gabrilovich et al., 1998). The nature of the genes responsible for these effects remains unknown.

SUMMARY For 10 years since its discovery, VEGF has made the long journey from obscurity to the front line of research. It is considered now as one of the major factors responsible for physiological and pathological angiogenesis and increasing evidence now indicates a prominent role for VEGF in the regulation of hematopoiesis and immune response. Despite the wealth of information many questions about the mechanisms of VEGF action remain unanswered. Our knowledge of signal transduction pathways for VEGF-R1 and VEGF-R3 are very sketchy and require more studies, as well as the molecular mechanisms of VEGF effects on hematopoietic cells. The significance

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of other members of the VEGF family needs elucidation. Angiogenic potential of VEGF stimulated use of this growth factor in the treatment of ischemic diseases, such as stroke, coronary heart disease, etc. In preclinical experiments VEGF was delivered either as a protein or DNA in a model of rabbit hind limb ischemia. Significant increase in collateral vessel formation has been detected (Takeshita et al., 1994). These animal studies encouraged clinical trials. Expression of VEGF165 DNA after intramascular gene transfer promoted collateral vessel formation in patients with critical limb ischemia (Baumgartner et al., 1998). In several clinical trials the positive effect of recombinant VEGF was detected in patients with myocardial ischemia. However, a placebo-controlled phase II study has not revealed any clinical benefit (Henry, 1999). VEGF’s role in tumor angiogenesis stimulated attempts to neutralize its effect in cancer. Several clinical trials are under way with neutralizing humanized monoclonal antibody and various small molecules inhibiting signal transduction. First results of these trials are encouraging. However, much more information is needed to clarify the possible therapeutic utility of VEGF-based therapy.

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not migration, through phosphatidylinositol 3-kinasedependent pathways. J. Biol. Chem. 276, 26969–26979. Ziche, M., Maglione, D., Ribatti, D. et al. (1997). Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic. Lab. Invest. 76, 517–531. Ziegler, B.L., Valtieri, M., Porada, G.A. et al. (1999). KDR receptor: a key marker defining hematopoietic stem cells. Science 285, 1553–1558.

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Subsection F. The Chemokines and other Cell Migration Regulating Factors

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45 Macrophage migration inhibitory factor (MIF) Douglas A. Arenberg1 and Richard Bucala2 1

University of Michigan Medical Center, Ann Arbor, MI, USA 2

Yale University School of Medicine, New Haven, CT, USA

There is one thing even more vital to science than intelligent methods; and that is, the sincere desire to find out the truth, whatever it may be Charles Sanders Pierce

INTRODUCTION Macrophage migration inhibitory factor (MIF) is considered to be the first lymphokine activity to be described. The simultaneous original descriptions by Bloom and Bennett (1966) and David (1966) were of a soluble factor purified from supernatants of activated lymphocytes that inhibited the random migration of guinea pig peritoneal macrophages. The scientific literature concerning MIF from the 1960s onwards focused largely on this property of MIF as well as its role in lymphocyte responses (Remold et al., 1970; Granger et al., 1970; Bloom and Jimenez, 1970; Papageorgiou et al., 1972; Pelley and Schwartz, 1972; Rocklin et al., 1972), and in its effect on macrophage activity (Ward et al., 1970; Bartfeld and Atoynatan, 1971; Yoshida, 1971; Leu et al., 1972; Simon and Sheagren, 1972; Churchill et al., 1975). The cloning of the cDNA for human and mouse MIF permitted the discovery of a diverse array of previ-

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

ously unsuspected activities attributable to MIF (Weiser et al., 1989; Bernhagen et al., 1993). This resulted in a clearer understanding of the pathobiology of MIF in conditions as varied as sepsis (Bozza et al., 1999; Calandra et al., 2000), arthritis (Leech et al., 1998, 1999), asthma (Rossi et al., 1998; Korsgren et al., 2000), wound healing (Abe et al., 2000; SchillingSchon et al., 2000) and cancer (Chesney et al., 1999; Ogawa et al., 2000). Perhaps one of the more compelling findings was by Bernhagen and colleagues, who showed that pre-synthesized MIF was released from storage granules of cells in the anterior pituitary in response to endotoxin. This MIF is released from the same cells that release adrenocorticotrophic hormone (ACTH) in response to stress (Nishino et al., 1995). Furthermore, these authors found that MIF then acted to counterbalance the systemic antiinflammatory effects of endogenous steroid hormones (Bernhagen et al., 1993; Calandra et al., 1995; Bacher et al., 1996).

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GENE STRUCTURE, PROTEIN PRODUCTION AND REGULATION The gene for human MIF maps to the long arm of chromosome 22 (22q11.2), covers only approximately 1 kb, and is made up of three exons with two short introns (Paralkar and Wistow, 1994). The gene for murine MIF has also been cloned and genetically mapped to mouse chromosome 10 (Mitchell et al., 1995; Bozza et al., 1995). Unlike the human genome, the mouse genome contains several pseudogenes for MIF (Mitchell et al., 1995; Kozak et al., 1995). Initial characterization of the 5-flanking region of the MIF gene has revealed a number of potential regulatory elements within 1 kb of the start site, including binding sites for Sp-1, a cAMP-responsive element (CRE), an AP-2 site, and a potential negative glucocorticoid responsive element (nGRE). Additional consensus sequences for DNA binding transcription factors are located further upstream of this 1-kb stretch, including a cytokine-1 (CK-1) site and a nuclear factor jB (NFjB) site on the minus DNA strand (Mitchell et al., 1995). Waeber et al. have performed a functional analysis of the murine MIF promoter region using the AtT-20 pituitary cell line. The authors employed a reporter gene (luciferase) controlled by a fragment containing nucleotides 1033 to 63 of the MIF gene. Using both mutational analysis of the promoter and gel shift assays with nuclear extracts of CRF-treated AtT-20 cells, these investigators found that induction of MIF transcriptional activity by corticotrophinreleasing factor involved CRE-binding protein (CREB) interacting with the cAMP-responsive element located 41 bases upstream of the transcription start site (Waeber et al., 1998). Many other cell types express MIF in response to appropriate stimuli; monocytes/ macrophages (Calandra et al., 1994), T cells (David, 1966), eosinophils (Yamaguchi et al., 2000), fibroblasts (Abe et al., 2000) and keratinocytes (Shimizu et al., 1996). While the promoter element(s) required for the transcription of MIF in these cells are not yet defined, a powerful stimulus for MIF transcription in monocytes/macrophages is LPS (bacterial endotoxin), which induces gene transcription at a concentration which is 100-fold lower than that required to induce the transcription of TNFa (Calandra et al., 1994). Human skin fibroblasts express MIF mRNA and

protein in response to alpha-thrombin (Shimizu et al., 1999b). In response to glucose, Waeber et al. have shown that pancreatic islet b cells produce and release MIF, which in turn potentiates the release of insulin in an autocrine fashion (Waeber et al., 1999). Calandra et al. (1998) also showed that MIF was released by macrophages in response to low levels of Gram-positive bacterial exotoxins such as staphylococcal enterotoxin B, or toxic-shock syndrome toxin-1 (TSST-1). An interesting feature of the regulation of MIF expression is that, unlike any other ‘proinflammatory’ cytokines (IL-1, TNF, etc.), MIF is released by the presence of low concentrations (⬃ 1010 to 1014 M) of corticosterioids (Calandra et al., 1995).

PROTEIN STRUCTURE AND ACTIVITY MIF is 115 amino acids long, and the mouse and human proteins share 90% sequence homology, which may be the highest shared homology for a known mouse–human cytokine pair (Plate 45.1). The MIF monomer has molecular weight of approximately 12.5 kDa. The tertiary structure of human MIF, resolved by X-ray crystallography, has revealed a unique protein superfamily. The three-dimensional structure of MIF is a unique ‘barrel-shaped’ protein resulting from the non-covalent association of three MIF monomers into a homo-trimer. Each monomer consists of two a helices separated by three b strands. The NH3-terminal portion of the molecule forms a fourth b strand, with two additional b strands forming the COOH terminus. From the NH3 to the COOH ends, the configuration of the monomer is therefore babbbabb. The folding of the monomer results in four of the b-strands (b1, b2, b4 and b5) forming a b-sheet, which in turn forms part of the inner wall of the barrel. The outer portion of the barrel is comprised of the two a helices in antiparallel configuration. Three monomers are joined via hydrogen bonds, and the inside of the solvent accessible channel is formed by the b-sheets of the three monomers (each comprised of four b-strands). The outside of the channel is enclosed by three pairs of a-helices (one pair from each monomer, Plate 45.2) (Sun et al., 1996). While MIF does not possess structural homology

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with any other known cytokines, Zhang and Issekutz (2001) have shown that rat MIF shares 27% sequence identity with a rat enzyme (D-dopachrome tautomerase) that catalyzes the conversion of the D-isomer of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). This group later found that MIF also has D-dopachrome tautomerase activity (Rosengren et al., 1996). Therefore, MIF is not only a cytokine, but appears to have enzymatic activity as well. A phenylpyruvate tautomerase catalytic activity for MIF has also been described (Rosengren et al., 1997). Several other prokaryotic isomerases also show a high degree of three-dimensional structural similarity with MIF (Sun et al., 1996) (reviewed by Baugh, 2002). Each of these enzymes has a conserved N-terminal proline that resides in a hydrophobic catalytic pocket. A debate which has emerged from these observations is whether the cytokine bioactivity of MIF is related to its enzymatic properties. The lines of evidence which suggest that the cytokine activity is independent of its catalytic activity are as follows: First, the Km values describing the interaction of MIF with its ‘substrate(s)’ are probably not relevant when compared with the concentrations of these substrates that occur in vivo (Rosengren et al., 1997; Swope and Lolis, 1999). Second, Bendrat and colleagues (1997) showed that an enzymatically inactive mutant of MIF (P1S MIF) retained its steroid counter-regulatory activity in vitro. A second group has also generated proline-1 mutants of MIF (serine or phenylalanine substituted), both of which inhibited monocyte migration and chemotaxis, but lacked or had significantly reduced catalytic activity (Hermanowski-Vosatka et al., 1999). In distinct contrast to these findings, Swope and coworkers (1998) created a glycine-substituted mutant of MIF (with glycine substituted for the catalytic proline 1; P1G) that lacked catalytic activity, as well as the ability to prime neutrophils for superoxide generation. Interestingly, a recent study by Senter et al. (2002) has provided a new viewpoint. This demonstrated that N-acetyl-para-benzoquinoneimine (NAPQI), a metabolite of the widely used antipyretic and analgesic acetaminophen, binds covalently to the catalytic Pro-1 of the MIF protein, and inhibits both its enzymatic and cytokine activities. It was concluded that intact catalytic activity is not needed for the biologic activity of the cytokine, however, an intact catalytic domain is necessary.

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It is interesting to note that MIF homologs have been identified from parasitic nematodes (Pastrana et al., 1998), C. elegans (Marson et al., 2001), the Lyme disease tick (Jaworski et al., 2001), and the plant species Arabidopsis thaliana (Esumi et al., 1998). With the existence of conserved genes in such evolutionary diverse life forms, it is intriguing to speculate on the potential role of MIF in these organisms. Notably, studies in mice with targeted deletion of the MIF gene have not indicated a critical role for MIF in development or homeostasis, but do reveal a vital role for the gene in both systemic and local inflammatory responses (Bozza et al., 1999). The MIF homologs present in primitive organisms may play a comparable role in generalized microbial host defense.

CYTOKINE EXPRESSION, REGULATION AND DISEASE STATES Like interleukin-1 and basic fibroblast growth factor (bFGF), MIF lacks a classical N-terminal leader sequence. MIF secretion occurs via a nonconventional pathway, the details of which remain unexplored. MIF has been identified in storage vesicles in both anterior pituitary (Nishino et al., 1995) and rat epididymal epithelial cells (Eickhoff et al., 2001). In epididymal epithelial cells, MIF was concentrated at the apical cell surface and MIF-containing vesicles were observed to pinch off from the plasma membrane (Eickhoff et al., 2001). There is, as yet, no data to indicate whether this is a generalized mechanism of MIF release from other MIF-producing cells. A unique feature of MIF among cytokines is the existence of preformed stores of protein, which are released immediately upon appropriate stimulation from anterior pituitary cells (LPS, CRF), monocytes (LPS, corticosteroids), eosinophils (PMA, C5s, IL-5 (Rossi et al., 1998)), and T cells (anti-CD3 or antigen stimulation). Interestingly, electron microscopic studies have found that, in anterior pituitary cells, MIF localizes both to specific storage granules, as well as to granules containing ACTH (Nishino et al., 1995). Consistent with this physical co-localization was the observation that CRF was a potent stimulus for MIF secretion as well. However, MIF release was also

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observed at concentrations of CRF below those required for ACTH release (Nishino et al., 1995). In the case of pituitary cells (Waeber et al., 1998), macrophages (Calandra et al., 1994) and T cells (Bacher et al., 1996), the same signals which lead to the release of MIF also lead to increased mRNA levels, suggesting that sustained MIF release occurs, and/or that stores of preformed MIF are replenished, by gene transcription and new protein synthesis. To date, the most convincing role for MIF in the host response has been demonstrated in animal studies of sepsis. MIF was ‘rediscovered’ in the early 1990s as a cytokine/hormone released from the anterior pituitary in response to endotoxemia (Bernhagen et al., 1993). Pituitary MIF may act systemically to counterbalance the anti-inflammatory effects of corticosteroids. In support of this steroid counterregulatory activity are studies employing mice with deletions of the gene for MIF. These mice have reduced mortality in models of sepsis including endotoxemia, and reduced sensitivity to staphylococcal enterotoxin B (SEB). Importantly, however, the clearance of intratracheally administered Gram-negative pathogens was not only preserved, but slightly enhanced in MIF/ mice. Plasma levels of the critical mediator, tumor necrosis factor a (TNFa), were lower in MIF/ mice than in controls in response to septic insults (Bozza et al., 1999). In a compelling model from a clinical standpoint, Calandra and colleagues showed that antibody-mediated neutralization of MIF reduced mortality in two different models of bacterial peritonitis (Cecal ligation and puncture (CLP) or E. coli intraperitoneal injection) (Calandra et al., 2000). Furthermore, anti-MIF was protective even in TNFa knockout mice, confirming the central role of MIF in models of septic shock. The protective effect of MIF neutralization was preserved even when administration of the antibody was delayed more than 4 hours after peritoneal soiling. Equally as important was the finding that neutralization of MIF prior to intraperitoneal injection with E. coli did not impair clearance of the organism. This is an important contrast to findings in studies employing anti-TNF-based strategies, in which TNF blockade reduced mortality from endotoxemia, but increased the lethality of a bacterial challenge from CLP (Echtenacher et al., 1990). Collectively, these findings point to MIF as a valid target for intervention studies

in human sepsis. Plate 45.3 is a representative model of the role of MIF in the host response to infection. One mechanism by which MIF regulates the innate host response to Gram-negative pathogens has been illuminated by an elegant series of experiments by Roger and co-workers (2001). In these studies, macrophages (RAW 264.7) transfected with an antisense MIF plasmid were markedly hyporesponsive to E. coli endotoxin. The defective response was restored by cotransfecting the cells with a constitutive expression vector for toll-like receptor 4 (TLR4), the signal transducing molecule of the LPS receptor complex. The authors further demonstrated that deficient TLR4 expression in MIF antisense transfected macrophages was due to reduced activity of the transcription factor PU.1, which drives TLR4 promoter activity (Roger et al., 2001). Therefore, MIF increases LPS responsiveness by increasing TLR4 levels, through a pathway that involves the transcription factor PU.1. An additional mechanism for MIF’s role in sustaining monocyte/ macrophage activation and proinflammatory cytokine production in innate immune responses has been provided by Mitchell et al. (2002), who observed enhanced apoptosis of activated macrophages in MIF/ mice. In a clinical setting, it is likely that MIF regulates both early host responsiveness (via TLR4) and late phase toxicity to LPS (via enhanced macrophage activation and proinflammatory cytokine production). Given data that clearly demonstrate the role of MIF in modulation of the innate immune system, the original discovery of MIF as a lymphocyte-derived cytokine should not be forgotten. Indeed, there is ample evidence demonstrating a role of MIF in regulating the adaptive arm of the immune response. T cells stimulated with anti-CD3 or PMA/ionophore express MIF mRNA and release MIF protein in a timecourse that is slightly delayed relative to acute IL-2 induction (Bacher et al., 1998). Neutralization of MIF was associated with inhibition of T-cell proliferation in response to either anti-CD3 stimulation, or ex vivo antigen stimulation of primed cells. Anti-MIF neutralizing antibodies also inhibited the expression of IL-2 and IL-2R by anti-CD3-treated T cells. While these studies found that MIF was necessary for full T-cell activation in vitro, MIF alone was not sufficient to increase proliferation of either naive or CD3-activated T cells (Bacher et al., 1998). Interestingly, this study

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also found that T-cell expression of MIF after Con A stimulation was enhanced largely in TH2 T cell subsets. In vivo treatement of mice with anti-MIF during immunization with bovine RNase resulted in a markedly reduced antibody response to this protein antigen. This study suggests a critical role for MIF in humoral responses. The inciting antigen, RNase, was delivered together with Freund’s complete adjuvant, and there are few (if any) examples of antibodymediated neutralization of a cytokine circumventing the adjuvanticity of Freund’s emulsion (Bacher et al., 1996). Although not as clearly defined, MIF probably plays a role in other important disease states aside from sepsis. For example, a ‘leukocyte migration inhibitory’ activity was found in the synovial fluid of patients with rheumatoid arthritis as early as 1980 (Morgan et al., 1980). More specific findings implicating a pathologic role for MIF in inflammatory arthritis were seen in murine models of arthritis (Mikulowska et al., 1997; Leech et al., 1998). Synovial macrophages expressed MIF after induction of arthritis, and antiMIF treatment reduced the severity scores of joint inflammation in a dose-dependent fashion (Leech et al., 1998). In a subsequent study by the same group, MIF expression was seen in human rheumatoid synovial macrophages and fibroblasts (Leech et al., 1999), which consistent with previous studies, responded to low concentrations of dexamethasone (1010 to 1012 M) with increased MIF expression. Based on several lines of evidence, MIF has also been postulated to play a role in asthma (Yamaguchi et al., 2000; Rossi et al., 1998; Korsgren et al., 2000). Initially, circulating human eosinophils, which are pivotal effector cells in allergic disease, were identified as a source of MIF (Rossi et al., 1998). Yamaguchi and co-workers demonstrated elevated levels of circulating MIF in asthmatic patients compared with age- and sex-matched controls. Furthermore, MIF levels were higher in the serum of patients with symptomatic asthma, as compared with asymptomatic patients. Induced sputum samples were obtained in a subset of patients, and these demonstrated an increased level of MIF which correlated closely with the level of eosinophilic cationic protein, a product of degranulated eosinophils. The eosinophils present in induced sputum samples also showed immunoreactivity for MIF (Yamaguchi et al., 2000). Evidence

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contradicting a role for MIF in allergic asthma was provided by Korsgren and associates (2000), who administered rabbit anti-MIF serum to ovalbuminsensitized mice. Neutralization of MIF in this setting had no impact on eosinophilic airway inflammation brought on by ovalbumin inhalation. The lack of physiologic measurements in this study, nevertheless, precludes any final conclusions on the efficacy, or lack thereof, of MIF neutralization on airway hyperreactivity. Other studies have found that MIF is involved in the inflammation associated with the adult respiratory distress syndrome (ARDS). ARDS is an acute form of lung injury that is characterized by diffuse chest X-ray infiltrates, and profound hypoxia which often proceeds to respiratory failure with or without multiple organ failure. Despite aggressive supportive care, the mortality of ARDS is 33–40% (ATS/ERS, 1999). ARDS is characterized histologically by acute inflammatory infiltrates in the airspaces, with formation of hyaline membranes, due to the loss of integrity of the alveolarcapillary membrane. Donnelly and colleagues (1999) have reported significant levels of MIF in bronchoalveolar lavage (BAL) fluid and serum obtained from patients with ARDS. These authors found that an important source of MIF in BAL fluid was the alveolar macrophage. Alveolar macrophages responded in vitro to exogenously added MIF by producing two other cytokines involved in the pathogenesis of ARDS, IL-8 (Donnelly et al., 1993; Chollet-Martin et al., 1993) and TNFa (Strieter and Kunkel, 1994). A recent study by Beishuzen and co-workers has revealed several other clinically important findings. This group obtained serial serum samples from patients admitted with sepsis (n  32) or multiple trauma (n  8), and compared their MIF values with age-matched hospitalized control patients (n  41). First, they found that, on admission to the hospital, MIF was elevated in patients with septic shock relative to patients with multiple trauma. The significance of this finding is tempered by the fact that septic shock patients were ‘sicker’ than the traumatized patients, as measured by the Acute Physiology and Chronic Health Evaluation (APACHE) scores. Of note, in patients with septic shock, MIF levels were higher in non-survivors, and were also higher in patients who went on to develop ARDS than in those who did not. Finally, these investigators found that both high levels

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of MIF and the development of ARDS independently predicted mortality from sepsis (Beishuizen et al., 2001). MIF has also been implicated in the pathogenesis of a variety of other inflammatory conditions, including inflammatory bowel disease (de Jong et al., 2001), glomerulonephritis (Lan et al., 1996, 1997), kidney allograft rejection (Lan et al., 1998), and ocular inflammation (Kitaichi et al., 1999). These conditions would appear, on first approximation, to have very little in common from a pathophysiologic perspective. However, one feature that unites these diverse processes is that they are all regularly treated with pharmacologic corticosteroids. The role of MIF as an endogenous steroid ‘counterweight’ suggests that inhibition of MIF in these inflammatory diseases may permit the antiinflammatory activity of endogenous corticosteroids to prevail, or produce an increase in the effectiveness of exogenous (pharmacologic) steroid therapy. This possibility has generated great interest in MIF as a therapeutic target for chronic inflammatory and autoimmune conditions. In addition to these steroidtreated diseases, MIF has been described in association with atherosclerosis, which is characterized by infiltration of inflammatory macrophages in the vessel wall (Burger-Kentischer et al., 2002). Abe and colleagues have demonstrated that MIF is expressed in the basal keratinocytes of normal mouse skin, and that the degree of espression is up-regulated after experimental wounding. This increase was seen not only in the skin, but in the serum as well, with both compartments demonstrating a biphasic pattern that peaked at 3 hours and then again at 24 hours. They found that MIF inhibition with neutralizing antibodies resulted in a significant delay in closure of the excisional wounds (Abe et al., 2000). While the mechanism by which MIF promotes wound healing is not yet evident, this finding expands the physiologic role of MIF in host responses. This finding is also intriguing in light of a recent proposal in which the processes which promote wound healing are co-opted by a growing tumor (Dvorak, 1986; Whalen, 1990). This paradigm is based on a consideration of the cellular and molecular activities which underlie these seemingly unrelated processes: cell proliferation, stroma formation, invasion and angiogenesis. By this perspective, there is a growing body of literature to support the involve-

ment of MIF in oncogenesis and tumor progression (reviewed in Mitchell and Bucala, 2000). As far back as the early 1970s and before the molecular cloning of MIF, a macrophage migration inhibitory ‘activity’ was described in the serum of patients with cancer (Wolberg and Goelzer, 1971; Ablin et al., 1974). More evidence for the presence of migration inhibitory factor(s) in cancer was published by Fahlbusch and colleagues, who isolated and purified a protein of approximately 10 to 45 kDa from the ascites of patients with ovarian cancer (perhaps equivalent to the monomeric and trimeric molecular weight of recombinant MIF, respectively) which inhibited random migration of guinea-pig macrophages (Fahlbusch et al., 1986a; Fahlbusch and Tittel, 1986b). This activity was termed ovarian cancer MIF (OC-MIF), and the authors concluded that it had similar physicochemical properties to ‘conventional lymphocyte-derived MIF’. In 1996, a study using differential display to identify genes involved in metastases of prostate cancer identified one differentially expressed sequence that turned out to be MIF (Meyer-Siegler and Hudson, 1996). Subsequently, this laboratory confirmed that MIF was expressed in normal prostatic epithelium, and that its expression was increased in malignant prostate epithelium (MeyerSiegler et al., 1998; Meyer-Siegler, 2000). A study by Takahashi and colleagues (1998) found MIF mRNA and protein to be expressed in CT26 colon carcinoma cells. Transfection of these cells with a MIF antisense construct resulted in reduced cell proliferation. An attractive explanation for these observations has been provided by the work of Hudson and co-workers (1999). Working independently, they employed an epidermoid carcinoma cDNA library in a cell-based screen for genes that could functionally overcome p53-mediated growth arrest. Multiple clones with this activity were isolated and upon sequencing were found to encode human MIF. They then found that recombinant MIF, in an enzymatically inactive form, was sufficient to overcome p53-mediated growth arrest. This study found that MIF treatment did not reduce levels of p53 protein, nor did it interfere with nuclear localization of p53. MIF did, however, inhibit the transcription of genes under the control of p53, including p21, cyclin G and MDM2. Cells transfected with MIF were protected from stimuli that led to p53dependent apoptosis, albeit less efficiently than with

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the antiapoptotic protein Bcl2. Finally, addition of high concentrations (100 ng ml1) of exogenous MIF protected primary fibroblasts from in vitro senescence. These findings imply that MIF is likely to be important in tumor progression and oncogenesis, particularly in the setting of chronic inflammation, such as when colon adenocarcinoma develops in the setting of ulcerative colitis, or in the ‘scar carcinomas’ of lung cancer. Inhibition of p53 by MIF has implications not only for cancer, but also in the propagation of the inflammatory response by prolonging the lifespan of activated cells (Hudson, et al.,1999). This speculation proved to be true in a study by Mitchell and colleagues (2002), who demonstrated that inhibition of p53 restored proinflammatory function of monocytes/ macrophages from MIF knockout mice. While these data supporting a role for MIF in tumor biology are indirect, recent studies in animal models have more clearly demonstrated a mechanism for MIF in supporting tumor growth in vivo. Chesney et al. (1999) showed that immunoneutralization of MIF inhibited the growth of implanted lymphomas in mice. This antitumor effect was not related to inhibition of cell proliferation, but rather to inhibition of angiogenesis. They went on to show that MIF was mitogenic for endothelial cells in vitro and angiogenic in an in vivo Matrigel model. In a different model of tumor growth employing melanoma cells, Shimizu and co-workers (1999a) found that both normal melanocytes and melanoma cells expressed MIF. Expression was significantly higher in the malignant cells, and antisense MIF-transfected cells displayed reduced in vitro proliferation. These authors also found that MIF induced chemotaxis of melanoma cells in Boyden chambers. In vivo, mice treated with anti-MIF antibody displayed reduced angiogenesis in dorsal air sacs containing G361 melanoma cells. Similarly, Ogawa and associates (2000) demonstrated inhibition of tumor growth and angiogenesis of CT26 colon cancer cells in Balb/c mice. However, angiogenesis is an extremely complicated process comprised of cell proliferation, migration, specialized adhesion and tube formation. MIF has not yet been studied for the full spectrum of its potential angiogenic activities. Moreover, certain of its effects may be indirect via the expression of other mediators (unpublished observations and White et al., 2001). Evidence that MIF can induce IL-8 and other angio-

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genic CXC chemokines has been published by several groups (Donnelly et al., 1997; Makita et al., 1998), and a recent study by White and co-workers (2001) demonstrated that MIF, expressed by lung cancer cell lines in vitro, induced CXC chemokine-dependent angiogenic activity in co-cultured monocytes. This is particularly relevant because of the central role played by the CXC chemokine family in regulating angiogenesis in lung cancer (White et al., 2002). These observations also suggest a link between the inflammatory actions of MIF and its reported tumor growth promoting activity (Plate 45.4).

RECEPTOR EXPRESSION, REGULATION AND DISEASE STATES Despite the fact that MIF activity was described almost 40 years ago, little is known of the cellular receptor(s) for MIF. The existence of a specific cell surface receptor for MIF is inferred based on its mechanism of action, the size of the protein, the target cells upon which it acts, and the efficacy of immunoneutralization in vitro and in vivo. MIFbinding proteins which are not cell surface receptors have been described. For example, Kleeman and associates identified an intracellular binding protein for MIF using a yeast two-hybrid approach, with MIF as the ‘bait’ protein. These authors identified jun activation-domain binding protein (Jab1) which interacted with MIF. Jab1 is an activator of the AP-1 transcription factor complex. MIF co-localized with Jab1 in the cytosol, and inhibited Jab1-enhanced AP-1 activity. Interestingly, MIF antagonized Jab1dependent cell-cycle regulation, and Jab1-mediated rescue of fibroblasts from growth arrest was blocked by MIF. This is in contrast to the findings of others who have shown that MIF promotes cell proliferation and antagonizes p53-mediated apoptosis (Mitchell et al., 1999, 2002; Hudson et al., 1999). Jung and co-workers (2001), also working with a yeast two-hybrid screen using MIF as a bait, identified another MIF-binding partner, the thiol-specific antioxidant protein PAG. PAG (proliferation-associated gene) is a family member of a group of genes known as peroxiredoxins, which are ubiquitously expressed, but found at higher levels in tissues with a high rate of

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cell proliferation (Prosperi et al., 1993; Chae et al., 1994). The interaction of MIF with PAG was confirmed with co-immunoprecipitation, and was dependent on the redox status of the complex, as reducing agents inhibited the MIF–PAG interaction. The activity of both proteins was inhibited as a result of the interaction, with MIF interfering with the antioxidant activity of PAG, and PAG inhibiting the catalytic (tautomerase) activity of MIF. Whether MIF–PAG interaction modulates MIF’s cytokine activity was not explored (Jung et al., 2001). While the proteins that interact with MIF are intriguing from a biochemical perspective, the physiologic significance of these interactions needs to be determined, and these findings must be reconciled with what is known about MIF action in vivo. The unique tertiary structure of MIF suggests that a cell surface or soluble protein receptor for MIF, when found, will also possess unique structural features.

pholipase A2 activity by glucocorticoids was reversed by the addition of recombinant MIF (Mitchell et al., 1999). cPLA2 leads to arachidonic acid production, which significantly enhances the translational efficiency of the mRNA for TNFa. This regulatory node likely represents one mechanism by which MIF and glucocorticoids control the production of proinflammatory cytokines by cells such as monocytes/ macrophages. Along these same lines, Daun and Cannon (2000) investigated the effect of MIF on the glucocorticoid-induced increase in IjB. IjB is a cytosolic protein which binds to NFjB and retains it in the cytosol, preventing its translocation into the nucleus to activate the transcription of proinflammatory genes. Steroid-induced increases in cytosolic levels of IjB are one known mechanism of the antiinflammatory actions of steroids, and this increase was blocked by MIF. These studies provide an additional mechanistic link between signaling pathways initiated by MIF, and its known proinflammatory, steroid antagonistic properties.

RECEPTOR SIGNALING While definitive identification of a cell surface receptor for MIF has remained an elusive target, there are studies that have examined intracellular signaling pathways that could account for certain of the proinflammatory activities of MIF. Mitchell and colleagues showed that MIF stimulated proliferation of quiescent NIH/3T3 cells, and that this response was associated with phosphorylation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). This activation by MIF was sustained (24 hours), which is extremely unusual for ligandinduced activation of ERK. Furthermore, the authors showed that this sustained activation was dependent upon protein kinase A (PKA) activity, as specific inhibitors of PKA blocked MIF-induced activation of ERK. Carrying the study further, the authors made an important observation relating this signaling cascade to the known steroid counter-regulatory activity of MIF. MIF was found to activate cytosolic phospholipase A2 (cPLA2) activity via ERK-1/2. Importantly, inhibition of cPLA2 is a key mechanism of the anti-inflammatory activity of glucocorticoids, and suppression of cytokine-induced cytoplasmic phos-

SUMMARY MIF is a critical proinflammatory mediator that has been found to play a role in diverse conditions characterized both by inflammation and cellular proliferation. MIF has a number of unique structural, genetic and functional properties that distinguish it from other cytokines (Table 45.1). These properties include its unique tertiary structure, its catalytic activity, a non-conventional secretory pathway, its widespread expression and secretion from endocrine as well as immune cells, and its action as an endogenous counter-regulator of the anti-inflammatory and immunosuppressive properties of steroids. Also, MIF is unique in being a ‘proinflammatory’ mediator that is induced in cells by glucocorticoids. Consistent with the wide range of activities of MIF, it has been found to be involved in the pathogenesis of many conditions (Table 45.2). Important areas of future investigation will include identifying the nature and regulation of the MIF receptor, understanding the regulation of MIF at the genetic level, and designing clinically effective means of inhibiting its activity.

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TABLE 45.1 Cellular and molecular properties of MIF Properties of MIF (Refs) Inhibition of random macrophage migration (Bloom and Bennett, 1966; David, 1966) Antagonism of corticosteroid anti-inflammatory activity (Bernhagen et al., 1993) Catalytic activity (Zhang and Issekutz, 2001; Rosengren et al., 1996) Induction of CXC chemokines (Donnelly et al., 1997; White et al., 2001) Promotion of insulin release from pancreatic b cells (Waeber et al., 1997) Promotion of T-cell proliferation (Bacher et al., 1998) Promotion of tumor cell proliferation (Takahashi et al., 1998) Promotion of angiogenesis (Shimizu et al., 1999a) Antagonism of p53-mediated apoptosis (Hudson et al., 1999; Mitchell et al., 2002) Induction of metalloprotease activity (Meyer-Siegler et al., 2000; Onodera et al., 2000) Promotion of endothelial cell proliferation (Chesney et al., 1999; Ogawa et al., 2000) Modulation of AP-1 transcription factor activity (Kleeman, 2000) Interaction with thiol-protein oxidase (Jung et al., 2001) TABLE 45.2 Association of MIF with pathologic processes in vivo In vivo activity of MIF in animal models (Refs) Endotoxemia and exotoxemia (Bernhagen et al., 1993; Calandra et al., 1998; Bozza et al., 1999) Delayed-type hypersensitivity (Bernhagen et al., 1996) Antigen-dependent T-cell activation (Bacher et al., 1996) Glomerulonephritis (Lan et al., 1997) Arthritis (Mikulowska et al., 1997; Leech et al., 1998) Transplant rejection (Lan et al., 1998) Tumor growth and angiogenesis (Meyer-Siegler et al., 1998; Chesney et al., 1999; Ogawa et al., 2000; Shimizu et al., 1999) Anemia of malaria (Martiney et al., 2000) Uveitis, iridocyclitis (Kitaichi et al., 2000; Taguchi et al., 2001) Wound healing (Abe et al., 2000) Atherosclerosis (Burger-Kentischer et al., 2002)

ACKNOWLEDGEMENTS This work was supported by NIH 1R01 AR48135-01-A1 (R.B.), 1RO1 CA 94121-01 (D.A.A.).

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Rocklin, R.E., Remold, H.G. and David, J.R. (1972). Characterization of human migration inhibitory factor (MIF) from antigen-stimulated lymphocytes. Cell. Immunol. 5, 436–445. Roger, T., David, J., Glauser, M.P. and Calandra, T. (2001). MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature 414, 920–924. Rosengren, E., Bucala, R., Aman, P. et al. (1996). The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol. Med. 2, 143–149. Rosengren, E., Aman, P., Thelin, S. et al. (1997). The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase. FEBS Lett. 417, 85–88. Rossi, A.G., Haslett, C., Hirani, N. et al. (1998). Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma. J. Clin. Invest. 101, 2869–2874. Schilling-Schon, A., Pleyer, U., Hartmann, C. and Rieck, P.W. (2000). The role of endogenous growth factors to support corneal endothelial migration after wounding in vitro. Exp. Eye Res. 71, 583–589. Senter, P.D., Al-Abed, Y., Metz, C.N. et al. (2002). Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites. Proc. Natl Acad. Sci. USA 99, 144–149. Shimizu, T., Ohkawara, A., Nishihira, J. and Sakamoto, W. (1996). Identification of macrophage migration inhibitory factor (MIF) in human skin and its immmunohistochemical localization. FEBS Lett. 381, 199–202. Shimizu, T., Abe, R., Nakamura, H. et al. (1999a). High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis. Biochem. Biophys. Res. Commun. 264, 751–758. Shimizu, T., Ohkawara, A., Mizue, Y. and Nishihira, J. (1999b). Alpha-thrombin stimulates expression of macrophage migration inhibitory factor in skin fibroblasts. Semin. Thromb. Hemost. 25, 569–573. Simon, H.B. and Sheagren, J.N. (1972). Migration inhibitory factor and macrophage bactericidal function. Infect. Immun. 6, 101–103. Strieter, R.M. and Kunkel, S.L. (1994). Acute lung injury: the role of cytokines in the elicitation of neutrophils. J. Invest. Med. 42, 640–651. Sun, H.W., Bernhagen, J., Bucala, R. and Lolis, E. (1996) Crystal structure at 2.6–A resolution of human macrophage migration inhibitory factor. Proc. Natl Acad. Sci. USA 93, 5191–5196. Swope, M.D. and Lolis, E. (1999). Macrophage migration inhibitory factor: cytokine, hormone, or enzyme? Rev. Physiol. Biochem. Pharmacol. 139, 1–32. Swope, M., Sun, H.W., Blake, P.R. and Lolis, E. (1998). Direct

link between cytokine activity and a catalytic site for macrophage migration inhibitory factor. EMBO J. 17, 3534–3541. Taguchi, C., Sugita, S., Tagawa, Y. et al. (2001). Macrophage migration inhibitory factor in ocular fluids of patients with uveitis. Br. J. Ophthalmol. 85, 1367–1371. Takahashi, N., Nishihira, J., Sato, Y. et al. (1998). Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth. Mol. Med. 4, 707–714. Waeber, G., Calandra, T., Roduit, R. et al. (1997). Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor. Proc. Natl Acad. Sci. USA 94, 4782–4787. Waeber, G., Thompson, N., Chautard, T. et al. (1998). Transcriptional activation of the macrophage migrationinhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3,5monophosphate responsive element-binding protein CREB in pituitary cells. Mol. Endocrinol. 12, 698–705. Waeber, G., Calandra, T., Bonny, C. and Bucala, R. (1999). A role for the endocrine and pro-inflammatory mediator MIF in the control of insulin secretion during stress. Diabetes Metab. Res. Rev. 15, 47–54. Ward, P.A., Remold, H.G. and David, J.R. (1970). The production by antigen-stimulated lymphocytes of a leukotactic factor distinct from migration inhibitory factor. Cell. Immunol. 1, 162–174. Weiser, W.Y., Temple, P.A., Witek-Giannotti, J.S. et al. (1989). Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc. Natl Acad. Sci. USA 86, 7522–7526. Whalen, G.F. (1990). Solid tumours and wounds: transformed cells misunderstood as injured tissue? Lancet 336, 1489–1492. White, E.S., Strom, S.R., Wys, N.L. and Arenberg, D.A. (2001). Non-small cell lung cancer cells induce monocytes to increase expression of angiogenic activity. J. Immunol. 166, 7549–55. White, E., Strieter, R. and Arenberg, D. (2002). Chemokines as therapeutic targets in non-small cell lung cancer. Current Medicinal Chemistry – Anticancer Agents 2, 403–417. Wolberg, W.H. and Goelzer, M.L. (1971). In vitro assay of cell mediated immunity in human cancer: definition of leukocyte migration inhibitory factor. Nature 229, 632–634. Yamaguchi, E., Nishihira, J., Shimizu, T. et al. (2000). Macrophage migration inhibitory factor (MIF) in bronchial asthma. Clin. Exp. Allergy 30, 1244–1249. Yoshida, T. (1971). Studies on macrophage migration inhibitory factor. Jpn J. Med. Sci. Biol. 24, 190. Zhang, H. and Issekutz, A.C. (2001). Growth factor regulation of neutrophil–endothelial cell interactions. J. Leukoc. Biol. 70, 225–232.

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46 Interleukin-8 and other CXC chemokines Naofumi Mukaida1, Sergey A. Ketlinsky1 and Kouji Matsushima2 1

Kanazawa University, Kanazawa, Japan 2

University of Tokyo, Tokyo, Japan

I hate when people honk at me. Unless I’m taking a left turn – then I like it, because that’s how I know it’s time to turn Rita Rudner

INTRODUCTION Since the first decription of interleukin-8 (IL-8) as a neutrophil chemotactic cytokine in 1987 (Yoshimura et al., 1987), a family of structurally related cytokines has been identified. Because most of them exhibit chemotactic activity against a limited spectrum of leukocytes, they are now called as chemokines (chemotactic cytokine) (Baggiolini et al., 1997; Luster, 1998). Other terms are used in the past to designate chemokines are intercrines, the scy (small cytokine) family and SIS (small inducible, secreted) cytokines (Oppenheim et al., 1991; Baggiolini et al., 1994). Chemokines are characterized by the presence of four cysteines at the well-conserved positions, although the overall similarity at the amino acid level is not so high. Two disulfide bridges are formed between the first and the third, and between the second and the fourth cysteines. Chemokines are low molecular weight proteins with a basic nature and with affinity for heparin. It is believed that chemokines are preferThe Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

entially immobilized through binding to proteoglycans on the vascular endothelium cells and to extracellular matrix proteins in the tissues (Baggiolini et al., 1997). Chemokine receptors comprise a large branch of the rhodpsin family of cell surface G proteincoupled receptors (GPCR) with seven transmembrane domains (7TMD) (Murphy et al., 2000). Functional binding to the target cells and subsequent signaling is mediated by these receptors. Chemokines are divided into four subgroups: CXC, CC, C and CX3C. CXC, CC and CX3C chemokines have four cysteines, whereas C chemokines have only two, corresponding to the second and fourth cysteines in the other groups. CXC and CX3C chemokines are distinguished by the presence of one (CXC) and three amino acids (CX3C), while the first two cysteines are adjacent in CC chemokines. A large number of CXC (>15) and CC chemokines (>25) have been identified in humans and other species, but human lymphotactin a and b (Kelner et al., 1994) and fractalkine (Bazan et al., 1997) and their equivalents in other

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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species are the only examples of C and CX3C chemokines, respectively. Based on function and expression pattern, chemokines can be classified into two groups, although there are some overlaps: an inflammatory/inducible and a homeostatic/constitutive group (Cyster, 1999; Yoshie, 2000; Moser and Loetscher, 2001). Proinflammatory stimuli such as lipopolysaccharide (LPS), IL-1 and TNF regulate the production of the former group, which together orchestrate innate and immune responses. The latter is important for lymphocyte and dendritic cell trafficking into lymphoid organs. A novel nomenclature system has been proposed for chemokines and their receptors (Murphy et al., 2000; Zlotnik and Yoshie, 2000). Systematic chemokine names, shown in Table 46.1 with their common names, are built from cysteine subclass roots, followed by L for ligand. The numbers generally correspond to the same number used in the corresponding gene nomenclature. Because most chemokine receptors are restricted to a single chemokine subclass, the nomenclature system of chemokine receptors is rooted by the chemokine subclass specificity, followed by R for receptor and the number (Table 46.2). CXC chemokines are produced as precursor molecules containing a signal sequence of 17 to 34 amino acids. After cleavage, mature proteins of 70 to 103 amino acids are secreted except for CXCL16, which is a membrane-bound protein (Matloubian et al., 2000). Most human CXC chemokine genes are located as a cluster in the long arm of chromosome 4 (Table 46.1) except for stromal cell-derived factor-1/pro-B cell growth-stimulating factor (SDF-1/PBSF, CXCL12), breast and kidney/B-cell monocyte-activating chemokine (BRAK/BMAC, CXCL14) and CXCL16. The 5 flanking region of the chemokine genes contains regulatory sequences for gene transcription and their mRNA stability may be influenced by RNA instability elements in the 3-untranslated region. The genes for IL-8 (CXCL8) (Mukaida et al., 1989), gro (CXCL1, 2 and 3) (Baker et al., 1990), endothelial cell-derived neutrophil attractant-78 (ENA-78, CXCL5) (Walz et al., 1991a), granulocyte chemotactic protein-2 (GCP-2, CXCL6) (Rovai et al., 1997), interferon c-inducible protein-10 (IP-10, CXCL10) (Luster and Ravetch, 1987a), interferon-inducible T cell a chemoattractant (I-TAC, CXCL11) (Tensen et al., 1999) and B lymphocyte chemoattractant/B cell-attracting chemokine-1

(BLC/BCA-1, CXCL13) (Ansel et al., 2000) consist of four exons while the genes for platelet factor (PF-4, CXCL4) (Eisman et al., 1990), neutrophil activating protein-2 (NAP-2, CXCL7) (Majumdar et al., 1991), and SDF-1/PBSF (CXCL12) (Nagasawa et al., 1996) consist of three exons. However, the structures of the first and second intron–exon junctions are well conserved. CXC chemokines can be further subclassified by structure into ELR and ELR CXC chemokines, based on the presence or the absence of tripeptide motif glutamic acid–leucine–arginine (ELR) NH2-terminal to the first cysteine. This classification is well correlated with the functional differences. ELR CXC chemokines bind CXCR1 and/or CXCR2 with high affinity and show a potent chemotactic activity mainly against neutrophils (Murphy, 1994; Ahuja and Murphy, 1996). Moreover, ELR CXC chemokines can induce angiogenesis in several assays (Strieter et al., 1995a; Belperio et al., 2000).

PROTEIN STRUCTURE OF HUMAN CXC CHEMOKINES (TABLE 46.1 AND FIGURE 46.1) IL-8 (CXCL8) Based on the amino-acid sequence, IL-8 (CXCL8) cDNA was cloned from a lipopolysaccharide-induced human monocyte cDNA library (Matsushima et al., 1998). The human CXCL8 cDNA is identical to the previously cloned 3-10C cDNA with unknown function (Schmid and Weissmann, 1987). The CXCL8 cDNA encodes a 99-amino-acid precursor protein with a signal sequence of 27 amino acids, which is cleaved to yield the 72-reside mature protein (Matsushima et al., 1998). CXCL8 is further processed at the NH2terminus yielding different truncation analogs (77-, 72-, 71-, 70-, 69-amino-acid forms). The truncation is due to proteases that are released from CXCL8secreting cells or by accessory cells (Yoshimura et al., 1989; Hebert et al., 1990; Nakagawa et al., 1991; Padrines et al., 1994) and the occurrence of the NH2terminal forms depends on the producer cells and culture conditions (Van Damme et al., 1989a, 1989b, 1990). The two major forms are the 77- and the

THE CYTOKINES AND CHEMOKINES

TABLE 46.1 CXC chemokine family Protein (amino acids)

Database accession

Protein

Gene

Chromosome

Synonyms

Precursor Mature

Protein

Gene

Subclass

CXCL1

GRO1

4q21.1

107

73

P09341

X54489

ELR

CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7

GRO2 GRO3 PF4 SCYB5 SCYB6 PPBP

4q21.1 4q21.1 4q21.1 4q21.1 4q21.1 4q21.1

107 107 101 114 114 128

73 73 70 78 77 70

P19875 P19876 P02776 P42830 P80162 P02775

U03019 U03020 M20901 L37036 U83303 M11517

ELR ELR ELR ELR ELR ELR

CXCL8

IL8

4q21.1

99

72 (79, 77, 69) P10145

M28130

ELR

CXCL9 CXCL10 CXCL11

MIG INP10 NA

4q21.1 4q21.1 4q21.1

125 98 94

103 77 73

Q07325 P02778 O14625

NAc NA Y15221

ELR ELR ELR

CXCL12

SDF1

10q11.21

ELR

4q21.1

68 (a) 72 (b) 87

AL137026

NA

89 (a) 93 (b) 109

P48061

CXCL13

O43927

NA

ELR

CXCL14

NA

5q31.1

99

77

O95715

AC005738

ELR

CXCL16

NA

17p13

groa, melanoma growth-stimulating activity (MGSA), NAP-3, SCYB1 (mouse) KC/N51, macrophage inflammatory protein (MIP)-2a grob, SCYB2 (mouse) MIP-2, MIP-2a groc, SCYB3; (mouse) MIP-2b Platelet factor-4 (PF-4), SCYB4 Endothelial-derived neutrophil attractant-78 (ENA-78) Granulocyte chemotactic protein-2 (GCP-2) Platelet basic protein (PBP, 94 amino acids)b Connective tissue activating peptide-III (CTAP-III, 85 amino acids) b-thromboglobulin (b-TG, 81 amino acids) Neutrophil activating protein-2 (NAP-2, 70 amino acids) SCYB7, low affinity platelet factor-4 Interleukin-8 (IL-8), monocyte-derived neutrophil chemotactic factor (MDNCF), NAP-1, GCP-1, SCYB8 monokine induced by interferon c (Mig), SCYB9; (mouse) CRG-10 interferon-inducible protein-10 (IP-10), SCYB10; (mouse) CRG-2 interferon-inducible T cell a chemoattractant (I-TAC), IP-9, b-RI, H174 SCYB11 Stromal cell-derived factor-1 (SDF-1)a, SDF-1b Pro-B cell growth-stimulating factor (PBSF), SCYB12 B lymphocyte chemoattractant (BLC), B cell-attracting chemokine-1 (BCA-1), SCYB13 breast and kidney (BRAK), B cell- and monocyte-activating chemokine (BMAC), SCYB14, bloekine none

273

225

AAG34365 NA

Official name

a b c

KC and MIP-2 are mouse proteins with similar sequence relatedness to each of three human Gro proteins. Sequential N-terminal truncation of PBP produces chemokines shown here. Only NAP-2 exhibits leukocyte chemoattractant activity, especially for neutrophils. Not available.

ELR

TABLE 46.2 CXC chemokine receptors Previous names Chromosome a Amino acidsb Accession number Distribution on leukocytes Thymocytes CD341 cells NK cells B lymphocytes T lymphocytes Immature dendritic cells Mature dendritic cells Macrophages/monocytes Neutrophils Eosinophils Basophils Platelets Ligand specificity CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL16 a

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

IL-8RA, IL-8R-I 2q34–q35 (IL-8RA) 350 P25024

IL-8RB, IL-8R-II 2q34–q35 (IL-8RB) 360 P25025

IP-10/MigR, GPR9 Xq13 (GPR9) 368 P49682

HUMSTR, LESTER, fusin, HM89 2pter–qter (CXCR4) 352 P30991

BNLR-1, MDR15 11 (BLR) 372 P32302

STRL33, Bonzo, TYMSTR 3p21.3 342 O00574

   

 

 

  

 

     

 

    

   

  

   

   

  

The names in parentheses are the official names of the genes. b The numbers of the amino acids of the mature proteins are shown here.



 

MARAALSAAPSNPRLLRVALLLLLLVAAGRRAAGASVATELRCQCLQTLQGIHPKNIQSVN-VKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN MARATLSAAPSNPRLLRVALLLLLLVAASRRAAGAPLATELRCQCLQTLQGIHLKNIQSVK-VKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN MAHATLSAAPSNPRLLRVALLLLLLVG-SRRAAGASVVTELRCQCLQTLQGIHLKNIQSVN-VRSPGPHCAQTEVIATLKNGKKACLNPASPMVQKIIEKILNKGSTN MSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQVRPRHITSLE-VIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES MSLLSSRAARVPGPSSSLCALLVLLLLLTQPGPIASAGPAAAVLRELRCVCLQTTQGVHPKMISNLQ-VFAIGPQCSKVEVVASLKNGKEICLDPEAPFLKKVIQKILDGGNKEN MSLPSSRAARVPGPSGSLCALLALLLLLTPPGPLASAGPVSAVLTELRCTCLRVTLRVNPKTIGKLQ-VFPAGPQCSKVEVVASLKNGKQVCLDPEAPFLKKVIQKILDSGNKKN MSLRLDTTPSCNSARPLHALQVLLLLSLLLTALASSTKGQTKRNLAKGKEESLDSDLYAELRCMCIKTTSGIHPKNIQSLE-VIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLK VRKSQRSRQKKTT CXCL10 MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKEMSKRSP CXCL11 MSVKGMAIALAVILCATVVQGFPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLIIKKVERKNF CXCL12a MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVAR--ANVKHLKILNTPNCALQIVARLKNNN-RQVCIDPKLKWIQEYLEKALNK CXCL12b MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVAR--ANVKHLKILNTPNCALQIVARLKNNN-RQVCIDPKLKWIQEYLEKALNKRFKM CXCL13 MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP CXCL14 MRLLAAALLLLLLALYTARVDGSKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRRVYEE CXCL16 MSGSQSEVAPSPQSPRSPEMGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGKRISSDSPPSVQFMNRLRHLRAYHRCLYYTRFQLLSWSVCGGNKDPWVQELMSKLDLKECGHAYSGIVAHQKHLLPT SPPTSQASEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNETTIHTAGHSLAVGPEAGENQKQPEKNAGPTARTATVPVLCLLAIIFILTAALSYVLCKRRRGQSPQSSPDLPVHYIPVAPDSNT CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9

FIGURE 46.1 Amino-acid sequences of human CXC chemokines. The conserved cysteine residues and ELR motifs are indicated with grayed background and italic letters, respectively.

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72-amino-acid-proteins with a minor form of 69amino-acid protein. Fibroblasts and endothelial cells predominantly produce the 77-amino-acid form, whereas leukocytes mainly secrete the 72- or 69amino-acid forms. These three forms exhibit neutrophil chemotactic activities with distinct potencies; 69- > 72- >77-amino-acid form, in vitro. In vivo, the 77-amino-acid form is rapidly cleaved to yield the 72-amino acid form (Ko et al., 1993). Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography have revealed that CXCL8, in a concentrated solution and on crystallization, occurs as a dimer of two identical subunits (Baldwin et al., 1991). The monomer contains a disordered NH2-terminus, followed by a loop region, three antiparallel b strands and an a helix extending from amino acid 57 to the COOH-terminus. At concentrations above 100 lM, CXCL8 exists in a dimer stabilized by six hydrogen bonds between the first b strands of the partner molecules (residues 23–29) and by other side-chain interactions. The dimer consists of two antiparallel a helices lying on top of a six-stranded antiparallel b sheet. However, at nanomolar concentrations, which are physiologically relevant and induce maximal biologic activity, most CXCL8 occurs in its monomeric form (Burrows et al., 1994; Paolini et al., 1994). Moreover, a chemically synthesized CXCL8 analog with methylated Leu-25, exhibits equivalent neutrophil activation and receptor binding to natural CXCL8, without any dimer formation (Rajarathnam et al., 1994). These observations suggest that the monomer is a functional form. CXCL8 is stable at extremes of pH (pH 2.0 and 9.0) and is resistant to mildly oxidizing and reducing conditions. Heating to 100 C does not alter its activity. CXCL8 resists exposure to detergents and organic solvents. It is inactivated only slowly at 37 C by cathepsin G and proteinase 3 (Schroder et al., 1987; Padrines et al., 1994). These properties are presumed to be due to the conformation of the chemokine. Reduction of disulfide bonds leads to unfolding and inactivation of CXCL8, but its activity is regained after dialysis. CXCL8 can be frozen or lyophilized with little loss of activity. However, storage without any additional protein in phosphate-buffered saline at 4 C results in a drastic loss of activity, due to binding of CXCL8 to plastic materials.

Platelet factor-4 (PF-4, CXCL4) PF-4 is released from the a granules upon platelet activation, together with platelet-derived growth factor (PDGF), transforming growth factor (TGF)b, and b-thromboglobulin (b-TG). PF-4 binds heparin and promotes blood coagulation. The determination of the amino-acid sequence revealed a protein of 70 amino acids containing four cysteines (Deuel et al., 1977). PF-4 cDNA encodes a protein of 101 residues, yielding mature PF-4 after cleavage of the 31amino-acid signal sequence (Poncz et al., 1987). For PF-4 a similar three-dimensional structure has been described as for CXCL8. PF-4 tends to aggregate into dimers and tetramers, but at lower concentrations remains as monomers (Mayo and Chen, 1989; Zhang et al., 1994; Clark-Lewis et al., 1995). Although the dimers and tetramers are presumed to have more heparin-binding sites, their biologic relevance remains elusive.

Neutrophil activating protein-2 (NAP-2, CXCL7) Activated platelets secrete b-TG and its NH2truncated form with neutrophil chemotactic activities, which is designated CXCL7 (Van Damme et al., 1989b; Walz and Baggiolini, 1989). CXCL7 is more abundantly produced in the conditioned medium of blood platelets cultured in the presence of monocytes. b-TG itself is an NH2-terminally processed form of platelet basic protein (PBP). The cDNA for PBP encodes a protein of 128 amino acids and mature 94residue PBP is generated after cleavage of the signal peptide (Wenger et al., 1989). Processing of PBP at the NH2-terminus yields at least three distinct molecules: connective tissue activating protein-III (CTAP-III), also called low-affinity PF-4 (85 amino acids), b-TG (81 amino acids), and CXCL7 (70 amino acids). Among these molecules, only CXCL7 exhibits a potent chemotactic activity against neutrophils. PBP and CTAP-III are both stored in a granules of platelets (Van Damme et al., 1989b; Walz and Baggiolini, 1990). PBP and its derivatives can form dimers and tetramers. Removal of the NH2-terminal residues from PBP reduces dimer and tetramer formation. Under physiologic conditions, all forms tend to be present as a monomer. The tertiary structure of PBP is

THE CYTOKINES AND CHEMOKINES

PROTEIN STRUCTURE OF HUMAN CXC CHEMOKINES

similar to that of CXCL8. An a helix and b-sheet structure are conserved among PBP and its derivatives. There is additional a-helix formation within the elongated NH2-terminal segment. The NH2-terminal portions of PBP, CTAP-III and b-TG are presumed to fold over the CXCL7 structure important for biological functions, thereby blocking neutrophil chemotactic activity (Yang et al., 1994), and the removal of the NH2-terminal portion gives rise to a chemotactic activity.

Gro-a/melanoma growth-stimulatory activity (CXCL1), gro-b (CXCL2) and gro-c (CXCL3) cDNA encoding gro-a has been identified as the gene overexpressed in transformed Chinese hamster cells and human cells (Anisowicz et al., 1987). Richmond and her colleagues (1988) independently purified melanoma growth-stimulating activity (MGSA) from melanoma tumors and found it to be identical to gro-a. The cDNA encodes a precursor protein of 107 residues, yielding mature protein after cleavage of 34 amino acids. Two highly related cDNAs, gro-b and gro-c, have been isolated from a monocyte cDNA library by Haskill and colleagues (1990). Both cDNAs encode a precursor protein of 107 residues with a signal sequence of 34 residues. The three gro proteins show 84 to 88% sequence similarity at the amino-acid level. The three-dimensional structure is similar to that of CXCL8 (Fairbrother et al., 1994; Kim et al., 1994). However, the NH2-terminal portion is more restrained and the turn, which is connected to the NH2-terminal portion, is less severe in gro, compared with CXCL8. The structural differences may contribute to the different receptor binding patterns between gro and CXCL8.

Endothelial cell-derived neutrophil attractant-78 (ENA-78, CXCL5) and granulocyte chemotactic protein-2 (GCP-2, CXCL6) ENA-78/CXCL5 was originally isolated from the conditioned media of a human type II pulmonary epithelial cell line stimulated with either IL-1 or TNF (Walz and Baggiolini, 1990). The cDNA encodes a precursor protein of 114 amino acids and a signal peptide is pro-

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posed to be composed of 31 amino acids (Corbett et al., 1994) or 14 amino acids (Chang et al., 1994). Both groups suggested that CXCL5 is processed proteolytically after secretion to yield a 78-residue protein. GCP-2/CXCL6 was purified from the supernatants of osteosarcoma cells (Proost et al., 1993a). Mature CXCL6 consists of 77 amino acids with three additional NH2-terminal truncated forms (75, 72 and 69 residues) (Proost et al., 1993b) The cDNA encodes a precursor protein of 114 amino acids with a signal peptide of 37 residues (Froyen et al., 1997; Rovai et al., 1997). CXCL6 shows 77% identity with ENA-78 at the amino acid level. However, CXCL6 binds both CXCR1 and CXCR2 with high affinity whereas ENA-78 binds only CXCR2 with high affinity (Wuyts et al., 1997; Wolf et al., 1998). CXCL6-induced activation of CXCR1 appears to depend on the presence of a basic residue Arg20, which is adjacent to the second cysteine (Wolf et al., 1998).

Monokine induced by IFNc (Mig, CXCL9), IFNc-inducible protein-10 (CXCL10, CXCL10) and interferon-inducible T-cell a chemoattractant (I-TAC, CXCL11) Mig/CXCL9 cDNA was originally identified as the gene expressed selectively in IFNc-stimulated mouse macrophages (Farber, 1990). Subsequently, the cDNA encoding the human homolog was isolated (Farber, 1993). The cDNA encodes a precursor protein of 125 amino acids with a signal peptide of 23 residues (Farber, 1993). IFNc-stimulated peripheral blood mononuclear cells and THP-1 cells secrete multiple, identical CXCL9 species as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Chinese ovary cells transfected with human CXCL9 cDNA produced multiple species ranging from 78 to 103 residues, due to proteolytic cleavage at the COOH-terminal portion (Liao et al., 1995). COOH-terminal truncate forms, however, exhibit lower biologic activity in terms of calcium influx induction, compared with the fulllength one. IP-10/CXCL10 cDNA was originally cloned from IFNc-stimulated U937 cells (Luster et al., 1985) although CXCL10 is produced by a variety of cells including endothelial cells, monocytes, fibroblasts

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and keratinocytes in response to IFNc. The cDNA encodes a precursor protein of 98 amino acids with a signal peptide of 21 residues. The molecular mass of the mature CXCL10 protein is about 12.4 kDa but CXCL10 is rapidly cleaved probably at the COOHterminal portion, yielding a 6 to 7 kDa species (Luster and Ravetch, 1987b). I-TAC/CXCL11 cDNA was originally isolated from cytokine-activated astrocytes and encodes a precursor protein of 94 residues with a signal peptide of 21 residues (Cole et al., 1998).

Stromal cell-derived factor (SDF, CXCL12) Mouse SDF-1a/CXCL23a and SDF-1b/CXCL12b have been cloned from a cDNA library of a bone marrow stromal cell line (Tashiro et al., 1993). Nagasawa and his colleagues (1994) independently purified pro-B cell growth-stimulating factor (PBSF) and cloned its cDNA, which is identical to SDF/CXCL12. Human CXCL12b (93 residues) differs from CXCL12a (89 residues) by only four additional amino acids at the COOH-terminus. The determination of the human CXCL12 gene revealed that both proteins are encoded by a single gene and arise by alternative splicing (Shirozu et al., 1995). CXCL12a and CXCL12b are encoded by three and four exons, respectively. CXCL12 adopts a chemokine-like fold consisting of three antiparallel b strands and an overlying a helix as similarly observed on other chemokines including CXCL8 (Crump et al., 1997; Dealwis et al., 1998). However, in CXCL12, the a helix is aligned more parallel with the b strands, whereas it is orthogonal to the b strands in other chemokines. Moreover, both N-terminal 8 residues and residues 12–17 of the loop region are important for binding to its receptor, CXCR4.

B-lymphocyte chemoattractant/B cellattracting chemokine-1 (BLC/BCA-1, CXCL13) Two groups independently identified cDNA, which is strongly expressed in the secondary lymphoid organs (Gunn et al., 1998; Legler et al., 1998). Because the gene product exhibits a potent chemotactic activity against B cells, they are called B-lymphocyte chemoattractant (BLC) or B cell-attracting chemokine-1

(BCA-1). BLC/BCA-1/CXCL13 cDNA encodes a precursor protein of 109 amino acids with a signal peptide of 22 residues.

Breast and kidney/B cell- and monocyte-activating chemokine (BRAK/BMAC, CXCL14) and CXCL16 A search of the EST database identified a novel chemokine gene named as BRAK/BMAC (Hromas et al., 1999; Sleeman et al., 2000). A cDNA encoding human BRAK/BMAC/CXCL14 encodes a precursor protein of 99 residues with a signal peptide of 22 residues. The gene product exhibits a chemotactic activity against monocytes and B cells (Sleeman et al., 2000). In most squamous cell carcinomas and some cervical squamous cell carcinomas, CXCL14 mRNA expression was enhanced in infiltrating inflammatory cells with a reduction in its mRNA expression in tumor cells (Frederick et al., 2000). Human CXCL16 cDNA was originally identified from the EST database (Matloubian et al., 2000). It encodes a precursor protein of 273 residues with a signal peptide of 48 residues. It is a membrane-bound CXC chemokine containing mucin, transmembrane and intracytoplasmic domains in the COOH-terminal portion. It is expressed mainly on CD11c dendritic cells in the lymphoid organs and delivers signals to activated T cells and natural killer cells, which express a specific receptor, CXCR6/BONZO/STRL33 on their surface (Matloubian et al., 2000; Wilbanks et al., 2001). A soluble form can be generated by the proteolysis.

PRODUCTION OF HUMAN CXC CHEMOKINES: CELLULAR SOURCES AND INDUCERS CXCL8 (IL-8) Yoshimura and colleagues (1987) purified CXCL8 to a homogeneity, from LPS-stimulated peripheral blood mononuclear cell culture supernatants. Subsequently, several groups independently purified CXCL8 as a neutrophil chemotactic factor from the supernatants from stimulated peripheral blood monocytes (Schroder et al., 1987; Walz et al., 1987;

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Van Damme et al., 1988). CXCL8 is also produced by other leukocytic cells (T cells, neutrophils and natural killer cells), non-leukocytic somatic cells (endothelial cells, fibroblasts and epithelial cells), and tumor cells (Oppenheim et al., 1991; Mukaida et al., 1992). Most types of cells do not produce CXCL8 constitutively but CXCL8 production can be rapidly induced by a variety of stimuli including proinflammatory cytokines (e.g. IL-1, TNFa) (Matsushima et al., 1988), bacteria (e.g. Helicobacter pylori, Pseudomonas aeruginosa) (DiMango et al., 1995; Aihara et al., 1997), bacteria products (e.g. LPS), virus (e.g. cytomegalovirus, rhinovirus) (Murayama et al., 1994; Johnston et al., 1997) and viral products (e.g. X protein of human hepatitis virus B, Tax protein of human T cellleukemia type I virus) (Mahe et al., 1991; Mori et al., 1998). These stimuli activate various types of cells to produce CXCL8 by inducing CXCL8 gene transcription and stabilizing CXCL8 mRNA. Moreover, in most cases, the CXCL8 gene transcription occurs through cooperative activation of two distinct types of transcription factors, NFjB and AP-1 (Mukaida, 2000). Of interest is that hypoxic conditions induce several types of tumor cells to produce a large amount of CXCL8 through the cooperative activation of NFjB and AP-1 and that in tumor tissues, CXCL8 protein is frequently detected in the close vicinity of necrotic areas, where oxygen concentration is low (Desbaillets et al., 1997; Xu et al., 1999; Kunz et al., 1999). Because CXCL8 has potent angiogenic activities (Strieter et al., 1995b; Belperio et al., 2000), hypoxic conditions may induce the production of an angiogenic factor, CXCL8, which in turn may induce neovascularization. In addition, malignant transformation induces constitutive activation of NFjB and NF-IL6 in head and neck squamous carcinoma cell lines, or PEA3 and AP-1 in hepatoma cell lines. The constitutive activation of these transcription factors eventually results in constitutive production of CXCL8 (Ondrey et al., 1999; Iguchi et al., 2000). IFNc and glucocorticoids can inhibit CXCL8 gene transcription mainly by inhibiting the action of NFjB (Oliveira et al., 1994; Mukaida et al., 1994).

of gro, in response to several stimuli such as IL-1, TNFa, serum and phorbol acetate (Anisowicz et al., 1988; Anisowicz et al., 1991; Wood and Richmond 1995). This inducible gro production is mediated by the cooperative activation of NFjB and an unidentified transcription factor(s), which binds to the region immediately upstream of the NFjB binding site (Anisowicz et al., 1991; Wood and Richmond, 1995). Moreover, IL-1 stablizes mRNA of gro as well as CXL8 (Stoeckle, 1991). In addition to these stimuli, human immunodeficiency virus (HIV)-1 can induce CXCL1 production in monocyte-derived macrophages through HIV-1 gp120 engagement of CXCR4 (Lane et al., 2001). The CXCL2 produced can enhance HIV-1 replication in macrophages and lymphocytes. Melanoma cells start to produce gro proteins constitutively as melanocytes gain a malignant phenotype (Wood and Richmond, 1995; Luan et al., 1997; Devalaraja et al., 1999). The constitutive gene expression may arise from the constitutive and cooperative activation of NFjB and an unidentified transcription factor(s), which binds to the region immediately upstream of the NFjB binding site. In Hs294 T melanoma cells, IjB kinase activity and subsequent IjBa phosphorylation is constitutively enhanced, resulting in increased basal CXCL1 transcription (Devalaraja et al., 1999).

CXCL1, 2 and 3 (gro a, b, c)

CXCL5 is produced by a variety of cells including epithelial cells, keratinocytes, endothelial cells, fibroblasts, neutrophils and monocytes. IL-1, TNF and LPS are major inducers of CXCL5 (Strieter et al.,

Various types of cells including endothelial cells, fibroblasts and epithelial cells produce large amounts

CXCL4 (PF-4) and CXCL7 (NAP-2) Both PF-4/CXCL4 and PBP are synthesized by megakaryocytes (Greenberg et al., 1987; Holt et al., 1988). CTAP-III is generated by a limited proteolysis of PBP during megakaryocyte and platelet maturation (Holt et al., 1986). PF-4, PBP and CTAP-III are stored in the a granules of platelets. Upon platelet activation, b-TG is generated from PBP and CTAP-III through platelet-derived enzymes and is released together with CXCL4 (Brandt et al., 2000). CXCL7 is generated from the released b-TG by a limited proteolysis of neutrophils (Brandt et al., 1991).

CXCL5 (ENA-78) and CXCL6 (GCP-2)

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1992; Lukacs et al., 1995; Walz et al., 1997). The NFjB binding site in the promoter region mediates IL-1- or TNFa-induced ENA-78 gene transcription (Chang et al., 1994). CXCL6 mRNA expression is induced in fibroblasts and an osteosarcoma cell line stimulated with IL-1 and phorbol ester (Froyen et al., 1997).

CXL9 (Mig) and CXL10 (IP-10) Both CXCL9 and CXCL10 mRNA expression is induced in macrophages by IFNc treatment (Farber, 1997). IFNa/b and LPS can induce CXCL10 but not CXCL9 mRNA expression in macrophages. TNFa can induce CXCL9 mRNA expression in endothelial cells, whereas only IFNc is effective in keratinocytes and fibroblasts. The determination of the mouse and human CXCL10 gene revealed the presence of an interferon-responsive element (ISRE) and two NFjB binding sites within 250 nucleotides of the transcription start site (Vaguri and Farber, 1990). These elements contribute to IFNc- and LPS-induced CXCL10 gene transcription (Ohmori and Hamilton, 1993). The CXCL9 gene possesses a similar but distinct element to the IFNc activation site and this site is important for IFNc-induced gene expression (Wright and Farber, 1991; Wong et al., 1994; Guyer et al., 1995).

CXCL12 (SDF-1) CXCL12 has been identified as the factor expressed by bone marrow stromal cells (Tashiro et al., 1993; Nagasawa et al., 1994). Various types of tissues including neuronal, cardiac, vascular, and hematopoietic systems, express CXCL12 mRNA and protein constitutively in the absence of stimuli generated from viral or bacterial products (McGrath et al., 1999; Nagasawa, 2000). The absence of a TATA-like sequence in the promoter region suggests that the CXCL12 gene is a house-keeping gene (Shirozu et al., 1995). However, DNA-damaging agents caused an increase in CXCL12 expression in immature osteoblasts and endothelial cells in bone marrow, thereby inducing stem cell homing to bone marrow (Ponomaryov et al., 2000).

CXL13 (BLC/BCA-1) BLC/BCA-1/CXCL13 was identified from expressed tag DNA sequences (Legler et al., 1998; Gunn et al.,

1998). BLC/BCA-1 was then identified as the ligand for CXCR5 (Forster et al., 1996), which has a fundamental role in lymphoid follicle formation. In situ hybridization revealed that CXCL13 transcripts are detected in B cell-rich zones in spleen, germinal centers in Peyer’s patches, and follicles in lymph nodes (Gunn et al., 1998), showing a similar distribution to follicular dendritic cells. Thus, follicular dendritic cells are presumed to be a major cellular source. Of interest is that CXCL13 expression is highly enhanced in Helicobacter pylori-induced mucosa-associated lymphoid organs and gastric lymphoma tissues (Mazzucchelli et al., 1999), although the pathologic relevance remains elusive.

HUMAN CXC CHEMOKINE RECEPTORS (TABLE 46.2) Signal transduction mechanism All chemokine receptors are membrane-bound molecules composed of seven transmembrane domains and are coupled to G proteins at the carboxylterminal portion and possibly the third intracellular loop (Murphy et al., 2000; Murdoch, 2000). Following ligand binding, chemokine receptors are presumed to be internalized and subsequently recycled, reappearing on the cell surface within 60 minutes, as observed on the human CXCL8 receptor system (Samanta et al., 1990). In the case of the human CXCL8 receptor system, the inhibition of recycling reduced CXCL8mediated chemotaxis, suggesting its important role in chemokine-mediated chemotaxis. Moreover, the ligand binding activates pertussis toxin-sensitive and receptor-coupled G proteins, particularly Gai proteins (Murdoch, 2000). G proteins, upon conversion to the guanosine triphosphate (GTP)-bound form, dissociate into Ga and Gbc subunits. Generated Gbc remains near the receptor–ligand complexes, recruiting and activating phosphatidyl-3-kinase-c (PI-3Kc), which in turn generates phosphatidylinositol 3,4,5trisphophate (PIP3) (Servant et al., 2000). PIP3 activates protein kinase B (Akt) and GTPases, resulting in directed cell migration. Deficiency in phospholipase C (PLC)b23, a key enzyme in generating inositol 1,4,5-trisphosphate (IP3) and diacyl glycerol (DAG), abrogates CXCL8-

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HUMAN CXC CHEMOKINE RECEPTORS

induced increase in intracellular calcium concentrations, protein kinase C phosphorylation, p47 phox translocation, and superoxide anion generation but enhances chemokine-mediated chemotaxis (Li et al., 2000). PI-3Kc-deficient mice exhibit a reduction in CXCL8-mediated neutrophil migration, PIP3 generation, Akt phosphorylation, and respiratory burst with a robust increase in intracellular calcium concentration in response to CXCL8 (Hirsch et al., 2000). These results suggest that chemokine-induced chemotaxis is mediated through both PI-3Kc-independent and -dependent pathways. A human hematopoietic cell-specific CDM family protein, DOCK2, is indispensable for Rac activation and subsequent T and B lymphocyte migration in response to SDF-1/ CXCL12 and BLC/CXCL13 (Fukui et al., 2001). However, the roles of these family proteins in other chemokine receptor-mediated cell migration remains unclear. G protein-coupled receptor-mediated signals can be down-regulated by RGS (regulator of G protein signaling) proteins. RGS proteins appear to enhance endogenous GTPase activities and thus decrease the half-life of the active GTP-bound state. Among RGS proteins, RGS1, RGS3 and RGS4 reduce CXCL8mediated migration and adherence (Druey et al., 1996). Moreover, expression of RGS proteins also reduces CXCL8-induced mitogen-activated kinase activation (Bowman et al., 1998).

CXCR1 and CXCR2 CXCL8 binds to two distinct types of receptors, CXCR1 and CXCR2, with similar high affinity (Murphy and Tiffany, 1991; Holmes et al., 1991). CXCR1 and CXCR2 consist of 350 and 360 amino acids, respectively. Both genes are present in a cluster on human chromosome 2q34–35 with one pseudogene. CXCR1 and CXCR2 possess high-sequence homology exceeding 80% at the amino-acid level, except in their amino-terminal portions. These receptors can bind other CXC chemokines with ELR motif amino-terminal to the first cysteine. However, because of the differences in their amino-terminal portion, their binding specificities differ; CXCR2 binds CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL7 with high affinity while only CXCL5 and CXCL6 bind CXCR1, but with lower affinity than CXCL8 (Murphy et al., 2000). In leukocytes, CXCR1 and CXCR2 are coordinately

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expressed (Chuntharapai et al., 1994; Morohashi et al., 1995). Hence, there remains a question concerning the differential roles of CXCR1 versus CXCR2. CXCR1 exhibits a slightly lower affinity for CXCL8 in vitro and requires more CXCL8 for recycling than CXCR2 (Chuntharapai and Kim, 1995). Thus, CXCR1 may transduce CXCL8 signals at a higher concentration than does CXCR2. Moreover, CXCL8-induced activation of NADPH oxidase and phospholipase D is mediated in vitro mainly by CXCR1 (Jones et al., 1996). CXCR1- but not CXCR2-mediated signals attenuate the response to other types of chemoattractants, such as C5a and fMLP without any effects on their receptor expression (Richardson et al., 1998). In contrast, C5a and fMLP attenuate CXCR2- but not CXCR1-mediated signals (Richardson et al., 1995). Thus, CXCR1 and CXCR2 can tune the response to CXCL8 in a coordinate manner.

CXCR3 Human CXCR3 consists of 368 amino acids and its gene is localized on chromosome Xq13 (Loetscher et al., 1998). CXCR3 binds CXCL9, CXCL10 and CXCL11, all of which chemoattract and induce calcium flux in activated T cells and tumor-infiltrating lymphocytes. The rank order of binding affinity is CXCL11  CXCL9  CXC10 (Loetscher et al., 1998; Cole et al., 1998). Curiously, some human CC chemokines, CCL11/eotaxin and CCL13/MCP-4, can bind CXCR3-transfected cells but with much lower affinity (Weng et al., 1998). CXCR3 is expressed on a proportion of T cells, B cells and natural killer (NK) cells (Qin et al., 1998). CXCR3 expression on T cells is markedly enhanced upon T-cell activation, accompanied with enhanced responsiveness to the ligands (Rabin et al., 1999). Blood T cells expressing CXCR3 are mostly CD45RO memory cells and express high levels of b1 integrins. Moreover, several lines of evidence suggest that CXCR3 is mainly expressed on TH1 cells (Qin et al., 1998; Sallusto et al., 1998).

CXCR4 Human CXCR4 consists of 352 amino acids (Federsppiel et al., 1993; Nomura et al., 1993;

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Loetscher et al., 1994) and its gene is localized on human chromosome 2q21 (Herzog et al., 1993). CXCR4 binds CXCL12 exclusively with a high affinity and is expressed on hematopoietic progenitor cells, B cells, T cells, endothelial cells and dendritic cells (Bleul et al., 1996b; Oberlin et al., 1996). Ablation of CXCR4 gene results in embryonal or perinatal death with defective development of cardiac ventral septae, large vessels and cerebellar architecture (Tachibana et al., 1998; Zou et al., 1998), indicating the indispensable roles of the CXCR4–CXCL12 axis in organogenesis. However, the functions of CXCR4 in adults remain to be investigated. Some human immunodeficiency virus (HIV)-1 strains utilize CXCR4 along with the CD4 antigen (Berger et al., 1998). These strains are now called X4 strains and are typically isolated late in the course of the infection and correlate more or less with T-cell line tropism. gp120 from the HIV envelope glycoprotein can bind to CXCR4 in the presence of CD4 (Lapham et al., 1999), although CD4-independent association of gp120 with CXCR4 has been demonstrated. While purified X4 gp120 inhibits monocyte response to CXCL12 through the interaction with CXCR4 (Wang et al., 1998), it can induce the apoptosis of the human neuronal cell line through the interaction with CXCR4 on the cell surface (Hesselgesser et al., 1998; Zheng et al., 1999). These findings may be relevant to the pathogenesis of HIV encephalitis and AIDS dementia.

memory/effector T cells whereas IL-2 induces its down-regulation (Sallusto et al., 1999). Up-regulation of CXCR5 on activated CD4 T cells implies a role for the movement of helper T cells into B-cell follicles in the lymph nodes. In addition to lymphocytes, in situ hybridization detected CXCR5 transcript in brain, specifically in the granule and Purkinje cell layer of mouse cerebellum (Kaiser et al., 1993).

CXCR6 Human CXCR6, previously called BONZO or STRL33, consists of 342 amino acids and binds CCL16 exclusively (Matloubian et al., 2000). Its gene is localized on chromosome 3p21.3. CXCR6 is expressed on activated and unstimulated CD8 T cells, activated and naive CD4 T cells, NK cells, natural killer T (NKT) cells and CD19 B cells (Liao et al., 1997; Deng et al., 1997; Sharron et al., 2000). Moreover, CXCR6 expression is presumed to define type I polarized T cells with extralymphoid tissue homing potential (Kim et al., 2001). Furthermore, CXCR6 can function as a co-receptor for the entry of HIV-1 and simian immunodeficiency virus (SIV) (Liao et al., 1997; Deng et al., 1997).

BIOLOGICAL PROPERTIES OF HUMAN CXC CHEMOKINES

CXCR5

CXCL8 (IL-8) and other ELR CXC chemokines

Independent groups have cloned two cDNAs encoding human CXCR5 and they are monocyte-derived receptor 15 (MDR15) and Burkitt’s lymphoma receptor 1 (BRL1) (Dobner et al., 1992; Barella et al., 1995). The open reading frame of MDR15 has 325 amino acids and is 45 codons shorter at the amino-terminus, than BRL1, due to alternative splicing of the gene. However, both bind CXCL13 exclusively (Legler et al., 1998; Gunn et al., 1998). CXCR5 is detected on all peripheral blood and tonsillar B cells but only on a fraction of cord blood and bone marrow B cells (Forster et al., 1994). CXCR5 is expressed by a small subset of peripheral blood CD4 (14%) and CD8 (2%) T cells with a memory phenotype. After T-cell receptor stimulation, CXCR5 is up-regulated on

CXCL8 was first purified and molecularly cloned as a neutrophil chemotactic factor (Yoshimura et al., 1987). Subsequent studies demonstrated that CXCL8 exerts a wide variety of actions on neutrophils, including degranulation, increase in intracellular calcium concentrations, respiratory burst, and adherence to endothelium cells (Oppenheim et al., 1991; Mukaida, 2000). CXL8 can enhance intracellular killing of Mycobacterium fortuitum (Nibbering et al., 1993) and Candida albicans (Djeu et al., 1990). CXCL8 activates 5-lipoxygenase with release of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) in the presence of exogenous arachidonic acid (Schroder, 1989) and induces the synthesis of platelet-activating factor (PAF) in neutrophils (Bussolino et al., 1992).

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Moreover, CXCL8 induces the shedding of L-selectin and up-regulates b2 integrins (CD11b/CD18 and CD11c/CD18) and complement receptor type 1 (CR1/CD35) on neutrophils, and alters the avidity of the constitutively expressed integrin molecules (Carveth et al., 1989; Detmers et al., 1990; Huber et al., 1991). CXCL8 promotes adhesion of neutrophils to plastic, extracellular matrix proteins and unstimulated as well cytokine-stimulated endothelial monolayers through the interaction with CD11b/CD18 (Carveth et al., 1989). CXCL8 stimulates transendothelial migration of neutrophils (Huber et al., 1991; Smith et al., 1991). Other ELR CXC chemokines, including CXCL1, 2, 3, 5, 6 and 7 can also induce chemotaxis, degranulation, an increase in intracellular calcium concentration and respiratory burst of neutrophils (Baggiolini et al., 1997). CXCL1 and CXCL8 but not CXCL7 elicit a weak increase in intracellular calcium concentrations and a weak respiratory burst in monocytes (Walz et al., 1991b). Moreover, in the presence of the shear stress, monocyte adherence to endothelium cells is markedly increased in response to CXCL8 (Gerszten et al., 1999), suggesting a potential role of CXCL8 also in in vivo macrophage migration. In addition, CXCL1, 2 and 3 caused monocyte adherence to endothelium stimulated with minimally modified low-density lipoprotein (Schwartz et al., 1994). However, the effects of other ELR CXC chemokines on monocytes have not yet been clarified. CXCL8 induces basophils to exhibit chemotaxis (Geiser et al., 1993), adhere to endothelium cells (Bacon et al., 1994), and increase intracellular calcium concentrations (Kriegler et al., 1992). CXCL8 induces a release of histamine and leukotrienes from IL-3-pretreated basophils at higher concentrations (Dahinden et al., 1989) and inhibits histamine release induced by IL-3 at lower concentrations (Bischoff et al., 1991; Kuna et al., 1991). CXCL7 also exhibits similar effects on basophils as CXCL8 (Bischoff et al., 1991; Kriegler et al., 1992). CXCL1, 2 and 3 are also chemotactic for basophils and induce an increase in intracellular calcium concentrations in basophils (Geiser et al., 1993). CXCL8 induces an increase in intracellular calcium concentration, shape change, and release of eosinophil peroxidase in eosinophils isolated from patients with hypereosinophilic syndrome (Kernen

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et al., 1991). Although CXCL8 does not exhibit any chemotactic activity against normal eosinophils (Schroder et al., 1987), it induces chemotaxis of eosinophils obtained from atopic patients’ peripheral blood (Bruijnzeel et al., 1993; Warringa et al., 1993) or those pretreated with IL-3 or granulocyte–macrophage colony-stimulating factor (GM-CSF) (Warringa et al., 1991). No chemotactic activities against eosinophils have been detected in CXCL1, 2, 3 and 6 (Geiser et al., 1993; Wuyts et al., 1997). Intradermal injection of CXCL8, CXCL1, CXCL6 and CXCL7 induces neutrophil infiltration (Foster et al., 1989; Rampart et al., 1989; Van Damme et al., 1990; Van Osselaer et al., 1991; Proost et al., 1993a, 1993b; Wuyts et al., 1997). Upon co-injection with vasodilators such as prostaglandin E2, plasma extravasation is observed at the injected sites. Intravenous injection of CXCL8 and CXCL7 results in an immediate leukopenia followed by a profound neutrophilia (Van Damme et al., 1988, 1990). Although the kinetics are similar for CXCL8 and CXCL7, the effects with CXCL8 are more profound. Intradermal injection of CXCL8 induces T-cell migration into the injected site (Larsen et al., 1989). CXCL8 exhibits chemotactic activity against freshly isolated T lymphocytes, whereas incubation of purified T lymphocytes resulted in a progressive decrease in CXCL8-binding sites on T lymphocytes in association with reduced chemotactic response. Addition of a TH1 cytokine, IFNc, into cultured T lymphocytes, restored CXCR1 and CXCR2 expression on these cells accompanied by an almost complete restoration of their chemotactic response to CXCL8 (Tani et al., 1998). Moreover, IFNc and TNFa enhance CXCR1 and CXCR2 expression on both freshly isolated CD4 and CD8 T lymphocytes with enhanced chemotactic response to CXCL8 and CXCL1 (Jinquan et al., 1995). In contrast, TH2 cytokines, IL-4, IL-10 and IL-13 decrease CXCR1 and CXCR2 expression on both freshly isolated CD4 and CD8 T lymphocytes with reduced chemotactic response to CXL8 and CXCL1 (Jinquan et al., 1995). In addition to its direct effects on T cells, CXCL8 indirectly causes T-cell chemotaxis by inducing neutrophils to release from their granules several factors such as defensin-1, defensin-2 and CAP37/azurocidin with chemotactic activities against T lymphocytes (Chertov et al., 1996). Thus, a selective depletion of neutrophils inhibits the CXCL8-induced

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in vivo migration of CD4 but not CD8 lymphocyte in rats (Kudo et al., 1991). Human CXCL8 and CXCL1 are chemotactic for B cells (Jinquan et al., 1997) and CXCL8 selectively inhibits IL-4-induced immunoglobulin E (IgE) production (Kimata et al., 1992, 1995). Human CXCL8 and mouse CXCL2 induce rapid mobilization of hematopoietic progenitor cells in monkeys and mice, respectively (Laterveer et al., 1996; Wang et al., 1997). This mobilization occurs in a leukocyte function-associated antigen (LFA)-1dependent manner, probably by inducing gelatinase B (matrix metalloproteinase 9; MMP-9) from neutrophils (Pruijt et al., 1999). Moreover, CXCL8 suppresses colony formation of immature subsets of myeloid progenitor cells stimulated with GM-CSF and steel factor (Broxmeyer et al., 1993). CXCL8 and CXCL7 inhibit megakaryocyte colony formation (Gewirtz et al., 1995). Angiogenic activities have been documented on several ELR CXC chemokines, including CXCL1, 2, 5, 6, 7 and 8 (Koch et al., 1992; Strieter et al., 1995a, 1995b). These chemokines can induce migration and proliferation of endothelial cells, which express CXCR1 and CXCR2. Moreover, antibodies to CXCL8, CXCL1 or CXCL5 significantly reduce angiogenic activities present in tumor extracts (Smith et al., 1994; Norgauer et al., 1996; Arenberg et al., 1998). Thus, these chemokines may have crucial roles in tumor neovascularization and eventually tumor progression. CXCL1 has been identified as a melanoma growth factor. CXCL1 and CXCL2 induce melanoma cell proliferation through the interaction with CXCR2. Human CXCL8 also induces proliferation and haptotactic migration of melanoma cells (Wang et al., 1990; Schadendorf et al., 1993). However, recent analysis failed to detect CXCR2 transcripts in melanoma cell lines (Muller et al., 2001), raising questions about the roles of CXCR2 in the pathogenesis of melanomas. CXCL8 enhances replication of cytomegalovirus in fibroblasts (Murayama et al., 1994), while it induces a loss of focal adhesion in fibroblasts and promotes chemotaxis and chemokinesis of these cells (Dunlevy and Couchman, 1995). CXCL1 has growthstimulatory activity for fibroblasts and decreases collagen expression in rheumatoid synovial fibroblasts (Unemori et al., 1993). CXCL8 is chemotactic and mitogenic for keratinocytes (Michel et al., 1992;

Tuschil et al., 1992) and smooth muscle cells (Yue et al., 1994).

CXCL4 (PF-4) A biochemical analysis identifies CXCL4 receptor as a chondrotinsulfate proteoglycan (Petersen et al., 1998). CXCL4 is a selective inducer of secondary granule exocytosis of neutrophils without inducing chemotaxis, increase in intracellular calcium concentration or degranulation of lysosomal enzymes (Petersen et al., 1996). CXCL4 can reverse heparin anticoagulation without inducing thrombocytopenia, leukopenia, activation of complements and systemic hypotension (Cook et al., 1992). However, an autoantibody to the heparin–CXCL4 complex is responsible for heparininduced thrombocytopenia (Newman and Chong, 2000). CXCL4 has angiostatic effects through inhibiting endothelial cell proliferation (Strieter et al., 1995b; Luster et al., 1995) and/or inhibiting the action of vascular endothelial cell growth factors (Gengrinovitch et al., 1995). CXCL4 suppresses the mitogenic activity of basic fibroblast growth factor on fibroblasts (Watson et al., 1994). Moreover, CXCL4 inhibits hematopoiesis including megakaryocytopoiesis (Han et al., 1990; Lecomte-Raclet et al., 1998).

CXCR3 ligands: CXCL9 (Mig), CXL10 (IP-10) and CXCL11 (I-TAC) CXCL10 is chemotactic for monocytes and CD4 memory T cells and induces the adherence of antiCD3-stimulated T cells to human umbilical endothelial cells (Taub et al., 1993). CXCL10 chemoattracts NK cells and enhances their killing activity against K562 cells (Taub et al., 1995). CXCL9 as well as CXCL11 induces the chemotaxis of activated T cells (Liao et al., 1995; Cole et al., 1998). CXCR3, a receptor for these chemokines, is expressed mainly by memory/activated T cells, TH0 cells, and TH1 cells (Bonecchi et al., 1998; Sallusto et al., 1998). Several lines of evidence suggest that these chemokines regulate the trafficking of TH1 cells and elicit TH1 response (Qin et al., 1998; Gangur et al., 1998). However, at a single cell level, correlation between CXCR3 expression and IFNcproducing capacity of polarized T cells is poor (Nanki and Lipsky, 2000). In addition, CXCL10 as well as CXCL9 suppresses colony formation from hematopoietic

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MICE WITH CXC CHEMOKINE GENE MODIFICATION

progenitor cells in vitro (Sarris et al., 1993). CXCL10 inhibits CXCL8- or basic fibroblast growth factor-mediated angiogenesis (Luster et al., 1995; Angiolillo et al., 1995; Addison et al., 2000). Moreover, several lines of evidence suggest that inhibition of tumor angiogenesis by IL-12 is mediated by endogenously produced CXCL10 and that CXCL10 as well as CXCL9 inhibits non-small cell lung carcinoma tumor growth and metastasis by inhibiting neovascularization (Arenberg et al., 1996a; Addison et al., 2000). Romagnani and colleagues (2001) demonstrated that microvascular endothelial cells express CXCR3 at the S/G2–M phase of their cell cycle and that CXCL9, CXCL10 and CXCL11 inhibit endothelial cell proliferation through the interaction with CXCR3.

CXCL12 (SDF-1/PBSF) CXCL12 was first identified as the growth-stimulating factor for a stromal cell-dependent pre-B-cell clone DW34 (Nagasawa et al., 1994). CXCL12 elicits transendothelial chemotaxis of lymphocytes and monocytes, but not neutrophils in vitro, and is a potent mononuclear cell attractant in vivo (Bleul et al, 1996a). Moreover, CXCL12 is chemotactic for human CD34 hematopoietic progenitor cells (Aiuti et al., 1997), murine pro-B cells and pre-B cells, but not mature B cells (D’Apuzzo et al., 1997). Furthermore, CXCR4 is expressed on CD4 CD8 double-positive thymocytes and is down-modulated after a positive selection, suggesting that CXCL12 has an important role in topological distribution of thymocytes in thymus (Suzuki et al., 1999). CXCR4 has been identified as one of the co-receptors for HIV-1 entry (Feng et al., 1996) and CXCL12 can block HIV-1 entry through CXCR4 (Bleul et al., 1996b). Gene targeting of either CXCR4 or CXCL12 has revealed that they have major roles in organogenesis of various types of organs. Mice deleted in either CXCR4 or CXCL12 gene exhibit similar phenotypes including defects in B-cell lymphopoiesis and myelopoiesis, defective formation of large vessels supplying the gastrointestinal tract, and aberrant fetal cerebellar development (Nagasawa et al., 1996; Tachibana et al., 1998; Zou et al., 1998). The latter defects can be explained by the observation that CXCL12 can induce the migration of microglial cells and astrocytes in the brain (Tanabe et al., 1997).

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CXCL13 (BLC/BCA-1) CXCL13 exhibits a potent chemotactic activity against human blood B lymphocytes, but is not active on freshly isolated or IL-2-stimulated T lymphocytes, monocytes and neutrophils (Legler et al., 1998; Gunn et al., 1998). However, subsequent study demonstrated that CXCR5 expression was up-regulated on murine antigen-specific CD4 T cells in vivo when mice were immunized and that CXCR5-expressing T cells exhibited a vigorous chemotactic response to CXCL13 (Ansel et al., 1999). Moreover, gene targeting of either CXCR5 or CXCL13 results in defective germinal-center formation in lymphoid organs (Forster et al., 1996; Ansel et al., 2000). Thus, CXCL13 is presumed to direct the migration of both B cells and antigen-specific T cells into germinal centers.

MICE WITH CXC CHEMOKINE GENE MODIFICATION CXCL8, ELR CXC chemokines and CXCR2 Simonet et al. (1994) prepared mice overexpressing the human CXCL8 transgene ubiquitously. These mice exhibit an elevation in circulating neutrophils and a decrease in L-selectin expression on mature neutrophils but not neutrophil precursors. Neutrophils are accumulated in the microcirculation of the lungs, liver and spleen without any signs of neutrophil extravasation, plasma exudation or tissue damage. However, neutrophil migration into the inflamed peritoneal cavity is severely impaired in transgenic mice.We also observed that mice expressing the CXCL8 transgene exhibit exaggerated cadmiuminduced liver injury, with reduced neutrophil infiltration into the liver (Horiguchi et al., 2000). In contrast, in mice expressing KC (mouse gro-b) transgene in a tissue-specific manner, a large number of neutrophils accumulate in the corresponding tissues which leads to tissue damage (Lira et al., 1994; Tani et al., 1996). Lung-specific transgenic expression of KC induces neutrophil accumulation in the bronchi and terminal bronchioles (Lira et al., 1997) but enhances resistance to Klebsiella pneumoniae infection (Tsai et al., 1998).

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The discrepancies in the phenotypes between CXCL8 and KC transgenic mice may be explained by the different gene expression pattern or differences in species. As CXCR2 but not the CXCR1 gene has been identified in mice to date, mice deficient in CXCR2 have been prepared to elucidate the roles of ELR chemokines in physiologic and pathologic conditions. Mice deficient in the CXCR2 gene are born healthy, develop normally and are fertile (Cacalano, et al., 1994). However, these mice exhibit neutrophilia, splenomegaly (resulting from an increase in metamyelocytes and mature neutrophils), lymphadenopathy (resulting from an increase in B lymphocytes) and increased serum IgE levels, when they are kept under specific pathogen-free conditions. IgE production in allergen-induced pulmonary inflammation is also enhanced in CXCR2-deficient mice, compared with wild-type mice (De Sanctis et al., 1999). Under germfree conditions, CXCR2-deficient mice do not exhibit neutrophilia and the number of myeloid progenitor cells in CXCR2-deficient mice is comparable to that in wild-type mice (Broxmeyer et al., 1996). Broxmeyer and colleagues concluded that CXCL8 mediates environmentally inducible suppressive effects on myeloid progenitors. However, our immunofluorescence analysis failed to detect CXCR1 or CXCR2 expression on human and mouse hematopoietic progenitor cells (Morohashi et al., 1995; Wang et al., 1996), raising the possibility that CXCL8 exerts its effects indirectly on hematopoietic progenitor cells. CXCR2-deficient mice show reduced neutrophil migration into aseptically inflamed tissues followed by attenuated inflammatory reaction and tissue damage (Cacalano et al., 1994; Terketlaub et al., 1998; Brito et al., 1999; Becker et al., 2000). However, CXCR2deficient mice show an enhanced susceptibility to mucosal and systemic Candida albicans infection with reduced neutrophil infiltration into the infected tissues (Balish et al., 1999). Moreover, in an acute pyelonephritis model caused by Escherichia coli infection, neutrophils cannot transverse the mucosal barrier and eventually accumulate under the epithelium in CXCR2-deficient mice (Frendeus et al., 2000; Godaly et al., 2000; Hang et al., 2000). Subepithelial neutrophil entrapment results finally in renal scarring. In Listeria monocytogenes infection, CXCR2deficient mice often develop chronic infection

compared with wild-type mice (Czuprynski et al., 1998). These observations would imply that CXCR2 and its ligands have essential roles in the eradication of infectious microbes. Interestingly, in atherosclerosis-susceptible lowdensity lipoprotein receptor-deficient mice, the lack of CXCR2 expression on bone marrow-derived cells reduces macrophage accumulation in the intima (Boisvert et al., 1998). This may be explained by the observation that CXCL8 quickly causes rolling monocytes to adhere firmly on to endothelium cells through the interaction between E-selectin and b2 integrin under flow conditions (Gerstzen et al., 1999).

CXCR3 and CXCL10 (IP-10) Luster et al. (1998) created transgenic mice which constitutively express CXCL10 in keratinocytes. These mice develop normally with no leukocyte infiltration into any organs and have a normal cutaneous hypersensitivity response. However, the mice have a delayed wound healing response with impaired neovascularization, suggesting that CXCL10 can inhibit the neovascularization associated with a physiologic response even in vivo. Mice deficient in CXCR3 or CXCL10 are born healthy and develop normally. Acute allograft rejection does not occur in the absence of either CXCR3 in recipients (Hancock et al., 2000) or CXCL10 in donors (Hancock et al., 2001), with a marked reduction in CD45, CD4, CD8, or CD25 cells but not macrophages in the transplanted organ. These observations suggest that CXCL10–CXCR3 have major roles in the development of acute allograft rejection, through inducing lymphocyte migration into the transplanted organs.

CXCR4 and CXCL12 (SDF-1/PBSF) CXCL12 homozygously null mutant mice start to die in utero after day 15.5 of embryogensis and the surviving animals die within an hour of birth (Nagasawa et al., 1996). Numbers of B-cell progenitor cells in the mutant mice are severely reduced in fetal liver and bone marrow, while myeloid progenitors are reduced only in the bone marrow but not in fetal liver. In addition, the mutants have a cardiac ventricular septal

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defect. CXCR4-deficient mice show similar defects in B-cell lymphopoiesis and myelopoiesis and cannot survive beyond the neonatal period (Tachibana et al., 1998; Zou et al., 1998). The defects in B-cell lymphopoiesis and myelopoiesis can be explained by the observations that CXCL12 and CXCR4 have essential roles in the retention of B-cell precursor cells and granulocytic precursor cells in bone marrow (Ma et al., 1999; Hargreaves et al., 2001). An extensive and complementary CXCL12 and CXCR4 mRNA expression pattern is observed in the nervous, vascular, hematopoietic, cardiac and craniofacial systems during embryogenesis (McGrath et al., 1999), suggesting the involvement of CXCL12 and CXCR4 in organogenesis of nervous and vascular systems. This notion was corroborated by defective formation of large vessels supplying the gastrointestinal tract observed in CXCL12- or CXCR4-deficient mice (Tachibana et al., 1998), and aberrant fetal cerebellar development in CXCR4-deficient mice, which is characterized by many proliferating granule cells invading cerebellar anlage (Zou et al., 1998).

CXCL13 and CXCR5 CXCR5-deficient mice lack inguinal lymph nodes and possess no or few phenotypically abnormal Peyer’s patches (Forster et al., 1996). Moreover, the migration of lymphocytes into splenic follicles is severely impaired with morphologically altered primary lymphoid follicles. Furthermore, activated B cells fail to migrate from the T cell-rich zone into B-cell follicles of the spleen. However, when CXCR5-deficient mice were immunized with T cell-dependent antigen, ectopic germinal centers developed within the T-cell zone, accompanied by the affinity maturation of Ig genes (Voigt et al., 2000). These observations suggest that B cells in CXCR5-deficient mice are functionally competent in terms of Ig affinity maturation in the presence of T-cell help. Similar phenotypes are observed in CXCL13deficient mice (Ansel et al., 2000). A detailed analysis demonstrated that in addition to mediating B-cell chemoattraction, CXCL13 induces B cells to upregulate membrane lymphotoxin a1b2, a cytokine that promotes follicular dendritic cell development and CXCL13 production. Thus, CXCL13 and lymphotoxin a1b2 form a positive feedback loop, which is important

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for follicle development. This notion was further supported by the observation that in pancreatic islets ectopic CXCL13 expression results in formation of lymph node-like structures (Luther et al., 2000). The lymph node-like structures contain B- and T-cell zones, high endothelial venules, stromal cells and CC chemokine SLC (CCL21). Moreover, lymph node-like structure formation is reversed by blocking lymphotoxin a1b2.

THE ROLES OF CXC CHEMOKINES IN PATHOLOGY CXC chemokines and infection Recruitment of leukocytes to sites of infection is essential in host defense and is involved in the clearance of inciting factors. CXC chemokines, particularly CXCL8, are frequently detected at sites of infections. This may be explained by the observations that various types of microbes and their products can induce CXCL8 protein production in a variety of cell lines in vitro (Mukaida, 2000). Intravenous injection of LPS into normal human volunteers causes a rapid increase in plasma CXCL8 levels, peaking at 2 hours and returning to baseline levels within 5 hours of LPS injection (Martich et al., 1991). Moreover, in lethal and sublethal sepsis induced by infusing primates with live E. coli and LPS, respectively, CXCL8 is detected in the circulation. The CXCL8 levels are higher in animals with lethal bacteremia than in those with sublethal endotoxemia (Van Zee et al., 1991). Furthermore, an elevation in plasma CXCL8 levels precedes neutrophil accumulation and activation as evidenced by an elevated neutrophil-derived elastase level (Redl et al., 1991). Furthermore, similar results are also observed in patients with sepsis (Friedland et al., 1992; Hack et al., 1992). However, CXCR2 is presumed to be protective against local infection because CXCR2-deficient mice fail to control several types of local infections (Czuprynski et al., 1998; Balish et al., 1999; Frendeus et al., 2000; Godaly et al., 2000; Hang et al., 2000). In addition to bacterial infections, several lines of evidence suggest the potential role of CXCL8 in viral infection. Various types of viruses and viral products

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can induce CXCL8 production in a wide variety of types of cells (Mahe et al., 1991; Murayama et al., 1997; Mastronarde et al., 1998; Mori et al., 1998; Polyak et al., 2001a). CXCL8 can enhance human cytomegalovirus replication in fibroblasts. Among patients infected with hepatitis C virus, serum CXCL8 levels are inversely correlated with sensitivity to interferon treatment (Polyak et al., 2001b). This may be explained by the observation that CXCL8 inhibits the antiviral activities of interferon a or b (Khabar et al., 1997). CXCR4 has been identified as a co-receptor for the entry of HIV-1, particularly X4 strains (Feng et al., 1996), and CXCL12 blocks HIV-1 entry (Bleul et al., 1996b). However, because X4 strains are typically isolated late in the course of infection, the effectiveness of CXCL12 and/or a related molecule(s) against HIV-1 infection remains to be investigated. Accumulating evidence indicates that various types of viruses have molecules resembling chemokines and chemokine receptors. Herpesvirus saimiri, a T-lymphotropic virus of several non-human primates, harbors ECRF3 (Ahuja and Murphy, 1993). ECRF3 encodes a functional receptor for CXCL8, CXCL1 and CXCL7 but not for CC chemokines. UL146 and UL147 of human cytomegalovirus (HCMV) encode CXC chemokine-like molecules, vCXC-1 and vCXC-2, respectively (Penfold et al., 1999). vCXC-1 binds human CXCR2 and induces calcium mobilization, chemotaxis and degranulation in human neutrophils. Moreover, HCMV harbors US28, which encodes a functional receptor for CCL2, CCL3, CCL4 and CCL5 (Gao and Murphy, 1994), and is involved in chemokine-mediated smooth muscle cell migration (Streblow et al., 1999). Human herpesvirus 8 (Kaposi’s sarcoma herpes virus; KSHV) encodes two C–C chemokine-like proteins, viral macrophage inflammatory protein (vMIP)-I and vMIP-II (Boshoff et al., 1997). Both proteins inhibit HIV-1 infection of peripheral blood mononuclear cells by using CCR8 as a receptor (Sozzani et al., 1998; Dairaghi et al., 1999; Haque et al., 2001). It is interesting that both exhibit potent angiogenic activity in the chorioallantoic assay (Boshoff et al., 1997), suggesting a possible pathogenic role in abundant neovasularization frequently observed in Kaposi’s sarcoma. Moreover, KSHV open reading frame (ORF) 74 also encodes a chemokine

receptor-like protein. Human CXCR2 is the closest homolog to this protein (Burger et al., 1999). CXCL8 and CXCL1 activate the ORF74-mediated signaling pathway (Gershengorn et al., 1998) while CXCL10 inhibits it (Geras-Raaka et al., 1998). Among G proteincoupled receptors, a DRY sequence in the second intracellular loop is highly conserved, whereas ORF74 shows a VRY sequence. The exchange of Asp138 of the DRY sequence in the CXCR2 with a Val results in an enhanced transformation potential similar to ORF74 (Burger et al., 1999). Moreover, ORF74 constitutively activates phosphoinositideinositoltrisphosphate protein kinase C, c-jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, and eventually induces malignant transformation and angiogenesis (Arvanitakis et al., 1997; Bais et al., 1998). Furthermore, transgenic expression of ORF74 induces an angioproliferative disease resembling Kaposi’s sarcoma (Yang et al., 2000). Thus, ORF74 may be involved in the pathogenesis of angiogenesis, a characteristic pathological feature of Kaposi’s sarcoma.

CXC chemokines and reperfusion injury Reperfusion injury after a transient ischemia frequently causes greater injury than ischemia (Friedl et al., 1990; Welbourn et al., 1991) and is observed frequently after myocardial infarction, cerebral infarction and multiple traumas. Reperfusion of blood flow results in reoxygenation of ischemic tissue, leading to the generation of reactive oxygen intermediates (ROI) such as hydrochlorous acid and hydrogen oxide (McCord, 1987). These generated ROI cause damage to every component in tissue, including nucleic acids, membrane lipids, enzymes and receptors. Reperfusion injury is pathologically characterized by the adherence and emigration of neutrophils in postcapillary venules (Engler et al., 1986). Depletion of neutrophils or the prevention of neutrophil adherence with antibodies to leukocyte adhesion molecules abolished reperfusion-induced vascular dysfunction (Korthuis et al., 1988; Horgan et al., 1990). These results imply that neutrophil infiltration has a crucial role in the establishment of reperfusion injury. ROI can activate NFjB (Janssen-Heiniger et al.,

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2000), an essential transcription factor for gene transcription of several chemokines including CXCL8, CXCL1, CXCL2 and CXCL5. CXCL8 is largely produced by infiltrated cells as well as tissue resident cells, when blood flow is reperfused into ischemic lungs (Sekido et al., 1993), myocardium (Kukielka et al., 1995) and brain (Matsumoto et al., 1997). Moreover, an antiCXCL8 antibody prevents neutrophil infiltration and tissue. In rats or mice, which lack an ortholog of CXCL8, other ELR CXC chemokines such as CXCL1, CXCL2 and CXCL5 are involved in the establishment of reperfusion injuries (Colletti et al., 1995; Lentsch et al., 1998; Chandrasekar et al., 2001).

CXC chemokines and skin diseases Neutrophil accumulation in the epidermis is a histologic characteristic of psoriasis. Skin scales obtained from psoriatic lesions contain several CXC chemokines such as CXCL8, CXCL1 and CXCL2 (Schroder et al., 1992). Moreover, CXCL1 and CXCL8 mRNA expression is restricted to the suprapapillary layers of psoriatic lesions (Kulke et al., 1996). CXCR2 but not CXCR1 expression is observed in suprabasal lesional keratinocytes (Kulke et al., 1998) and CXCL8 promotes epidermal proliferation in vitro (Tuschil et al., 1992). This may account for keratinocyte hyperproliferation in psoriatic lesions. Immunosuppressive agents, cyclosporin A and FK-506 inhibit CXCL8 and CXCL1 expression along with the resolution of skin lesions (Elder et al., 1993; Kojima et al., 1993; Schulz et al., 1993; Lemster et al., 1995). In addition, CXCL10 is detected in keratinocytes and infiltrated cells (Gottlieb et al., 1988) while CXCL9 is detected in dermal microvascular endothelial cells and infiltrated macrophages (Goebeler et al., 1998). CXCR3 is expressed on CD3 and/or CD4 lymphocytes in psoriatic lesions (Wakugawa et al., 2001; Rottman et al., 2001). Thus, CXCR3 may be involved in T-cell trafficking to the psoriatic epidermis. In rabbits, infusion of an anti-CXCL8 antibody suppresses the development of a delayed-type hypersensitivity induced by purified protein derivative, with attenuated neutrophil and lymphocyte accumulation at the injected site (Larsen et al., 1995). In hapteninduced delayed-type hypersensitivities in mice, CXCL1 (KC) and/or CXCL2 (MIP-2) are responsible for neutrophil infiltration and subsequent elicita-

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tion of contact hypersensitivity (Dilulio et al., 1999; Biedermann et al., 2000). Moreover, mast cells are major source of these chemokines (Biedermann et al., 2000). In addition, several additional chemokines, such as CXCL9, CXCL10, CCL2 (monocyte chemoattractant protein-1, MCP-1), CCL17 (thymus and activation-regulated chemokine, TARC), CCL18 (pulmonary and activation-regulated chemokine, PARC) and CCL22 (macrophage-derived chemokine, MDC) are detected in the skin lesions during the elicitation of contact hypersensitivity (Gautam et al., 1994; Goebeler et al., 2001). The expression of these chemokines is presumed to be involved in T-cell accumulation observed in contact hypersensitivity. Moreover, CXCL10, CXCL9 and CXCL11 expression is detected in the lesions of several skin diseases such as lichen planus and chronic discoid lupus erythematosus and CXCR3 is expressed by infiltrating T cells (Flier et al., 2001). Thus, CXCR3 axis may have crucial roles in T-cell accumulation observed in various types of skin diseases.

CXC chemokines and pulmonary diseases Acute respiratory distress syndrome (ARDS) is identified as an acute respiratory failure associated with a non-hydrostatic pulmonary edema (Bernard et al., 1994). In acid aspiration-induced and endotoxemiainduced ARDS in rabbits, CXCL8 is produced in the lungs and treatment with anti-CXCL8 antibodies reduces neutrophil infiltration as well as tissue damage (Folkesson et al., 1995; Yokoi et al., 1997). Moreover, CXCL8 levels in bronchoalveolar lavage fluids (BALF) correlate with the development of ARDS in atrisk patient groups (Donnelly et al., 1993). Thus, CXCL8 may have a crucial role in the establishment of ARDS. Increased CXCL8 expression by alveolar macrophage is observed in idiopathic pulmonary fibrosis (IPF) (Carre et al., 1991). CXCL8 levels in both serum and BALF are increased significantly and serum CXCL8 levels indicate the disease activity IPF (Car et al., 1994; Ziehenhagen et al., 1998). In mice, administration with anti-mouse CXCL2 antibodies attenuate bleomycin-induced pulmonary fibrosis without any effects on neutrophil infiltration (Keane et al., 1999a). However, the antibodies significantly reduce

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angiogenesis. In contrast, CXCL10 also attenuates bleomycin-induced pulmonary fibrosis by inhibiting angiogenesis (Keane et al., 1999b). Because CXCL10 contents are decreased in IPF tissues (Keane et al., 1997), the imbalance between CXCL8 and CXCL10 may be responsible for the pathogenesis of IPF, by inducing angiogensis. Several lines of evidence suggest potential roles of neutrophils in asthma (Sampson, 2000). Inducedsputum analysis in persistent asthma identifies two different inflammatory patterns (Gibson et al., 2001). The most common pattern is non-eosinophilic, associated with neutrophil influx into the lungs and increased local CXCL8 production. Moreover, increases in sputum CXCL8, as well as CCL2 and CCL4 levels, precede the exacerbation of acute asthmatic attack (Kurashima et al., 1996). Furthermore, in noninfectious status asthmaticus, neutrophil number as well CXCL8 levels in BALF is markedly increased (Lamblin et al., 1998). These observations suggest that CXCL8 contributes to the pathogenesis of asthma at various stages by inducing neutrophil infiltration and activation. CXCL8 inhalation can directly provoke bronchoconstriction in guinea pigs (Fujimura et al., 1999), suggesting a direct contribution of CXCL8 in the establishment of asthma.

CXC chemokines and glomerulonephritis Glomerulonephritis is characterized by leukocyte infiltration, mesangial cell proliferation, and increased extracellular matrix deposition (Hricik et al., 1998). This will finally lead to impaired infiltration, proteinuria and glomerulosclerosis. Because mesangial cells and renal epithelial cells produce various ELR CXC chemokines, including CXCL8, CXCL1, CXCL2 and CXCL5 in response to lipopolysaccharide, IL-1 and TNFa (Brown et al., 1996), these chemokines are presumed to be involved in glomerulonephritis by inducing leukocyte infiltration. In a rabbit model of immune complex-mediated acute glomerulonephritis, CXCL8 is detected in glomeruli with proteinuria and neutrophil infiltration (Wada et al., 1994a). Anti-CXCL8 antibody reduces neutrophil infiltration and eventually proteinuria. Similarly, in rat models of immune complex-mediated and antiglomerular basement membrane antibody-

induced acute glomerulonephritis, neutrophils are infiltrated into glomeruli, and CXCL1 and CXCL2 are detected in kidney (Wu et al., 1994; Feng et al., 1995). Moreover, in both models, the administration of either anti-CXCL1 or anti-CXCL2 antibodies reduce neutrophil infiltration as well as proteinuria. These results suggest the potential involvement of ELR CXC chemokines in the pathogenesis of acute glomerulonephritis, by inducing neutrophil infiltration. Supporting this notion, CXCL8 expression is up-regulated locally and urinary CXCL8 levels are significantly increased in acute glomerulonephritis, acute exacerbation of IgA nephropathy, and active lupus nephritis where neutrophil infiltration is frequently observed (Wada et al., 1994b; Yokoyama et al., 1998).

CXC chemokines and rheumatoid arthritis (RA) CXCL8 and CXCL5 levels are increased in synovial fluids of patients with rheumatoid arthritis (Endo et al., 1991; Koch et al., 1994). Moreover, CXCL8, CXCL1, CXCL2 and CXCL5 expression is detected in synovial fluid mononuclear cells of RA patients (Hosaka et al., 1994; Koch et al., 2001). In a mouse type II collageninduced arthritis model, CXCL2 and CCL3 are increased in synovial fluids and the administration of either anti-CXCL2 or anti-CCL3 antibodies causes a decrease in the incidence of arthritis as well as in the degree of joint inflammation (Kasama et al., 1995). This observation suggests that ELR CXC chemokines contribute to the pathogenesis of RA by inducing neutrophil infiltration into synovium. In addition to leukocyte infiltration, panus formation is a characteristic pathologic change in RA joints. Accumulating evidence suggests that CXCL8 and CXCL5 are also involved in neovascularization, which is required for panus formation (Koch et al., 2001). Accumulation of CD4 memory T cells is observed in RA joints. CD4 memory T cells in RA joints express CXCR4 and CXCL12 is expressed in the RA synovium (Nanki et al., 2000). Moreover, CXCL12 stimulates the migration of rheumatoid synovial T cells and inhibits activation-induced apotosis of T cells. In addition, a unique feature in inflammatory tissue of RA is the formation of ectopic lymphoid aggregates with geminal center (GC)-like structures. These structures are considered to contribute to the pathogenesis of RA, by

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inducing local production of the autoantibody. CXCL13 is predominantly detected in follicular dendritic cells in the GC and B cells are aggregated in regions of CXCL13 expression (Shi et al., 2001; Takemura et al., 2001). These results suggest that CXCL13 contributes to the formation of GC-like structures in RA joints. Similarly, in a lupus-prone mouse strain, dendritic cells in thymus and kidney aberrantly express a high level of CXCL13 and autoantibodyproducing B1 cells accumulate to CXCL13 (Ishikawa et al., 2001). Hence, aberrant CXCL13 expression may recruit autoantibody-producing B cells in the organs and eventually result in the development of autoimmune diseases.

CXC chemokines and tumors The roles of leukocyte infiltration in cancer still remain unclear despite a long history of investigation. There are at least two models for the functions of tumor-associated leukocytes. On one hand, tumorassociated leukocytes may reflect the host’s ineffective attempt to reject tumors. In order to augment leukocyte-mediated antitumor activity, several CXC as well as CC chemokines have been used in animal models to elicit tumor rejection and subsequent specific tumor immunity. These CXC chemokines include CXCL10 and CXCL8. CXCL10 inhibits tumor growth by inducing specific cytototoxic T-cell generation (Luster and Leder, 1993) and inhibiting neovacularization (Sgadari et al., 1996). CXCL8 induces the accumulation of neutrophils, which can directly kill tumor cells (Lee et al., 2000). On the other hand, tumorassociated leukocytes, particularly macrophages, may be a potential source of growth factors for tumor cells and endothelial cells. Thus, chemokines with chemotactic activities against monocytes/macrophages, particularly CCL2, are presumed to be involved in tumor progression by recruiting and activating macrophages to produce growth factors (Walter et al., 1991). The potential role of chemokines in malignancy extends beyond their roles in leukocyte recruitment. Most ELR CXC chemokines are chemotactic for endothelial cells. These include CXCL1, CXCL2, CXCL5, CXCL6, CXCL7 and CXCL8 (Koch et al., 1992; Strieter et al., 1995a, 1995b). All of these chemokines are angiogenic in the rat corneal vascularization

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assay. In some tumor tissues such as non-small cell lung cancer and gastric cancer tissues, CXCL8 expression correlates better with the degree of neovascularization at the tumor tissue, than other angiogenic factors such as vascular endothelial cell growth factor and basic fibroblast growth factor (Smith et al., 1994; Kitadai et al., 1998). In contrast, non-ELR CXC chemokines, including CXCL4, CXCL9 and CXCL10 can inhibit the angiogenic effects of ELR CXC chemokines and basic fibroblast growth factor (Watson et al, 1994; Luster et al., 1995; Angiolillo et al., 1995; Addison et al., 2000). Thus, the balance of ELR versus non-ELR CXC chemokines produced in tumor tissues may dictate the degree of angiogenesis, and eventually tumor aggressiveness (Strieter et al., 1995). This assumption is partly supported by the observations that the administration of anti-ELR CXC chemokine antibodies, reduces tumor growth in nude or severe combined immunodeficient mice (Arenberg et al., 1996b, 1998). Several lines of evidence demonstrate that CXCR1 or CXCR2 is expressed by several types of cancer cell lines such as melanoma, pancreatic carcinoma, colon carcinoma and gastric cell lines (Loukinova et al., 2000; Takamori et al., 2000; Brew et al., 2000; Kitadai et al., 2000). In vitro studies demonstrate that either CXCL8 or CXCL1 stimulates the proliferation of melanoma, pancreatic carcinoma and colon carcinoma cell lines, while CXCL8 induces the expression of metastasis-related genes such as matrix metalloproteinases by a gastric carcinoma cell line. However, it remains to be investigated whether these CXC chemokines directly modulate tumor growth in vivo. It has been demonstrated that some chemokine receptors, for example CXCR4, are involved in morphogenetic movements during organogenesis (Tachibana et al., 1998; Zou et al., 1998). Thus, malignant transformation may induce the reappearance of such chemokine receptors involved in this process earlier in ontogeny and eventually enhance metastatic potential. Supporting this assumption, CXCR4 and CCR7 are abundantly expressed in human breast cancer cells, although not in normal mammary gland epithelial cells (Muller et al., 2001). The cognate ligands for these receptors, CXCL12 and CCL21, exhibit peak levels of expression in regional lymph nodes and lungs, where breast cancers frequently metastasize. The signaling through CXCR4 or CCR7 mediates actin

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polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. Moreover, neutralizing the interaction of CXCL12/CXCR4 significantly reduces metastasis of breast cancer cells to regional lymph nodes and lungs, in a SCID mouse model. Furthermore, several lines of evidence suggest the involvement of CXCL12/CXCR4 in the development of bone marrow metastases in neuroblastoma and peritoneal dissemination of ovarian cancer (Geminder et al., 2001; Scotton et al., 2001). However, the pathological relevance of these observations to human neoplasms still remains to be investigated.

PERSPECTIVE AND CONCLUDING REMARKS In 1987, CXCL8/CXCL8 was discovered as a neutrophil chemotactic factor (Yoshimura et al., 1987). Since the discovery, the universe of chemokines has rapidly expanded and it presently consists of about 50 ligands and 20 receptors. Initially, chemokines were presumed to be important mainly for inflammatory reactions. However, subsequent studies have provided definitive evidence that chemokines coordinately regulate the trafficking of every type of hematopoietic cells including granulocytes, monocytes/ macrophages, lymphocytes, other immune cells and megakaryocytes. Thus, it has become evident that chemokines are one of the master players in innate and acquired immunity as well as hematopoiesis. The discovery that chemokine receptors are a co-receptor for HIV-1 entry has paved the way for developing a novel strategy in HIV-1 infection. Moreover, the fact that various types of viruses encode chemokineand/or chemokine receptor-like molecules, suggests the potential roles of chemokines in host defense against virus infection. Finally, several lines of evidence demonstrate that some chemokines play crucial roles in organogenesis and angiogenesis. These observations suggest that chemokines have profound activities against diverse types of cells beyond hematopoietic cells. However, the signaling mechanisms mediated by chemokines still remain unclear, due to the lack of chemokine-dependent cell lines and complicated receptor systems. Clarification on these points will lead to the development of treatment

for various types of diseases by targeting a selective set of chemokines.

ACKNOWLEDGEMENTS This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology and Yasuda Medical Foundation.

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47 CC chemokines Alberto Mantovani1,2, Massimo Locati2 and Silvano Sozzani1,3 1

Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 2 University of Milan, Milan, Italy 3 University of Brescia, Brescia, Italy

To travel is to take a journey into yourself. Dena Kaye

INTRODUCTION CC chemokines are the most numerous and diversified family of the four subgroups defined, based on the Cys motif (CC, CXC, C, CX3C). In humans, it includes at least 25 members interacting with at least 11 signaling receptors (Rollins, 1997; Mantovani, 1999; Zlotnik and Yoshie, 2000). CC chemokines have been discovered following different pathways, ranging from biological and biochemical identification to direct cDNA cloning to, more recently, in silico cloning by gene bank mining. For instance, in the case of CCL2 (monocyte chemotactic protein 1, MCP-1), it had already been noted in the early 1970s that supernatants of activated blood mononuclear cells contained attractants active on monocytes and neutrophils. Subsequently, a chemotactic factor active on monocytes was identified in culture supernatants of mouse (Meltzer et al., 1977) and human (Bottazzi et al., 1983) tumor lines and was called tumor-derived chemotactic factor (TDCF) (Bottazzi et The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

al., 1983; 1985). TDCF was at the time unique in that it was active on monocytes but not on neutrophils (Bottazzi et al., 1983) and had a low (12 kDa) molecular weight (Bottazzi et al., 1983). Moreover, correlative evidence suggested its involvement in the regulation of macrophage infiltration in murine and human tumors (Bottazzi et al., 1983; Mantovani et al., 1992). A molecule with similar cellular specificity and physicochemical properties was independently identified in the culture supernatant of smooth muscle cells (SMDCF) (Valente et al., 1984). The JE gene has been identified as an immediate–early PDGF-inducible gene in fibroblasts (Zullo et al., 1985; Rollins et al., 1988). Thus, in the mid-1980s a gene (JE) was in search for function and a monocyte-specific attractant was waiting for molecular definition. In 1989, MCP-1 was successfully purified from supernatants of a human glioma (Yoshimura et al., 1989a), a human monocytic leukemia (Matsushima et al., 1989) and a human sarcoma (Graves et al., 1989; Van Damme et al., 1989; Zachariae et al., 1990): sequencing and Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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molecular cloning revealed its relationship with the long-known JE gene (Furutani et al., 1989; Yoshimura et al., 1989b; Bottazzi et al., 1990). CC chemokines act on a most diverse spectrum of target cells, mostly but not exclusively in the hematopoietic system. Non-hematopoietic elements, including epithelial cells, fibroblasts and vascular elements have been shown to express receptors for and respond to CC chemokines, although the actual in vivo importance of these responses remains to be defined. The action of CC chemokines is regulated at the level of agonist production and processing as well as at the level of receptor expression and coupling. Therefore an analysis of ligands must necessarily consider receptors. In this chapter we will summarize the general features of CC chemokines and their receptors. Certains aspects of the pathophysiology of CC chemokines will be discussed. In particular, their role in the transition from innate to acquired immunity and in amplification of polarized responses in more detail and emphasis will be on selected molecules and pathologies used as a paradigm.

AGONISTS AND RECEPTORS Chemokines represent a large superfamily of small proteins classified into four distinct families according to cysteine residues’ relative position in the N-terminal portion of the molecule. In CC or b chemokines, the most abundant family with 25 molecules in humans, the first two Cys residues are adjacent (Table 47.1). Although the overall sequence identity of chemokines is less than 20%, these molecules share a common structural organization constrained by disulfide bonding between the first and third and second and fourth cysteine residues, with a structurally disordered N-terminal loop followed by three antiparallel b sheets and a C-terminal a helix (Zlotnik and Yoshie, 2000). The first set of CC chemokines identified represented a functional homogeneous group of molecules, including the monocyte chemotactic proteins (CCL2, CCL8, CCL7 and CCL13), the macrophage inflammatory proteins (CCL3, CCL3L1 and CCL4), CCL11 and CCL5. These molecules, clustered on chromosome 17, shared inducibility in inflammatory

conditions and chemotactic activity on monocytes, T lymphocytes and eosinophils but not neutrophils, and for these reasons they were traditionally associated with leukocyte infiltrate in chronic inflammation. At the other site of the system, CXCL8 and related molecules were associated with neutrophil recruitment during acute events (Rollins, 1997). This simplified scheme has been overcome by the recent discovery of a second set of CC chemokines, such as CCL19, CC21, CCL20, and others. These constitutive chemokines, encoded by different loci, are active on different leukocyte populations and have a role in physiologic events during the development of the immune system, such as thymocyte development, lymphocyte homing and dendritic cell migration to secondary lymphoid organs. Consequently, the spectrum of target leukocytes for CC chemokines now includes virtually all leukocyte populations with the possible exception of neutrophils (Mackay, 2001). In general, CC chemokines are redundant in their action on leukocytes. Any given leukocyte population usually responds to different molecules, in most cases acting on different receptors, and most chemokines are active on multiple leukocyte populations (Table 47.2). The interaction of chemokines is also characterized by considerable promiscuity, with most receptors interacting with multiple ligands, and most ligands with more than one receptor. These properties, more pronounced for inflammatory CC chemokines involved in pathologic conditions, confer robustness to the system (Mantovani, 1999) and account for the relatively faint phenotype of knockout mice of these sets of chemokines, when compared with constitutive chemokine knockout mice. The eponymous function of chemokines is to elicit directional migration of leukocytes, and the traditional view of chemokines as mediators of leukocyte accumulation at sites of inflammation is still valid. However, it is now clear that CC chemokines also affect leukocyte functions other than directional migration, as discussed later on, and also act on other cell types, such as fibroblasts and endothelial cells, affecting functions of relevance both in normal tissue growth and disease, such as collagen production, angiogenesis and proliferation of hematopoietic precursors (Lloyd et al., 1997; Broxmeyer et al., 1999). Chemokines elicit their biological activities through

THE CYTOKINES AND CHEMOKINES

TABLE 47.1 CC chemokines and their receptors

Systematic name

Human chromosome

Human ligand

Mouse ligand

Chemokine receptor(s)

CCL1 CCL2 CCL3 CCL3L1 CCL4 CCL5 (CCL6) CCL7 CCL8 (CCL9/10) CCL11 (CCL12) CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

17q11.2 17q11.2 17q12 17q12 17q12 17q12

I-309 MCP-1/MCAF/TDCF MlP-1a/LD78a LD78b MIP-1b RANTES Unknown MCP-3 MCP-2 Unknown Eotaxin Unknown MCP-4 HCC-1 HCC-2/Lkn-1/MlP-1d HCC-4/LEC/LCC-1 TARC DC-CK1/PARC/AMAC-1 MIP-3b/ELC/exodus-3 MIP-3a/ARC/exodus-1 6Ckine/SLC/exodus-2 MDC/STCP-1 MPIF-1/CKb8/CKb8-1 Eotaxin-2/ MPIF-2 TECK Eotaxin-3 CTACK/ILC MEC

TCA-3/P500 JE? MlP-1a Unknown MlP-1b RANTES C10/MRP-1 MARC? MCP-2? MRP-2/CCF18/MIP-1 Eotaxin MCP-5 Unknown Unknown Unknown Unknown TARC/ ABCD-2 Unknown MIP-3b/ELC/exodus-3 MIP-3a /LARC/exodus-1 6Ckine/SLC/exodus-2/TCA-4 ABCD-1 Unknown MPIF-2 TECK Unknown ALP/CTACK/ILC/ESkine

CCR8 CCR2 CCR1, CCR5 CCR1, CCR5 CCR5 CCR1, CCR3, CCR5 Unknown CCR1, CCR2, CCR3 CCR3, CCR5 CCR1 CCR3 CCR2 CCR2, CCR3 CCR1, CCR5 CCR1, CCR3 CCR1,CCR2 CCR4 Unknown CCR7d CCR6 CCR7d CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR3/CCR10

17q11.2 17q11.2 17q11.2 17q11.2 17q12 17q12 17q12 16q13 17q12 9p13.3 2q36.3 9p13.3 16q13 17q12 7q11.23 19p13.3 7q11.23 9p13.3 5p12

Based on the report by the IUIS /WHO Nomenclature Committee chaired by Robin Thorpe (2001): Kevin Bacon (Japan); Marco Baggiolini (Switzerland); Hal Broxmeyer (USA); Richard Horuk (USA); Ivan Lindley (Austria); Alberto Mantovani (Italy); Kouji Matsushima (Japan); Philip Murphy (USA); Hisayuki Nomiyama (Japan); Joost Oppenheim (USA); Antal Rot (Austria); Thomas Schall (USA); Monica Tsang (USA); Robin Thorpe (Chairman) (UK); Jo Van Damme (Belgium); Meenu Wadhwa (UK); Osamu Yoshie (Japan); Albert Zlotnik (USA); Kathy Zoon (USA).

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interaction with seven transmembrane domain proteins which form a distinct group of structurally related proteins within the GTP-binding proteincoupled receptor superfamily. The sequences of chemokine receptors show 25 to 80% amino acid identity, suggesting the presence of a common ancestor belonging to the family of chemotactic receptors. Indeed, the first chemokine receptors were identified in the early 1990s using reverse transcriptase-PCR strategies with degenerate primers designed on conserved regions of classical chemoattractant receptors. However, many other G protein-coupled peptide receptors also have approximately 25% amino acid identity with chemokine receptors, indicating that the structural boundary is not clear and that more than overall sequence identity, relatedness is established by structural features more frequently observed in chemokine receptors than the unrelated seven transmembrane domain receptors. These include a length of 340–370 amino acid, an acidic N-terminal segment, a conserved sequence (LxxLxxDLLF) in the second transmembrane domain, a conserved sequence (DRYLAIVHA or subtle variations of it) in the second intracellular loop, a short basic third intracellular loop and the presence in most cases of one cysteine residue for each of the four extracellular domains (Murphy et al., 2000). Eleven CC chemokine receptors have been molecularly defined to date, with a promiscuous pattern of

ligand recognition and differential expression and regulation in leukocytes (Table 47.2). Receptors for inflammatory chemokines (CCR1 to CCR5 and CCR8) were first identified. This set of receptors is encoded by genes in a cluster on chromosome 3p21, recognizes CC chemokines associated with monocyte and lymphocyte recruitment in chronic inflammation, and includes most of the HIV co-receptors described (Berger et al., 1999). CCR1 was the first CC receptor identified, described originally as the MIP-1a/RANTES receptor. Expressed on a particularly large panel on different leukocytes, it is in fact one of the most promiscuous chemokine receptors, with at least nine ligands identified. A clear indication of CCR1’s role in disease has still to be established. Although the knockout mouse has increased susceptibility to pathogens, the fact that in the mouse this receptor is associated with neutrophil recuitment, as substitution of CXCR1, is a confounding element (Gao et al., 1997). On the other hand, CCR2 is the only CCL2 receptor identified so far and the only receptor for all four monocyte chemotactic proteins. It presents a more restricted leukocyte expression profile, and its role in monocyte recruitment in pathologic conditions and in atherosclerosis has been clearly defined in knockout models (Boring et al., 1998). CCR3 expression on basophils, eosinophils and TH2 and the selective interaction with the three eotaxins (CCL11, CCL24 and CCL26) are a clear indication of the poten-

TABLE 47.2 Ligand and leukocyte specificities for human chemokine receptors Systematic name

Chromosome

Main ligands

Main leukocytes

CCR1

3p21

CCR2 CCR3

3p21 3p21

NK, TH1, TH2, iDC, Mo, Ba, Eo, Neu NK, TH1, TH2, Mo, Ba TH2, Ba, Eo

CCR4 CCR5

3p22 3p21

CCR6 CCR7 CCR8 CCR9 CCR10 CCR11

6q27 17q12–21 3p22–p23 3p21.3–22 17q21.1–q21.3 3p22

CCL3, CCL3L1, CCL5, CCL7, CCL8, CCL14, CCL15, CCL16, CCL23 CCL2, CCL7, CCL8, CCL13, CCL16 CCL5, CCL7, CCL11, CCL13, CCL15, CCL24, CCL26 CCL17, CCL22 CCL3, CCL3L1, CCL4, CCL5, CCL8, CCL14 CCL20 CCL19, CCL21 CCL1 CCL25 CCL27, CCL28 CCL19, CCL21, CCL25

Thy, NK, TH2, TC2, TM, Ba, Thy, TH1, TC1, iDC, Mo TM, B, iDC Thy, TN, B, mDC Thy, TH2, Mo Thy, TM, B TM T, iDC

NK, natural killer cells; Mo, monocyte–macrophages; Ba, basophils; Eo, eosinophils; Neu, neutrophils; Thy, thymocytes; TH, T helper; TC, T cytotoxic; TM, T memory; TN, T naive; iDC, immature dendritic cells; mDC, mature dendritic cells. THE CYTOKINES AND CHEMOKINES

REGULATION OF PRODUCTION : CONSTITUTIVE VERSUS INDUCIBLE CC CHEMOKINES

tial role of this receptor in allergic inflammation, including asthma and antihelminthic host defence. CCR3, CCR4 and CCR8 characterize TH2 cells, as opposed to CCR5 expressed preferentially on TH1 cells. These receptors are involved in lymphocyte traffic and polarized responses, as will be discussed later on. Owing to its predominant role in HIV infection, CCR5 is at present the best-defined CC receptor. Ligands and expression profile have been defined both in the human and mouse setting, and appear to be similar. Other than TH1 cells, CCR5 is also expressed in immature dendritic cells, being down-regulated during maturation, as for all the CC receptors for inflammatory chemokines, and in monocytes. The presence in the general population of an allelic variant with a 32-bp deletion in the ORF, resulting in the absence of CCR5 expression in homozygotes, which appear healthy, indicates that CCR5 functions are probably performed by other receptors. However, its role in disease has been demonstrated in knockout mice, which show subtle defects under specific pathologic conditions (Huffnagle et al., 1999). A second set of CC receptors includes molecules encoded outside the 3p21–22 cluster that are mainly involved in regulation of leukocyte trafficking in physiologic conditions. CCR6 and CCR7 play a crucial role in different steps of dendritic cells, the first being crucial for skin homing of immature Langerhans-type dendritic cells, the second being up-regulated after dendritic cell activation and driving the mature cell to lymph nodes. Sharing ligands with CCR7, CCR11 might also have a role in this process (Gosling et al., 2000). CCR9 and CCR10 are associated with T-cell development in the thymus and with mature T-cell homing in different regions (Wurbel et al., 2001). Two CC chemokine receptors, termed DARC and D6, have been reported to bind chemokines with high affinity with no subsequent signaling activity. DARC binds most CC and CXC chemokines, while D6 binding properties are restricted to CC inflammatory chemokines. Both receptors have a typical seven transmembrane domain architecture, with low but significant sequence homology to chemokine receptors. Interestingly, alignment of D6 and DARC with functional chemokine receptors highlighted few distinct residues conserved in all signaling chemokine receptors and modified in both binding molecules involved in ligand-dependent receptor activation

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(Mantovani et al., 2001). Although in the absence of signaling and chemotactic activity these molecules cannot be included in the list of CCRs, the structural properties and binding profiles indicate that they can be viewed as ‘silent’ receptors. Different functional hypotheses for these receptors have been formulated, including presentation, transport or scavenging of the ligands. In consideration of the high expression level in erythrocytes and the wide range of ligands, DARC was first proposed to be involved in maintaining a tissue-to-blood chemokine gradient by clearing chemokines from the blood stream (Horuk et al., 1993). However, the existence of an erythroid-specific DARC promoter variant allele resulting in the absence of DARC expression on erythrocytes of healthy DARC-negative subjects argued against this hypothesis. It was subsequently found that DARC is expressed in all individuals under the action of a different promoter in various nonerythroid regions, and that it can act as a transendothelial transporter for the ligand. A similar hypothesis has also been formulated for D6, in particular after demonstration of D6 expression on lymphatic vessels (Nibbs et al., 2001). However, we observed that D6 interaction with ligands results in chemokine internalization and degradation (manuscript in preparation). For this reason, we propose that D6, and possibly other chemokine orphan receptors, might functionally act as ‘structural decoy receptors’, being able to interact with and scavenge the ligand without giving rise to activating intracellular signals but competing with signaling-competent receptors and removing chemokines from the system when no longer required.

REGULATION OF PRODUCTION: CONSTITUTIVE VERSUS INDUCIBLE CC CHEMOKINES A useful classification of chemokines, and more so of the most numerous CC family, distinguishes between molecules that are produced in a tonic way (constitutive) and those produced in response to diverse signals (inducible or inflammatory CC chemokines) (Mantovani 1999; Zlotnik and Yoshie 2000) (Figure

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CC CHEMOKINES

Constitutive

Inducible CCL 2

CCL14 CCL15 CCL17 CCL16

CCL18 Normal traffic

CCL27

CCL 4

CCL 8 Infection Inflammation CCL 11 Immunity

CCL20 CCL22

CCL25

CCL3

CCL 7

CCL19 Neoplasia

CCL21

CCL1

CCL 3L1 CCL 5

CCL 13

CCL 23

CCL 24 CCL 28

CCL 26

FIGURE 47.1 Functional classification of CC chemokines.

47.1). Prototypic molecules of the constitutive CC chemokine realm are CCL14, present in normal plasma, and CCL18, constitutively expressed by dendritic cells. Prototypic molecules of the inducible CC chemokine realm are CCL2, CCL3, CCL4, CCL5 etc. The general significance of constitutively expressed CC chemokines is to guide the normal traffic of leukocytes under normal conditions. For instance, CCL27 is probably important for regulation of traffic to cutaneous sites. CCL21 and CCL19, expressed by lymphatic endothelium and by high endothelial cells respectively, are important for guiding migration of lymphocytes and dendritic cells in lymphoid organs. The presence in normal plasma of substantial amounts of CCL14, CCL15 and CCL16 (the HCCs), interacting with receptors widely expressed on peripheral blood leukocytes (CCR1, CCR5) has as yet unknown significance. The general significance of inducible chemokines is to regulate recruitment of leukocytes on demand, in response to immunological, inflammatory and infectious signals. These two realms overlap in terms of molecules and pathology. As discussed below, in neoplastic disorders constitutive expression of inducible chemokines is observed. Moreover, a number of molecules behave both as constitutive and inducible chemokines. For instance, CCL22 was initially described as a chemokine constitutively expressed in certain cell types, most notably dendritic cells, and in certain lymphoid organs, in particular the thymus (Mantovani et al., 2000a). Subsequent work,

prompted by the recognition that this molecule preferentially attracted polarized type II T cells, has shown that CCL22 is expressed in a regulated way. Expression of inducible chemokines is stimulated by signals which interact with diverse cellular receptors, including receptors involved in innate immunity, pattern recognition receptors such as members of the Toll family, the T-cell receptor and co-stimulatory molecules for lymphocytes. As discussed below, chemokines are part of the circuits that are involved in the generation and amplification of polarized type I and type II responses. It is therefore not surprising that master cytokines that activate polarized responses differentially regulate chemokine production (Plate 47.2 see Plate section). For instance, the type II master cytokines, IL-4 and IL-13, induce production of agonists which interact with receptors that are preferentially expressed on polarized type II T cells, including CCL22 and CCL17 (agonists for CCR4), CCL11 (agonist for CCR3) and CCL1 (CCR8). Conversely, IFN as expected inhibits production of CCL22 in different cell types and induces expression of CXCR3 agonists (Rollins 1997; Mantovani 1999; Zlotnik and Yoshie, 2000) as well as of CX3CL1. CX3CL1 expression, the last identified chemokine involved in polarized responses (Fraticelli et al., 2001), is induced by IFN and TNF and inhibited by IL-4 and IL-13. Hence, using CCL22 as a paradigm, this chemokine is induced by generic stimuli representatives of interaction with microbial pathogen (LPS) or immunocompetent cells (CD40L). In addition, the master cytokines, IL-4/IL-13 and IFN, have reciprocal and divergent effects on production of this chemokine involved in the amplification of polarized type II responses (Mantovani et al., 2000b).

ROLE OF CD26/DPP IV IN CHEMOKINE PROCESSING An additional level of regulation of chemokine function is represented by modifications at the NH2terminus. Chemokines of both CXC and CC subfamilies are naturally posttranslationally modified. For instance, two predominant forms of CXCL8 were described to possess a different activity in neutrophil activation, and truncated forms of several CC chemokines were purified by cell supernatants and

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REGULATION OF RECEPTOR EXPRESSION AND COUPLING

the clarification of the role of CD26 in chemokine biology.

REGULATION OF RECEPTOR EXPRESSION AND COUPLING CC chemokines are regulated not only by changing the levels of agonist production, but also by regulating expression of the appropriate receptors. One of the first observations made on this point was that monocytes exposed to bacterial LPS showed a dramatic down-regulation of the receptor for CCL2, CCR2 (Sica et al., 1997; Moser and Loetscher, 2001). This effect was associated with destabilization of the transcript, and was not dependent on induction of the agonist. Subsequent work has extended this observation to other proinflammatory signals, including TNF, IL-1 and IFN as well as to other cell types such as dendritic cells (see below) and certain chemokine receptors on activated T and NK cells. These results suggest that down-regulation of certain inflammatory chemokine receptors (CCR2 most dramatically, CCR5, CCR1) may deliver a stop signal to recruited mononuclear phagocytes to focus their action at sites of infection and inflammation (Figure 47.3). When these proinflammatory signals leak into the systemic circulation, by down-regulating CCR2 expression, they may provide a negative signal to inhibit excessive mononuclear phagocyte recruitment at sites of inflammation and tissue damage.

CCL2

ADHESION

MIGRATION

STOP!

CCL2 

CCL2

CCL2

CCL2

TNF

CCL2

biological fluids (Van Damme et al., 1999). These truncations are due to the action of several proteases including attractin, plasmin, urokinase plasminogen activator and CD26 (Van Damme et al., 1999). The role of dipeptidyl-peptidase IV, also known as CD26, in chemokine processing has recently been the focus of great interest (DeMeester et al., 1999; Van Damme et al., 1999). CD26 was originally identified as a marker of activated memory T lymphocytes (Morimoto and Schlossman, 1998; DeMeester et al., 1999). However, CD26 is expressed in several cell types, including endothelial and epithelial cells. CD26 is a cell membrane-associated protein that is also found in a soluble form in seminal fluid, urine and plasma. This protein exerts a unique peptidase activity, it cleaves dipeptides from the NH2-terminus of proteins having a Pro or Ala residue at the penultimate position. Several chemokines possess a proline residue in the NH2 region in position 2. However, some of them, like MCP-1, -2, -3 and -4 are protected from CD26 degradation by a pyroglutamate at the NH2-terminus which protects the protein by degradation. In contrast, other chemokines can be effectively processed by the enzyme (Lambeir et al., 2001), and the biological output of this cleavage is unpredictable. CXCL6 a CXC chemokine, is cleaved, but its biological activity remains unaffected. However, for most chemokines (CCL5, CXCL12, CCL22 and CCL11) truncation by CD26 is accompanied by reduced, or somewhat altered, receptor binding and signaling. For all these chemokines a Pro and not Ala residue at the penultimate position is involved. An exception is CCL22 which is processed beyond this cleavage site. Very often, CD26-processed chemokines (e.g. CXCL12 and CCL11) not only lose their biological activity but also start to function as receptor antagonists being able to inhibit the biological activity of intact proteins. Occasionally, the processed chemokine has a more complex biological behavior. CCL5 (3–68) functions as a receptor antagonist for CCR1 and CCR3, but shows an increased affinity for CCR5 (DeMeester et al., 1999). Similarly, CCL3L1 (3–70) was 10-fold more efficient and 30-fold less efficient than full-length protein for binding to CCR5 and CCR3, respectively (Struyf et al., 2001). The availability of new analytical techniques able to identify full-length proteins, as well as processed chemokines in biological fluids, will allow



LPS

IFNc IL-1



FIGURE 47.3 Down-regulation of inflammatory chemokine receptors by primary proinflammatory molecules as a stop signal. For simplicity only CCR2 is shown.

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Reciprocally, anti-inflammatory signals such as glucocorticoid hormones and IL-10 were shown to increase expression of certain chemokine receptors, including CCR2 and CCR5 (Sozzani et al., 1998a; Penton-Rol et al., 1999; Moser and Loetscher, 2001). Hence, pro- and anti-inflammatory signals have reciprocal and divergent effects on expression of certain chemokine receptors in human mononuclear phagocytes and this may serve as a strategy to finely tune the action of chemokines. The results described so far refer to canonical cellular targets for CC chemokines, mononuclear phagocytes. It was also found that non-canonical cellular targets are rendered responsive to chemokines by microenvironmental signals. IFN rendered neutrophils responsive to a series of CC chemokines, in particular CCR1 agonists (Bonecchi et al., 1999). Mononuclear phagocytes express CXCR1 and CXCR2 but show little functional response. When cells are exposed to IL-4 and IL-13, increased receptor expression and coupling render these cells extremely sensitive to CCL8 and related CXC chemokines. Therefore, microenvironmental signals tune and shape the action of CC chemokines, by regulating receptor expression and coupling. IL-10 tends to increase expression of certain inflammatory chemokine receptors, such as CCR2 and CCR5 (Sozzani et al., 1998a). The significance of this observation may in fact relate to the effect of this cytokine when combined with primary proinflammatory signals (D’Amico et al., 2000). It was observed that IL-10 blocks down-regulation of inflammatory chemokine receptors induced by LPS alone or in combination with IFN in monocytes and dendritic cells. However, monocytes and dendritic cells exposed to a combination of IL-10 and LPS, while showing high levels of CCR2 and CCR5, did not migrate in response to appropriate agonist and showed defective activation of signal transducing events. Inflammatory chemokine receptors in cells exposed to a combination of LPS and IFN retain the ability of binding and sequestering agonists. Evidence was obtained that chemokine scavenging may also occur in vivo in tissues where dendritic cells exposed to a combination of IL-10 and TNF are present. Hence, in an inflammatory environment dominated by IL-10, cells expressing inflammatory chemokine receptors are set in a chemokine scavenging, anti-inflammatory mode. It was therefore suggested that these chemokine recep-

tors act as functional decoy receptors for chemokines. More generally it has been suggested that nonsignaling ‘silent’ receptors (see above) are decoy receptors for chemokines, including CC molecules (Mantovani et al., 2001).

TRANSITION FROM INNATE TO ADAPTIVE IMMUNITY: DENDRITIC CELLS Dendritic cells (DC) are potent antigen-presenting cells with unique ability to capture antigens and to induce T- and B-cell response (Banchereau et al., 2000). Chemokines, and especially CC chemokines, regulate DC trafficking and localization. Immature myeloid DCs express functional CCR1, CCR2, CCR5, that are probably responsible for their migration to the site of immune response. Blood plasmacytoid DCs express a pattern of chemokine receptors similar to that of myeloid DCs. However, most chemokine receptors of plasmacytoid DCs, in particular those for inflammatory chemokines, are apparently not functional in circulating cells (Penna et al., 2001). Lymphoid cells purified from skin or generated in vitro from CD34 precursors are characterized by the expression of CCR6, the receptor for CCL20 in addition to the receptors expressed by mono-DCs (Power et al., 1997). CCR6 also interacts with defensins which have agonist activity for DCs (Yang et al., 2000). In the mouse, myeloid DCs express CCR6 and localize to the subepithelial dome of Peyer’s patches in response to CCL20 which is strongly expressed by the overlying epithelium. CCR6/ mice have defective accumulation of DCs in gut mucosal tissues and defective response to orally administered antigens. Thus, increasing evidence points to a distinct role of CCL20/CCR6 in recruitment of DC towards the mucosal surfaces (Cook et al., 2000). Monocyte DCs also express two orphan receptors ChemR23 (Samson et al., 1998), and HCR (Otero and Sozzani, unpublished). The role of these two receptors in DC biology is still unknown. Maturation of DCs is associated with the inhibition of chemotactic response to inflammatory chemokines and the up-regulation of CCR7. CCL19 and CCL21, the ligands of CCR7, are specifically expressed in T cell-rich areas of tonsils, spleen and LN, where mature DCs home, to become inter-

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LYMPHOCYTE TRAFFIC AND POLARIZED RESPONSES

digitating DCs (Cyster, 1999; Allavena et al., 2000). The crucial role of CCR7 is clearly reflected in mice deficient for this receptor and the two ligands. Overall, these findings provide a model for DC trafficking in which inflammatory chemokines acting through CCR1 and CCR5 or CCR6, function as signals to localize DC precursors to peripheral tissues. After antigen uptake, immune/inflammatory stimuli induce DC maturation and loss of responsiveness to the inducible cytokines locally produced. This unresponsiveness may play a permissive role for DCs to leave peripheral tissues. Meanwhile, the slower upregulation of CCR7 prepares the cells to respond to CCL19 and CCL21 expressed in lymphoid organs. DCs also represent a source of chemokines in vitro and in vivo. In vitro, immature DCs constitutively produce CCL22, CCL9/10 and CCL18 (Allavena et al., 2000). Thymic DCs selectively express CCL25, a CC chemokine active on thymocytes, macrophages and DCs. Production of chemokines by DCs is strongly increased when these cells are induced to differentiate by proinflammatory stimuli (e.g. LPS, TNF) or engagement of CD40. In vitro, mature DCs produce conspicuous amounts of CCL2, CCL3, CCL5 and CXCL8 (Sallusto et al., 1998; Vulcano et al., 2001). Mature DCs also produce very high concentrations of MDC and TARC (Vulcano et al., 2001). In vivo, MDC was detected by in situ hybridization in mature DCs and the protein is strongly produced by CD83 cells in the skin of atopic dermatitis patients (Vulcano et al., 2001). CX3CL1 and CXCL16, two chemokines present in both membrane-anchored and soluble forms are produced by mature DCs (Kanazawa et al., 1999; Matloubian et al., 2000). CXCL13 is produced by follicular DCs and germinal center DCs and is likely to play a role in DC interaction with B and T lymphocytes (Vissers et al., 2001). DC-derived chemokines are believed to contribute to the recruitment of precursor cells and immature DCs at peripheral sites of inflammation (Sallusto et al., 2000; Cyster, 1999; Allavena et al., 2000). Furthermore, within lymph nodes, chemokines may also play a role in T- and Bcell localization and in DC–T cell interaction (Cyster, 1999; Banchereau et al., 2000).

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LYMPHOCYTE TRAFFIC AND POLARIZED RESPONSES Homeostatic CC chemokines play a role in regulating the normal traffic of lymphocytes in lymphoid organs (Luther and Cyster, 2001; Moser and Loetscher, 2001). The CCR7 receptor is expressed on mature DCs (see above), naive T cells and a subset of memory T cells (central memory T cells) (Sallusto et al., 1999). CCR7 is recognized by two ligands, CCL21, present in two forms in the mouse differing by one amino acid, serine–leucine at position 65, and CCL19. CCL21 is expressed on lymphatic endothelial cells whereas CCL19 is expressed by various cell types, including high endothelial venules. Analysis of the mouse mutant plt and of CCR7/ mouse has given results consistent with a nonredundant role of this CC chemokine receptor in regulating traffic of lymphoid cells and DC in lymphoid organs (Förster et al., 1999; Gunn et al., 1999). As discussed above, gene targeting has also revealed unequivocally a role for CCR6 in the regulation of DC traffic in the mucosal compartment. The CC chemokines CCL17 and CCL27 are important for the traffic of skin-homing T lymphocytes (Campbell et al., 1999; Morales et al., 1999). CCL17 recruits CLA T cells, which are the predominant type present in the skin. CCL17 is expressed on skin endothelial cells. Moreover, CCL27, a chemoattractant expressed widely in the skin, being produced by keratinocytes, is active on CLA T cells. Therefore, this tissue-specific CC chemokine can direct migration of the CLA T-cell subset to this anatomical site. There is compelling evidence that a set of chemokines are involved in polarized TH1/TH2 responses (Mantovani, 1999; Luther and Cyster, 2001; Moser and Loetscher, 2001). Polarized TH1 and TH2 cells express differential chemokine receptors. Typically, the CC chemokine receptors CCR3, CCR4 and CCR8 have been associated with a TH2 phenotype whereas functional CXCR3 and CCR5 are preferentially expressed on polarized type I T cells (Plate 47.2). CCR3 ligands also attract eosinophils and basophils, crucial for polarized type II responses. Differentially expressed chemokine receptors are not markers for polarized T cells, in that there is no absolute association between chemokine receptor expression and cytokine repertoire of polarized T cell populations. In contrast,

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CC CHEMOKINES

the seven transmembrane domain receptor CRTH2 has been shown to be more closely associated with the TH2 phenotype than are chemokine receptors. For instance, CCR4, while expressed at much higher levels in polarized TH2 cells, is induced in TH1 cells following activation and is also expressed in non-polarized T-cell populations. Moreover, CCR4 has been suggested to be involved in homing to the skin. As discussed above and described in Plate 47.2 consistent with the role of polarized T-cell responses, agonists for receptors expressed differentially in polarized T cells are differentially induced/inhibited by IL-4, IL-13 and IFN. For instance, the CCR4 agonists CCL17 and CCL22 are induced by IL-4 and IL-13 and are inhibited by IFN. A CCR4/ mouse has been generated, but no evidence for alteration in polarized responses has been observed. However, antibody against MDC blocked recruitment of T cells in a classic model of airway hyperreactivity. No abnormalities in airway hyperreactivity were observed in CCR3/ mice. In constrast, CCR8/ mice were protected against higher airway hyperreactivity. Recent careful analysis of chemokine receptors expressed in T cells in asthma has yielded results consistent with a role of this molecule in allergic inflammation (Panina-Bordignon et al., 2001). It was found that in allergic asthma CCR3 is rarely expressed in T cells, unlike CCR4 and CCR8. Collectively, these results suggest that chemokines play a key role in the induction and amplification of polarized responses and may represent an important target for therapeutic intervention. In addition to playing a role in the effector phase of polarized responses, chemokines may be important in their induction. Analysis of responses to a parasite component has suggested a key role for CCR5 expressed on DCs not only in their migration but also in the induction of IL-12 (Aliberti et al., 2000). Studies in CCL2/ mice have indicated that this chemokine may be important for the induction and expression of polarized type II responses. Interestingly, data in CCR2/ mice have pointed in the opposite direction as far as polarized responses are concerned (Bonecchi et al., 1999; Gu et al., 2000). In contrast, consistent results between gene targeting of the agonist and of the receptor have been obtained in terms, for instance, of susceptibility to atherosclerosis . Indeed, in a redundant and promiscuous system in which several agonists interact with one receptor, inactivation

of one of them is not necessarily equivalent to receptor blockade. Receptor inactivation may block one pathway which scavenges the agonists. Therefore, in the MCP system, this may result in increased concentration of members of a family such as MCP-3, which in addition to interacting with CCR2 interacts with CCR3, a receptor expressed on a subset of polarized TH2 cells as well as on eosinophils and basophils. This may explain the apparent discrepancy in terms of influence on polarized responses obtained between studies in agonist CCL2 and receptor (CCR2) genetargeted mice.

TUMORS AS A PARADIGM OF THE ROLE OF CC CHEMOKINES Most, if not all, tumors produce chemokines of the two major groups, CC and CXC (Balkwill and Mantovani, 2001). Compelling evidence in murine models and in human tumors suggests that CCL2 and related CC chemokines such as CCL5 are major determinants of macrophage and lymphocyte infiltration in melanoma, in carcinoma of the ovary, breast and cervix, and in sarcomas and gliomas (Sciacca et al., 1994; Negus et al., 1995; Riethdorf et al., 1996; Luciani et al., 1998; Valkovic et al., 1998; Luboshits et al., 1999). In Hodgkin’s disease the malignant Reed–Sternberg cells express the TH2 attracting chemokines CCL22 and CCL17 (Cossman et al., 1999; van den Berg et al., 1999) and the chemokine eotaxin, produced by stromal elements, correlates with eosinophil infiltration (Jundt et al., 1999; Mazzucchelli et al., 1999). Chemokine production by tumor cells may reflect constitutive gene expression or activation by autocrine/paracrine loops, as shown for Fas/FasL in brain tumors and IL-6 in cervical neoplasia (Hess et al., 2000; Choi et al., 2001). Chemokine receptors are expressed by both infiltrating leukocytes and tumor cells. The former may lose receptor expression once they are exposed to cytokines in the tumor microenvironment, as shown for CCR2 on TAM in ovarian cancer (Sica et al., 2000). In the murine and human tumors studied, TAM have a skewed type II phenotype. They do not produce IL-12 and NFB activation is lacking. TAM spontaneously release conspicuous amounts of IL-10 and TGFb (Kim et al.,

THE CYTOKINES AND CHEMOKINES

TUMORS AS A PARADIGM OF THE ROLE OF CC CHEMOKINES

1995; Maeda and Shiraishi, 1996; Sica et al., 2000a). Autocrine production of IL-10 may be responsible for the phenotype of TAM (Sica et al., 2000b). The mechanisms responsible for the polarization of TAM to a type II phenotype are a matter of speculation. A TH2/TC2 dominated T-cell response in situ, with production of IL-4 and IL-13 (Huang et al., 1995; Sozzani et al., 1998b; van den Berg et al., 1999), would drive a type II inflammatory response. Some chemokines, including CCL2, induce IL-10 in macrophages. Moreover, data in knockout mice suggest that CCL2 polarizes immunity in a TH2 direction (Gu et al., 2000). Chronic exposure to high chemokine concentrations in the tumor microenvironment may set in motion a chemokine-centred vicious circuit, leading to skewing towards a type II inflammatory response (Sica et al., 2000). Some viruses encode chemokines, chemokine inhibitors and chemokine receptors. Of particular interest is human herpesvirus 8 (HHV8), which is involved in the pathogenesis of Kaposi’s sarcoma (KS) and body cavity lymphoma. In addition to encoding a constitutively active chemokine receptor, which acts as a dominant oncogene (Cesarman et al., 2000; Yang et al., 2000), HHV8 contains three chemokines, called vMIP-I, vMIP-II and vMIP-III. These three chemokines are selective attractants of polarized type II T cells and interact with chemokine receptors (CCR3, CCR4, CCR8) expressed on this population (Sozzani et al., 1998b; Endres et al., 1999; Stine et al., 2000; Weber et al., 2001). T cells infiltrating KS, which are predominantly CD8, have a skewed TH2 phenotype (Sozzani et al., 1998b). HHV-8 encoded chemokines thus might be a strategy to subvert immunity, by activating type II responses and diverting effective TH1 defense mechanisms (summarized in Plate 47.2). In terms of inflammatory reactions, neoplastic disorders constitute an apparent paradox. As discussed above, many, if not all, tumors produce inflammatory cytokines, chemokines and are infiltrated by leukocytes. However, neoplastic disorders are associated with a defective capacity to mount inflammatory reactions at sites other than tumors, and circulating monocytes from cancer patients are defective in their capacity to respond to chemoattractants (Snyderman and Cianciolo, 1984; Balkwill and Mantovani, 2001). Various factors originating in the tumor microenvironment may contribute to the systemic anti-

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inflammation associated with cancer. Chemokines leaking into the systemic circulation are likely to desensitize circulating leukocytes (Rutledge et al., 1995); elevated levels of TNF receptors and the type II decoy IL-1 receptor may buffer inflammatory cytokines; and tumors also produce anti-inflammatory cytokines such as IL-10 or TGFb (Kim et al., 1995; Maeda and Shiraishi, 1996; Sica et al., 2000b). On this basis, a defective capacity to mount a systemic inflammatory response in cancer patients coexists with continuous leukocyte recruitment at the tumor site. CC chemokines induce production of proteases, such as MMP and urokinase-type plasminogen activator in tumor cells and macrophages. These enzymes are important for invasion and it has been suggested that monocytes infiltrating the tumor tissue provide cancer cells with a ready-made path for invasion (counter current invasion theory) (Opdenakker and Van Damme, 1992). As described above, tumor cells may also express chemokine receptors. Appropriate chemokine agonists induce migration or proliferation of tumor cells, raising the interesting possibility that tumor cells may use chemokine gradients to spread around the body (Wang et al., 1990, 1998; Singh et al., 1994; Prest et al., 1999; Haghnegahdar et al., 2000; Muller et al., 2001). It has recently been found that breast carcinoma cells and melanoma cells express CCR7 and CXCR4 and these may be important for metastasis (Muller et al., 2001). Receptors such as CCR7, essential for lymphocyte and DC homing to lymph nodes, could play a similar role for lymphatic dissemination of certain carcinomas. Direct evidence for chemokines guiding the secondary localization of cancer has been obtained (Wang et al., 1998; Muller et al., 2001; Scotton et al., 2001; Schutyser et al., 2002). The finding of constitutive production of chemokines in tumors is consistent with the view that recruited leukocytes, macrophages in particular, provide a mechanism for tumor promotion directly or indirectly (Balkwill and Mantovani, 2001). The protumor function of chemokines is strictly related to the amounts produced. It has been shown that lowlevel CCL2 production renders non-tumorigenic melanoma cells capable of forming progressing tumor lesions (Nesbit et al., 2001). In contrast, forced expression of high levels of CCL2 and other CC

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chemokines has caused tumor inhibition (Rollins, 1997; Mantovani, 1999; Nokihara et al., 2000; Zlotnik and Yoshie, 2000). In this context, expression of CC chemokines capable of attracting DCs such as CCL7 and CCL21 was associated with DC recruitment, activation of specific immunity and tumor regression (Fioretti et al., 1998; Vicari et al., 2000; Kirk et al., 2001; Nomura et al., 2001).

THE TRANSCRIPTIONAL PROGRAM ACTIVATED BY CC CHEMOKINES Various indications in the literature suggest that chemokines have a role in the development of different biological responses that goes beyond cell recruitment. For example, CXCR2 agonists support tumor growth through growth factor induction in malignant cells (Balentien et al., 1991), as also demonstrated for the related constitutively active GPCR ORF74 receptor encoded by HHV-8 (Sodhi et al., 2000). Chemokines have also been demonstrated to play a direct role in the definition of the cytokine milieu during both inflammatory (Jiang et al., 1992) and immune responses (Bandeira-Melo et al., 2000; Braun et al., 2000). Thus, chemokines not only support differential leukocyte recruitment, but also directly affect target cell functions. Gene chip-based gene expression profile analysis in chemokine-activated monocytes revealed that CC chemokines induce specific transcriptional programs in target cells (Table 47.3, and unpublished results), demonstrating that chemokine effects on target cells include induction of transcriptional events. CCL5 induced a significant fraction of TABLE 47.3 Comparison of transcriptional profile induced by CCL5 and LPS in monocytes  LPS

  LPS

 LPS

 CCL5

10 (0.2%)

16 (0.3%)

16 (0.3%)

  CCL5

84 (1.7%)

1385 (27.7%)

10 (0.2%)

0 (0%)

0 (0%)

0 (0%)

 CCL5

Monocytes were stimulated in non-adherent conditions for 2 hours with indicated agonists and transcriptional profile was analyzed using Affymetrix HuGene FL arrays, interrogating 5200 human genes. Percentages indicate the fraction of cell transcriptome regulated. , upregulated; , downregulated;  , not regulated.

transcripts, which included inflammatory cytokines, indicating an active role for chemokines in the organization of the inflammatory reaction, and chemokines and their receptors, as previously reported (Fischer et al., 2001), suggesting an amplification and diversification cascade for leukocyte recruitment. CCL5activated transcriptional program also supports cell migration and tissue penetration, as demonstrated by induction of proteases and adhesion molecules acting on subendothelial structures. In contrast, consistent gene suppression was not observed after exposure to CCL5. Comparison with the classic inflammatory mediator LPS demonstrated that chemokines activate a specific transcriptional program partially overlapped with but clearly distinct from that induced by LPS.

CC CHEMOKINES AS THERAPEUTIC TARGETS Classic immunosuppressive and anti-inflammatory drugs are potent inhibitors of the production of certain chemokines. Active molecules include glucocorticoid hormones, FK506 and cyclosporin A (Poon et al., 1991; Zipfel et al., 1991; Mukaida et al., 1992; Wertheim et al., 1993; Loetscher et al., 1994; Sozzani et al., 1996). The compound 2-methyl-2[[1-(phenylmethyl)-1Hindazol-3yl]methoxy] propanoic acid (bindarit) was recently found to inhibit chemokine production in a relatively specific way, although at high concentrations (Sironi et al., 1999). Bindarit inhibited MCP-1 production in human monocytes, with no effect on production of IL-8, RANTES and MIP-1a. Bindarit also blocked synthesis of TNF to some extent, but not of IL-1 or IL-6. Although recent results indicate that it inhibits p38 MAP kinase, the molecular mechanisms responsible for selectivity have not been defined. Agents that affect cholesterol metabolism can affect MCP-1 production. Lovastatin and mevastatin (two inhibitors of cholesterol production which block 3hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibit MCP-1 production in mononuclear phagocytes and smooth muscle cells (Romano et al., 2000). The effect of lovastatin was reversed by the addition of mevalonate, as expected, considering its activity on HMG-CoA. Given the fundamental role of MCP-1 in the pathogenesis of atherosclerosis, it is

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tempting to speculate that inhibition of chemokine synthesis, in addition to cholesterol lowering, is in fact a major part of these agents’ activity in the prevention of acute myocardial infarction. NH2-terminal truncation or modification also resulted in the generation of potent antagonists for CC chemokine receptors. This is true for molecules such as CCL5, CCL2 and CCL7. Removal of eight residues at the N-terminus of MCP-1 produces an antagonist (Gong and Clark-Lewis 1995). CCL2 (9–76) is of special interest, as it has been used in models of diseases such as arthritis, with appreciable therapeutic results (Gong et al., 1997). Expression of CCL5 in E. coli produced Met-CCL5, a functional antagonist or weak partial agonist for chemoattractant receptors. Similar results were obtained when CCL7 was expressed in Pychia pastoris. Met-CCL5 has been investigated in vivo in experimental models, such as arthritis and glomerulonephritis, and in models of airway inflammation (Plater-Zyberk et al., 1997). Aminoxypenthane modified CCL5 (AOP-RANTES) was an even better antagonist for several CC receptors (Simmons et al., 1997). AOP-CCL5 is internalized but does not elicit signal transduction and the receptor is prevented from being recycled. Peptide antagonists have proved valuable tools for chemokine pharmacology. They have provided structure–activity information essential for understanding ligand–receptor interactions. In addition, before the availability of knockout mice they validated chemokines as targets for pharmacological intervention in disease models. Finally, the discovery of N-terminal processing of chemokines by enzymes such as CD26 (see above), suggests that the generation of peptide antagonists may offer a strategy for fine tuning the chemokine system. Efforts have been made to develop small antagonists directed against HIV fusion co-receptors, particularly those most frequently used, CXCR4 and CCR5. TAK-779 antagonized CCR5, to a lesser extent CCR2B, and had no activity on CCR1, CCR3 and CCR4 (Baba et al., 1999). Small antagonists for CCR3 and CCR1, CCR2 and CCR5 (Hesselgesser et al., 1998; Dragic et al., 2000; Forbes et al., 2000; Huang et al., 2000; Liang et al., 2000; Mirzadegan et al., 2000; Proudfoot et al., 2000; Maeda et al., 2001; Naya and Saeki, 2001; Tagat et al., 2001) competitive or non-competitive, have been developed (e.g. see Sabroe et al., 2000;

White et al., 2000). At least one CCR5 antagonist is currently undergoing clinical evaluation. It should be noted that studies appearing in the peer-reviewed literature in the field of chemokine antagonists represent the tip of the iceberg, most efforts not having surfaced as yet because of industrial strategies. Major stumbling blocks in the development of CCR antagonists include species specificity of receptor recognition or receptor function, crossreactivity with other GPCR, conformational heterogeneity of receptors. In addition to being drug candidates for diverse disorders ranging from allergy to HIV infection, small antagonists hold promise as tools to dissect the action of chemokines in vitro and in vivo.

CONCLUDING REMARKS CC chemokines are a complex system of molecules which affect a variety of hematopoietic and nonhematopoietic cell types. At least some of these molecules and their cognate receptors are interesting targets for pharmacological intervention (Mantovani et al., 2000a). Validation as pharmacological targets includes gene targeting (e.g. CCL2/CCR2, CCR1, CCR7, CCR8), usage of antibodies or antagonists (e.g. anti-CCL22, Met-CCL5), expression in human pathology (e.g. CCR4 and CCR8 in asthma), as discussed in this chapter. CC chemokine receptor antagonists have been developed (e.g. CCR5, CCR1) and are at various stages of preclinical or clinical development. The development of efficacious CC chemokine antagonists remains a ‘holy grail’ for the general field of cytokine pharmacology.

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48 Fractalkine Violetta Zujovic and Jeffrey K. Harrison University of Florida College of Medicine, Gainesville, FL, USA

Dendrograms depicting evolutionary relationships in the growing array of chemokines and their receptors are beginning to resemble elegant Mandlebrot patterns – those eerily beautiful, iterative and self-referential tracings that have been used to describe the geometry of everything from cauliflowers to continents. Thus, it seems fitting to invoke such a complex metaphor to describe the latest layer in the baroque system controlling leukocyte trafficking, inflammation and infectious processes. ‘Fractalkine’ therefore seems an appropriate name for a novel class of chemokine that adds a new twist to models of cellular trafficking and immune regulation. T.J. Schall, Immunology Today

INTRODUCTION Indeed, the discovery of fractalkine (CX3CL1) as a new class of chemokine has reshaped the thinking of the field of chemokine biology along multiple fronts. Fractalkine represents the sole member of the CX3C chemokine subfamily and its unique structural, functional and localization pattern suggest interesting new roles for chemokines. A primary structural hallmark of the chemokine family includes the presence of conserved cysteine residues, the first two of which are separated by a variable number of amino acids. The expansion of the chemokine family to include a CX3C member affords further philosophical fodder regarding the structural evolution of the chemokine gene family if only to raise questions regarding limitations of the number of residues that are flanked by these conserved cysteines and the effect on their

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

structural and/or biological properties. Of greater significance, the extended structure of fractalkine, including most notably its membrane association and role as an adhesion molecule, furnishes a new model of chemokine-dependent regulation of cellular trafficking, one that is independent of the physical association of other chemokine binding proteins and the dependence of activation of other classes of adhesion molecules, most notably the integrins. Finally, the prominent expression of fractalkine and its receptor CX3CR1 in normal CNS provides a new ligand–receptor pair to consider in unraveling the complexities of nervous system cell–cell communication. In the previous edition of this book, the fractalkine and fractalkine receptor (CX3CR1) molecules were introduced to the community of cytokine biologists. The number of publications, since the last edition, examining aspects of this ligand–receptor pair has

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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increased nearly two orders of magnitude. Here, we summarize this information with the intention of getting closer to understanding the function(s) of this chemokine. Emerging data on the function of fractalkine and CX3CR1 are not beyond the realm of a concept in dualism as it pertains to pitting good versus evil. Like the dual nature of the fractalkine protein itself, existing as a soluble chemoattractant molecule and as a membrane attached pro-adhesive substance, issues relevant to the function of this molecule in the periphery and CNS beg discussion of whether this chemokine serves similar or diverse functions in these two distinct areas of the body. Data supporting roles for fractalkine as a proinflammatory cytokine in peripheral system pathologies, including glomerulonephritis, inflammatory bowel and cardiovascular diseases are clearly established. On the other hand, a neuroprotective role for fractalkine in the CNS is also supported by evidence derived from studies of this molecule in the central nervous system. Therefore, the framework in which we present information regarding this chemokine system will be based upon discussing contrasts and similarities of the function of these genes in the periphery and the CNS.

FRACTALKINE GENE STRUCTURE/CHROMOSOMAL LOCALIZATION DNA sequences comprising the fractalkine gene have been identified in human, murine and rat genomes (Nomiyama et al., 1998; Rossi et al., 1998; Greaves et al., 2001; Hiroyama et al., 2001; Harrison and Luo, unpublished). The fractalkine gene consists of three exons and two introns. The positions of the intron/ exon borders are conserved in the three species and are located at similar positions as is found in other chemokine genes. Exon 1 contains DNA sequences encoding the signal peptide while exon 2 contains DNA sequences encoding three of the four conserved cysteine residues. Notably, the DNA sequences encoding the mucin stalk, TM domain and intracellular C-terminal region are contained within exon 3, i.e. region containing the distal portion of the chemokine domain. A separate exon, containing the sequences encoding this extended portion of fractalkine, would probably have been met with greater prediction. The

pathway of molecular evolution of this molecule, from chemokine gene family members and other mucin-like molecules, is open to a classic chicken versus egg proposition. A schematic of the relationship of the exons and introns, as well as the size, and splice donor/acceptor sequences of the various exons and introns of the human, murine and rat fractalkine genes are shown in Plate 48.1 (see Plate section). The fractalkine gene is clustered with two other CC chemokine genes, namely macrophage-derived chemokine (MDC/CCL22) and thymus and activationregulated chemokine (TARC/CCL17). In the human genome, these genes are found on chromosome 16, specifically 16q13 (Nomiyama et al., 1998). The three genes are arranged in a head-to-tail manner in the order MDC, fractalkine and TARC with 6.3 kbp and 28.5 kbp of intervening DNA sequences separating the individual genes. In the mouse genome, the genes are clustered on chromosome 8 (Rossi et al., 1998). Evidence for the expression of alternatively spliced forms of fractalkine and TARC exists. Specifically, two ‘hybrid’ forms of mRNA are suggested from RT-PCR analysis of various mouse tissues, with the first exon of FKN (containing the signal peptide) and exons 3 and 4 of TARC (encoding the mature form of the protein) as well as an unusual form of FracTARC in which exons 1 and 2 of FKN and 3 and 4 of TARC form an mRNA (Hiroyama et al., 2001). This latter RNA species would be predicted to encode a six cysteine chemokine molecule. Identification of expressed protein will be required with further characterization of the binding and functional properties of this potential chemokine hybrid. If this protein does exist, it will be interesting to determine if it acts as a ligand at both CX3CR1 and CCR4 (primary receptor for TARC and MDC) or whether it serves as a selective ligand for an as yet identified chemokine receptor. Elements in the promoter of the fractalkine gene have not been extensively characterized although as discussed below, given its regulation by proinflammatory molecules such as IL-1, TNFa, and IFNc, it is certainly expected that NFjB and interferon response elements are present in the promoter that probably control expression. Additional cis-acting elements are also certain to be identified. Interestingly, ectopic expression of p53 (in p53-deficient cells) has been shown to induce fractalkine expression (Shiraishi et al., 2000). A potential p53-binding sequence has

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been identified in the promoter region of the human fractalkine gene. This p53-dependent expression of fractalkine has been hypothesized to target these cells for destruction by CX3CR1 expressing NK cells. Predictably, cancer cells expressing mutant forms of p53 may evade immunosurveillance as a result of their inability to up-regulate fractalkine on their cell surface.

CX3CR1 GENE A cDNA encoding CX3CR1 was originally identified in rat and was followed shortly thereafter by identification of cDNAs from human tissue sources (Harrison et al., 1994; Raport et al., 1995; Combadiere et al., 1995). These initial reports only documented characterizations of the DNA and protein sequences as well as the respective tissue distributions of these orphan G-protein coupled receptors. Their formal identifications as fractalkine receptors came shortly after the discovery of the fractalkine molecule (Imai et al., 1997; Combadiere et al., 1998a; Harrison et al., 1998). The CX3CR1 gene is localized near a group of genes coding for CC chemokine receptors on human chromosome 3p21–3pter (Raport et al., 1995; Maho et al., 1999). The gene consists of at least two exons, with the entire protein-coding sequence found in the second exon. Receptor cDNAs have also been characterized in the mouse (Combadiere et al., 1998b).

FRACTALKINE PROTEIN SEQUENCE/STRUCTURE The primary structures of human, murine and rat fractalkine proteins have been deduced from characterizations of the cognate cDNA sequences. The proteins exhibit considerable amino-acid sequence similarities. Plate 48.2 depicts a multiple alignment of the three orthologous proteins. The regions of greatest identities are the chemokine, transmembrane and intracellular domains. The least similar region of the proteins are the mucin-like stalks. Of notable difference, is the tri-amino acid motif separating the first two conserved cysteines. Human and rat fractalkine chemokine domains contain an Asn–Ile–Thr (NIT)

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sequence while the cognate region of murine fractalkine is Glu–Ile–Met (EIM). The functional significance of this unique N-linked glycosylation site seems insignificant as it does not contribute to fractalkine:CX3CR1 interactions as defined by studying the effect of mutation of this tri-amino acid motif on binding, signaling and cell adhesion properties (Harrison et al., 2001). Biophysical measurements of the chemokine domain of human fractalkine have been made. Data derived from solution NMR (Mizoue et al., 1999) as well as X-ray crystallography (Hoover et al., 2000) has revealed that the structure of the fractalkine chemokine domain is similar to other chemokines, particularly members of the CC chemokine subfamily. Fractalkine chemokine domain monomers form dimers in the crystalline environment via intermolecular interactions involving residues 8–14 of the mature protein (Hoover et al., 2000). However, there is no evidence of dimer formation in solution, even at relatively high concentrations (Mizoue et al., 1999). Regardless, the relevance of dimer formation in the context of the full-length, membrane attached form of the protein is questionable. The structures of extended forms of fractalkine, i.e. membrane-attached as well as forms of fractalkine containing any portion of the mucin stalk are far less refined. Rotary shadowing electron microscopy of a form of fractalkine containing the chemokine domain and the entire mucin stalk indicates that the chemokine portion is globular in structure while the mucin-like domain projects as a long stalk (Fong et al., 2000). These data suggest that the role of the stalk is to act as a molecular extender of the chemokine domain to CX3CR1-expressing cells. In support of this, replacement of the mucin stalk with the rod-like segment of E-selectin yielded a molecule just as capable of capturing CX3CR1-expressing cells under conditions mimicking blood flow (Fong et al., 2000). Thus, specific primary sequences of this stalk region do not necessarily dictate the ability of fractalkine to capture CX3CR1-expressing cells, but rather the role of the stalk is to provide an extender of the chemokine domain away from the surface of the cell. Its slow dissociation rate from CX3CR1 also contributes to the adhesion properties of fractalkine (Haskell et al., 2000; Fong et al., 2002). As a signaling ligand, the chemokine domain is also all that is

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necessary for receptor activation. The chemokine domain alone (and likely with any length of the mucin stalk) is sufficient for activation of various signaling cascades including calcium mobilization, Akt and ERK activation, as well as mechanisms involved in the chemotaxis of CX3CR1-expressing cells (Bazan et al., 1997; Fong et al., 2000; Harrison et al., 2001). Furthermore, specific residues within the chemokine domain, namely K7, K14, M15, R47 and F49 play varying roles in binding of fractalkine to CX3CR1 as well as receptor activation (Harrison et al., 2001; Mizoue et al., 2001). Plate 48.3 summarizes data from independent studies analyzing site-directed mutants of the fractalkine chemokine domain. These results provide revealing first glimpses into the receptor-binding domain. Previous studies indicated that soluble forms of fractalkine could be detected in the conditioned media of cells, whether they were stimulated endothelial cells or cells expressing recombinant forms of fractalkine (Bazan et al., 1997; Chapman et al., 2000a). A dibasic site proximal to the transmembrane domain has been proposed as a possible cleavage site, although no protease has been identified that will carry out this specific proteolysis. However, two groups have identified TNF alphaconverting enzyme (TACE or ADAM17) as a metalloprotease that can cleave fractalkine from the cell surface (Tsou et al., 2001; Garton et al., 2001). The specific cleavage site(s) are not exactly known, but clearly are at a position in the fractalkine molecule that is distinct from the dibasic juxtamembrane motif. The involvement of a metalloprotease in fractalkine shedding was also suggested by an earlier report on the effect of batimastat, a metalloprotease inhibitor, to inhibit fractalkine shedding from endothelial cells (Chapman et al., 2000a). Whether or not additional proteases are involved in fractalkine cleavage remains to be determined. None the less, the identification of enzymes capable of modifying the levels of membrane-bound and shed forms of fractalkine indicates that potential mechanisms are in place that could dynamically alter the balance of pro-adhesive and pro-chemotactic forms of this chemokine. As a consequence of high proteolytic cleavage, dramatic increases in the levels of shed fractalkine might be predicted to inhibit adhesion mediated by the membrane-anchored form.

FRACTALKINE EXPRESSION: REGULATION AND DISEASE Periphery Beside its particular structure, fractalkine presents other singular properties. Fractalkine is detected in various tissues such as heart, lung, kidney and testis (Bazan et al., 1997) and is principally localized to endothelial, epithelial and dendritic cells unlike other chemokines that are expressed predominantly by hematopoietic or immune cells. In peripheral tissues, fractalkine expression is generally up-regulated in inflammatory conditions. In endothelial cells, its expression is increased after treatment with proinflammatory molecules such as TNFa, IL-1, LPS and IFNc (Bazan et al., 1997; Harrison et al., 1999; Garcia et al., 2000; Imaizumi et al., 2000). This proinflammatory factor regulation of endothelial cell expression of fractalkine has been convincingly demonstrated from both in vitro and in vivo analysis. The recruitment of leukocytes from the circulation, which necessitates the slow down and the arrest of these cells at the vascular endothelium interface, is coordinated by local expression of both chemokines and adhesion molecules. Since fractalkine is a membrane-bound chemokine, it can directly, in a selectin- and integrinindependent manner (Goda et al., 2000), mediate the firm adhesion of a subset of leukocytes under physiological flow conditions (Fong et al., 1998; Haskell et al., 1999). Thus, proinflammatory-induced endothelial cell-expressed fractalkine likely mediates leukocyte recruitment from the blood into the tissue interstitium. Consistent with a role for fractalkine in leukocyte recruitment, an up-regulation of fractalkine expression on endothelial cells is found in numerous inflammatory diseases including crescentic glomerulonephritis (Feng et al., 1999; Furuichi et al., 2001), atherosclerosis (Greaves et al., 2001), cutaneous inflammatory disease (Raychaudhuri et al., 2001) and Crohn’s disease (Muehlhoefer et al., 2000) (see Table 48.1). Glomerulonephritis is characterized by an influx of leukocytes within the glomeruli; fractalkine expression is induced in the endothelium of rat glomeruli in a model of crescentic glomerulonephritis (Feng et al., 1999) and an increased number of fractalkine-positive cells is detected in the interstitium

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TABLE 48.1 Fractalkine and CX3CR1 expression in different pathological conditions Disease

Species

Fractalkine RNA

Periphery Rheumatoid arthritis Crescentis glomerulonephritis Atherosclerosis HIV Crohn’s disease Lung inflammatory disease Heart allograft rejection MTGL Psoriasis Atopic dermatitis Castelman’s disease Reactive lymph node hyperplasia

Rat, human Rat, human Human Human Human Human Mouse Human Human Human Human Human

CNS Facial nerve axotomy EAE Ischemia HIV-associated dementia

Rat Rat, Mouse Rat Human

Protein

CX3CR1 RNA

Protein ND

ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND

ND ND

References

ND ND ND ND ND ND ND

Ruth et al., 2001; Volin et al., 2001 Feng et al., 1999 Furuichi et al., 2001 Greaves et al., 2001 Foussat et al., 2001 Muehlhoefer et al., 2000 Fujimoto et al., 2001 Robinson et al., 2000 Fracticelli et al., 2001 Fracticelli et al., 2001; Raychaudhuri et al., 2001 Fracticelli et al., 2001 Fracticelli et al., 2001 Fracticelli et al., 2001

ND ND ND ND

Harrison et al., 1998 Scwaeble et al., 1998; Jiang et al., 1998 Chapman et al., 2000a Tong et al., 2000; Pereira et al., 2001

ND

ND, not determined; HIV, human immunodeficiency virus; MTGL, mycobacterium tuberculosis granulomatous lymphadenitis; EAE, experimental allergic encephalomyelitis.

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of patients with glomerulonephritis (Furuichi et al., 2001; Cockwell et al., 2002). Similarly, in Crohn’s disease, an inflammatory intestinal disease, endothelial cell-expressed fractalkine mRNA and protein levels are up-regulated (Muehlhoefer et al., 2000). The expression of fractalkine on endothelial cells is differentially regulated by cytokines associated with polarized T-cell response. IFNc, a TH1 cytokine, stimulates fractalkine expression while the TH2 cytokines, IL4 and IL13, are without effect (Fraticelli et al., 2001). Furthermore, these TH2 cytokines inhibit TNFaand IFNc-dependent induction of fractalkine. IL-1-, TNFa- and IFNc-dependent stimulation of endothelial cell-expressed fractalkine has also been shown to be inhibited by soluble IL-6 receptor alpha (Matsumiya et al., 2001). Cultured smooth muscle cells also express fractalkine in a TNFa- and IFNcdependent manner. Unlike TH1 cytokine-stimulated endothelial cell expression of fractalkine, expression in smooth muscle cells requires co-application of TNFa and IFNc (Ludwig et al., 2002). The participation of fractalkine in TH1-dependent processes is further supported by documented increases in endothelial cell-expressed fractalkine in TH1-dependent pathologies including psoriasis, Mycobacterium tuberculosis granulomatous lymphadenitis (Fraticelli et al., 2001), and cardiac allograft rejection (Robinson et al., 2000). On the other hand,TH2-driven responses, including atopic dermatitis, reactive lymph node hyperplasia and Castelman’s disease, display no changes in fractalkine expression (Fraticelli et al., 2001). Bronchial and intestinal epithelial cells are also capable of expressing fractalkine (Muehlhoefer et al., 2000; Fujimoto et al., 2001). In bronchial epithelial cells, the level of fractalkine is enhanced after IFNc stimulation (Fujimoto et al., 2001). Up-regulation of fractalkine in bronchial epithelium of patients with lung inflammatory disease has also been documented (Fujimoto et al., 2001) along with an associated rise in soluble fractalkine in bronchoalveolar lavage fluid. Consistent with these in vivo observations, IFNc (Fujimoto et al., 2001) treatment of cultured epithelial cells elevates levels of soluble form(s) of fractalkine. Expression of fractalkine is also increased in an epithelial cell line stimulated with IL-1 and increased levels of fractalkine protein and mRNA are found in intestinal mucosa of active Crohn’s disease (Muehlhoefer et al., 2000).

The expression of fractalkine by dendritic cells has been identified and confirmed in several paradigms (Papadopoulos et al., 1999, 2000; Kanazawa et al., 1999). These cells play a sentinel role after stimulation by an antigen and maturation, acting efficiently as antigen-presenting cells. Increased expression of fractalkine mRNA and protein levels correlate with maturation of dendritic cells, suggesting that fractalkine is involved in the interaction between mature dendritic cells and lymphocytes (Papadopoulos et al., 1999). In papillary dermis of psoriasis tissue, dendritic cell-expressed fractalkine is augmented (Raychaudhuri et al., 2001). Increased expression of fractalkine in dendritic cells of HIV-infected individuals has also been shown (Foussat et al., 2001). More recent studies indicate that fractalkine is also expressed by peripheral immune cells. RT-PCR and immunohistochemical analysis of fractalkine in human atherosclerotic lesions indicated expression by macrophages (Greaves et al., 2001). Fractalkine expression in macrophages was found to be influenced by TH2-type cytokines like IL-4 and IL-13. Immune cell expression of fractalkine is evident in rheumatoid arthritis (RA) as well as in rat adjuvantinduced arthritis (Ruth et al., 2001). In the rat model, fractalkine was localized to endothelial and dendritic cells as well as macrophages and fibroblasts. Similar findings were noted in human RA patients where increased levels of the soluble form of fractalkine were detected in synovial fluid (Ruth et al., 2001).

CNS While many other chemokines are detected in the brain only in inflammatory conditions, fractalkine is expressed in a robust and constitutive manner in the CNS (Pan et al., 1997; Nishiyori et al., 1998; Harrison et al., 1998). Fractalkine is localized in many specific regions including cortex, hippocampus, caudate putamen, thalamus and olfactory bulb (Harrison et al., 1998, Nishiyori et al., 1998). The primary source of this constitutive expression in the CNS is neurons and its presence in these cells has been demonstrated by several groups (Harrison et al., 1998; Nishiyori et al., 1998; Schwaeble et al., 1998; Zujovic et al., 2000; Chapman et al., 2000a). However, glial cell expression of fractalkine has also been documented. Initial immunohistochemical analysis of experimental aller-

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gic encephalomyelitis (EAE) mice demonstrated that microglia cells were the main source of fractalkine (Pan et al., 1997) and this chemokine has been detected in cultured microglial cells (Nishiyori et al., 1998; Zujovic et al., 2000). Although the presence of fractalkine on astrocytes is controversial (Boddeke et al., 1999; Zujovic et al., 2000), treatment of human astrocytes with TNFa and IFNc increase fractalkine expression in these cells (Yoshida et al., 2001). In vivo studies of fractalkine expression in different models of brain inflammation have not provided clear and convincing evidence of altered expression of fractalkine during inflammatory processes involving the CNS. Multiple sclerosis is an autoimmune demyelinating disease characterized principally by infiltration of T lymphocytes and macrophages to the CNS. EAE is an animal model of demyelinating diseases induced by the activation of T cells after injection of MBP, PLP or MOG protein composing the myelin. A critical role for MCP-1 and MIP-1a, in the disease process is clearly established (Kennedy et al., 1998). However, studies of fractalkine expression in EAE have been met with mixed results. As stated, the first detailed report of fractalkine expression in the CNS localized the expression to microglial cells and an increase in fractalkine immunostaining in EAE mice was noted (Pan et al., 1997). Subsequent studies revealed that the pattern of neuronal expression of fractalkine in EAE was not affected (Schwaeble et al., 1998). Studies of EAE mice treated with an altered peptide ligand of PLP (known to prevent the induction and the development of EAE) which present with a decrease in the expression of many chemokines, show reduced fractalkine (Fischer et al., 2000). Lesions due to brain ischemia clearly display inflammatory components and multiple chemokines are increased after an ischemic episode. However, after middle cerebral artery occlusion no changes of fractalkine mRNA are seen (Chapman et al., 2000a). Cultured neurons exposed to an excitotoxic dose of glutamate also do not manifest changes in fractalkine mRNA or protein levels. However, an increase in the level of a soluble form of fractalkine in the medium, which was dependent on metalloprotease activation, was seen after an excitotoxic insult (Chapman et al., 2000a). Two studies demonstrate increased fractalkine expression in CNS inflammation induced by HIV,

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although the source of fractalkine was found to be different. In one study of HIV-infected patients, fractalkine was localized in neurons and neuropils and an increase of its expression in demented pediatric patients compared with non-demented HIV patients was seen (Tong et al., 2000). On the other hand, increases in fractalkine associated with astrocytes was found in patients with HIV-associated dementia (Pereira et al., 2001). Lesioning of the facial motor nerve in the rat is accompanied by a strong microglial cell response with little, if any, peripheral cell inflammatory infiltrate (Kreutzberg, 1996; Streit, 1996). In this model an increase in fractalkine consistent with soluble form(s) is seen despite a clear reduction in motor neuron-expressed fractalkine mRNA after nerve injury (Harrison et al., 1998). A prominent role for fractalkine and CX3CR1 in microglial proliferation is not likely as mice deficient in CX3CR1 (Jung et al., 2000) display similar nerve injury-dependent microglial cell changes, both in terms of the kinetics and extent of the response. Thus, more subtle roles for this chemokine are likely. Regardless of clear changes in the expression of fractalkine in CNS pathologies, a role for this chemokine in any aspect of disease initiation, progression or resolution is still possible. The development of specific fractalkine receptor antagonists may aid in discerning what role, if any, fractalkine plays in CNS inflammation.

CX3CR1 EXPRESSION AND REGULATION Periphery In the periphery, immune cells including lymphocytes, monocytes, natural killer cells and mastocytes express CX3CR1 (Imai et al., 1997; Al-Aoukaty et al., 1998; Kanazawa et al., 1999; Jung et al., 2000; Chapman et al., 2000b; Foussat et al., 2000; Papadopoulos et al., 2000; Cambien et al., 2001; Inngjerdingen et al., 2001). Leukocyte migration toward the endothelium is a multistep process that includes leukocyte rolling, activation and firm adhesion followed by extravasation into the tissue parenchyma. Chemokines including fractalkine participate, first in the attraction and the migration of leukocytes. Subsequently, the

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adhesion of leukocytes requires the coordinate and local expression of both chemokines and adhesion molecules. However, membrane-bound fractalkine can directly induce the firm adhesion of leukocytes even in physiological flow conditions. The specific targets of endothelial cell-expressed fractalkine in the periphery include monocytes, T cells and natural killer (NK) cells. While constitutive expression of CX3CR1 in NK cells has been reported (Imai et al., 1997; Campbell et al., 2001), other investigators have detected CX3CR1 expression only in IL-2-activated NK cells (Inngjerdingen et al., 2001). This latter group has also provided evidence for a second fractalkine receptor. They identified a population of fractalkineresponsive NK cells which did not express CX3CR1. Functional consequences of an endothelial cell-NK cell interaction mediated by fractalkine and CX3CR1 include NK cell cytotoxicity directed toward endothelial cells expressing fractalkine (Yoneda et al., 2000). A role for fractalkine in the control of NK cell cytolytic activity is implied from these findings. Recall that the expression of fractalkine on endothelial cells is differentially regulated depending on the cytokine stimulus: IFNc, a TH1 cytokine, induces fractalkine expression while the TH2 cytokines IL-4 and IL-13 have no effect (Fraticelli et al., 2001). Not surprisingly, TH1-polarized cells preferentially express CX3CR1 as compared with cells with a TH2 phenotype (Fraticelli et al., 2001). In parallel to the increase of fractalkine in pathologies with a TH1 bias, e.g. Crohn’s disease (Muehlhoefer et al., 2000), glomerulonephritis (Feng et al., 1999) and HIV (Foussat et al., 2001), there is an enhanced expression of CX3CR1 in leukocytes (Table 48.1). Mastocytes, immune cells that detect a foreign antigen in the interstitial space, express CX3CR1 (Papadopoulos et al., 2000). Unlike other chemokines that induce migration and activation of these cells, fractalkine stimulates only migratory events. Thus the role of fractalkine is limited to homing of mastocytes. Human dermal microvascular endothelial cells have also been shown to express CX3CR1 and fractalkine induces their chemotaxis and tube formation in vitro (Volin et al., 2001). These data suggest a potential role for fractalkine in angiogenesis.

CNS Microglial cells are the principal cells of the CNS that express CX3CR1 (Harrison et al., 1998; Nishiyori et al., 1998; Boddeke et al., 1999). Classical chemokine properties of fractalkine on microglial cells include induction of chemotaxis (Harrison et al., 1998; Maciejewski-Lenoir et al., 1999). Other signaling mechanisms shared by chemokine receptor activation include increased intracellular calcium mobilization (Harrison et al., 1998; Boddeke et al., 1999), activation of ERK pathways, and stimulation of Akt/PKB (Maciejewski-Lenoir et al., 1999). This latter protein kinase is involved in cell survival. Consistent with this, fractalkine has been shown to inhibit Fas/Fas ligand-induced microglial cell death (Boehme et al., 2000). Two studies demonstrate both in vitro and in vivo that fractalkine controls microglial activation; fractalkine inhibits the production of two markers of microglial activation, namely TNFa and free radicals (Zujovic et al., 2000, 2001). The expression of CX3CR1 appears to be dependent on the state of microglial cell activation. Two studies demonstrate that CX3CR1 mRNA and protein expression is down-regulated in LPS-activated microglia (Boddeke et al., 1999; Zujovic et al., 2000). On the other hand, an up-regulation of CX3CR1 expression in microglial cells was observed in the rat facial motor nucleus after peripheral nerve injury (Harrison et al., 1998). This latter model is characterized by an absence of classical proinflammatory mediators. Since fractalkine is predominantly expressed by neurons and CX3CR1 localized to microglial cells, it seems clear that fractalkine provides a communication link between neurons and microglia. However, the presence of CX3CR1 in both cultured neurons and astrocytes indicates additional cell–cell networks utilize fractalkine-dependent signaling mechanisms. Numerous studies demonstrate the presence of CX3CR1 in cultured astrocytes (Jiang et al., 1998; Maciejewski-Lenoir et al., 1999; Dorf et al., 2000). Fractalkine treatment stimulates calcium mobilization in these cells but does not promote their migration (Maciejewski-Lenoir et al., 1999). Perhaps other physiological endpoints are regulated by fractalkine in this glial cell subtype. For instance, MaciejewskiLenoir et al., (1999) demonstrated that conditioned

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medium of fractalkine-treated astrocytes increased microglial cell survival suggesting that fractalkine promotes the astrocytic secretion of a microglial cell trophic factor(s). Proinflammatory cytokines such as TNFa and IL-1b enhance the expression of CX3CR1 in astrocytes (Maciejewski-Lenoir et al., 1999). The presence of CX3CR1 in neurons is subject to controversy. Multiple methods have been applied to demonstrate the presence or absence of this receptor in cultured neurons. In support of neuronal expression, evidence from several approaches including RT-PCR, Western blot analysis and functional responsiveness to fractalkine (including stimulation of intracellular calcium mobilization and activation of excitatory responses as measured from electrophysiological recordings), has been put forth (Meucci et al., 1998, 2000). Furthermore, evidence for a role for fractalkine in neuronal survival also exists (Meucci et al., 1998, 2000; Tong et al., 2000). In these studies, fractalkine has been shown to inhibit neuronal cell death induced by glial cell feeder layer removal (Meucci et al., 1998). Fractalkine also prevented neuronal death induced by HIV gp120 (Meucci et al., 1998), Tat and Paf (Tong et al., 2000). As with fractalkine effects on microglia, this neuronal protective effect is probably due to the activation of Akt/PKB kinase (Meucci et al., 2000). Despite the clear and convincing demonstration of functional CX3CR1 on both cultured astrocytes and neurons, the relevance of fractalkine receptor localization to either of these cells awaits identification of the expression of CX3CR1 on these cells in vivo. Analysis of CX3CR1 in various pathologies is far more limited as compared with studies of fractalkine. Increased CX3CR1 mRNA in the lumbar spinal cord of EAE rats has been documented (Jiang et al., 1998). Similarly, increased expression of CX3CR1 in the facial motor nucleus after peripheral nerve injury has also been demonstrated (Harrison et al., 1998). These specific increases are probably due to a combination of increased numbers of CX3CR1-expressing cells associated with the lesioned tissue as well as upregulated expression within specific cells. The functional significance of increased receptor expression is not known, although enhanced sensitivity to fractalkine and/or more robust activation of downstream signaling events is a probable consequence of receptor up-regulation.

CX3CR1 SIGNALING PATHWAYS CX3CR1 shares common signaling pathways with other chemokine receptors, including stimulation of intracellular calcium mobilization (Imai et al., 1997; Harrison et al., 1998; Combadiere et al., 1998a, 1998b; Meucci et al., 1998; Al-Aoukaty et al., 1998; Boddeke et al., 1999; Maciejewski-Lenoir et al., 1999), tyrosine kinase (Cambien et al., 2001), MAP kinase (Meucci et al., 1998; Maciejewski-Lenoir et al., 1999; Cambien et al., 2001; Kansra et al., 2001) and phosphoinositide 3 kinase (PI-3K) activation (Maciejewski-Lenoir et al., 1999; Kansra et al., 2001; Cambien et al., 2001). Furthermore, actin polymerization and cytoskeletal rearrangement (steps required for chemotaxis) occur in fractalkine-stimulated CX3CR1-expressing cells such as microglia (Maciejewski-Lenoir et al., 1999) and lymphocytes (Foussat et al., 2000). Plate 48.4 highlights all of the signaling pathways known to be activated or regulated by CX3CR1. In all studies realized in CX3CR1-expressing cells (natural killer cells, microglia, astrocytes and neurons), a rapid increase of calcium is observed after addition of fractalkine (Al-Aoukaty et al., 1998; Harrison et al., 1998; Meucci et al., 1998; Maciejewski-Lenoir et al., 1999; Boddeke et al., 1999). The origin of this calcium increase differs depending on the CX3CR1-expressing cell type analyzed. For instance, in microglial cells, depletion of extracellular calcium weakly affects fractalkinestimulated calcium mobilization while thapsigargin or ryanodine strongly inhibits calcium signaling (Boddeke et al., 1999). On the other hand, the main source of fractalkine-stimulated calcium mobilization in CX3CR1-transfected CHO cells is extracellular (Kansra et al., 2001). Fractalkine induces activation of mitogen-activated protein kinase (MAPK) family members such as MEK (Maciejewski-Lenoir et al., 1999), ERK1/2 (Meucci et al., 1998; Maciejewski-Lenoir et al., 1999; Cambien et al., 2001; Kansra et al., 2001), SAPK1/JNK, and SAPK2/p38 (Cambien et al., 2001). PI3K activation is also a downstream component of CX3CR1 signaling (Meucci et al., 1998; Maciejewski-Lenoir et al., 1999; Kansra et al., 2001; Cambien et al., 2001). Whether or not PI-3K lies upstream of MAP kinase activation also depends upon the cell type examined. In CHO cells engineered to express CX3CR1, both calcium and

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MAPK signaling events are inhibited by the PI-3K specific inhibitor, LY29402 (Kansra et al., 2001). However, in MonoMac6 cells PI-3K does not control ERK, JNK or p38 activation (Cambien et al., 2001). One of the known downstream components of the PI-3K signaling cascade is phosphorylation of Akt/PKB. Akt/PKB is known for its role in cell survival notably by controlling the activation of caspases and the level of bcl-2 family members. Fractalkinedependent activation of this kinase has been demonstrated in monocytes (Cambien et al., 2001), microglial cells (Maciejewski-Lenoir et al., 1999), neuronal cells (Meucci et al., 2000) and recombinant CX3CR1-expressing CHO cells (Kansra et al., 2001). Treatment of microglial cells with fractalkine results in an up-regulation of the anti-apoptotic members Bcl-xL, and a down-regulation and inhibition of Bax and Bad, two proapoptotic proteins (Boehme et al., 2000). Enhanced neuronal survival, after fractalkine treatment, notably due to PI-3K mediated NFjB translocation is also supported by experimental data (Meucci et al., 2000). Non-receptor tyrosine kinases have been implicated in signaling pathways of various other chemokine receptors. Monocytic cells treated with fractalkine, rapidly activate Src and Syk kinases (Cambien et al., 2001). Inhibitors of Src and Syk, respectively, leads to an inhibition of SAPK and ERK1/2 activation, suggesting that in monocytes, these two kinases are upstream of the MAPK and SAPK activation. A common characteristic of chemokine receptor signaling is coupling to pertusis toxin-sensitive G proteins. In this regard, CX3CR1 is not unique, as chemokine receptor-stimulated responses are largely coupled to Gai (Imai et al., 1997; Al-Aoukaty et al., 1998; Maciejewski-Lenoir et al., 1999; Cambien et al., 2001). However, in NK cells CX3CR1 is also coupled to Gaz, a G protein that can directly induce cellular proliferation and NK cell cytotoxicity (Al-Aoukaty et al., 1998). In the majority of cells studied, CX3CR1 signaling is only sensitive to pertusis toxin. However, in MonoMac6 cells cholera toxin blocks fractalkine stimulation of ERK and p38 but not JNK or PI-3K activation, suggesting an involvement of the stimulatory G-protein, Gs, in some aspects of CX3CR1 signaling (Cambien et al., 2001).

CX3CR1 POLYMORPHISMS Polymorphisms in the protein-coding sequence of CX3CR1 have been identified with allelic differences at amino-acid positions 249 and 280 being reported (Faure et al., 2000). A French cohort of HIV-infected individuals homozygous for the I249/M280 form of CX3CR1 were found to progress more quickly to AIDS as compared with individuals carrying other haplotypes (Faure et al., 2000). No such association was found upon examining these polymorphisms in North American cohorts, indicating that CX3CR1 probably does not play a major role in HIV pathogenesis (McDermott et al., 2000). On the other hand, a reduced risk of coronary artery disease is associated with allele I249 (McDermott et al., 2001; Moatti et al., 2001). Individuals carrying this allele present less severe disease than others, possibly due to a greater capacity of the coronary vessels to undergo endothelial-dependent vasorelaxation (McDermott et al., 2001).

FUNCTION OF FRACTALKINE AND CX3CR1 REVEALED FROM STUDYING GENE-DISRUPTED MICE Three reports on the phenotype(s) or lack thereof, of either fractalkine (Cook et al., 2001) or CX3CR1 (Jung et al., 2000; Haskell et al., 2001) gene-disrupted mice are present in the literature. The fractalkine knockout mice present no phenotype and develop normal inflammatory processes (Cook et al., 2001). While a different set of cells seems to express CX3CR1 in vitro, immunohistologic analysis of mice engineered to express green fluorescent protein under control of the CX3CR1 promoter revealed that, in vivo, the primary CX3CR1-expressing cells are monocytes, NK cells and microglia (Jung et al., 2000). These receptor knockout mice presented no phenotypes, even after various inflammatory challenges. However, independently generated CX3CR1/ mice displayed a significant increase of graft survival in a model of cardiac allograft rejection (Haskell et al., 2001). This phenotype confirms an earlier report showing similar effects of anti-CX3CR1 containing rabbit sera in this model (Robinson et al., 2000). Thus, future development

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of CX3CR1 antagonists may hold promising new therapeutics for the treatment of transplantation rejection.

VIRALLY ENCODED FRACTALKINE AND CX3CR1-BINDING PROTEINS: VMIPII, RSV G GLYCOPROTEIN AND US28 Viruses have evolved molecular mechanisms to thwart detection by the immune system, and viral pirating of chemokine and chemokine receptor homologs is a well-represented example of this scheme (Murphy, 2001). One such chemokine analog is vMIPII, which is encoded by human herpesvirus-8 (HHV8). This protein binds to several chemokine receptors including CX3CR1 (Kledal et al., 1997; Feng et al., 1999). Another potential virally encoded protein–CX3CR1 interaction has been described for the G glycoprotein of respiratory syncytial virus (RSV). HEK293 cells expressing CX3CR1 bind RSV G glycoprotein (Tripp et al., 2001). Furthermore, anti-CX3CR1 antibodies prevented RSV infectivity and RSV G glycoprotein induced similar chemotactic responses as was observed for fractalkine. US28 is an open reading frame found in human cytomegalovirus (CMV) and encodes a seven transmembrane spanning G protein-coupled receptor (GPCR). US28-expressing cells bind RANTES, MCP, MIP-1a (Gao and Murphy, 1994) as well as fractalkine (Kledal et al., 1998). Residues highlighted in Plate 48.3, as critical determinants of fractalkine binding to CX3CR1 have also been shown to be important in its interactions with US28 (Mizoue et al., 2001). Of particular interest, while chemokines such as RANTES and MCP-1 activate US28, fractalkine inhibits US28 constitutive phospholipase C activation, identifying that chemokines possess inverse agonist activity (Casarosa et al., 2001). This range of efficacy for a virally encoded GPCR may prove useful in the development of novel antiviral agents. Of course, the involvement of either vMIPII/CX3CR1 or fractalkine/ US28 interactions in the pathogenesis of HHV8 or CMV remains to be determined.

CXCL16: A SECOND MEMBRANE-BOUND CHEMOKINE A second membrane-bound chemokine has been recently identified, termed CXCL16 (Matloubian et al., 2000; Wilbanks et al., 2001). Like fractalkine, CXCL16 contains a mucin-like stalk that tethers a chemokine domain to a single transmembrane spanning region. The CXCL16 chemokine domain contains six cysteine residues, with the first two separated by a single amino acid, thus finding its place within the CXC subfamily. The gene is expressed in a variety of tissues including lung, liver, spleen, kidney, thymus, small intestine and pancreas, as well as on the surface of monocytes, dendritic and B cells. Both shed and membrane-attached forms of CXCL16 have been detected. A previously identified orphan receptor termed Bonzo/STRL33/ TYMSTR is a receptor for CXCL16. Unlike fractalkine, membrane-bound CXCL16 dependent cell adhesion has not been formally demonstrated.

SUMMARY AND FUTURE INQUIRY The expansion of the chemokine gene family has clarified aspects of immune system cell–cell communication and in particular has enhanced our understanding of the specificity of leukocyte chemotactic responses. The discovery of fractalkine (CX3CL1), and more recently CXCL16, has added further dimensionality towards understanding functional roles of chemokines. While fractalkine and CXCL16 share several characteristics of chemokine family members, they are unique in their dualistic properties. Significantly more information has been gathered regarding the biology of fractalkine, as compared with CXCL16, and as such fractalkine has become the prototypical dual functional chemokine. The dual properties of these chemokines are characterized along multiple fronts and include attributes related to both their physical and functional properties. Fractalkine exists in both membrane-attached and shed forms, with the former mediating adhesion and the latter promoting chemotaxis of CX3CR1expressing cells. It is expressed in both peripheral and CNS tissues and differentially regulated in various

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diseases. While significant advances in understanding the function of fractalkine have been made, further inquiry is needed in order to fully understand the biology of this interesting chemokine system. The field of chemokinology has witnessed great strides in clarifying additional roles of chemokines, beyond classically described properties of these molecules as simple chemoattractants, and the discovery of fractalkine has certainly contributed to this multidimensionality. From this information explosion, an additional level of classification has emerged, categorizing members as inflammatory or homeostatic chemokines. Once more fractalkine may present singular properties. While in the periphery fractalkine plays a role as an inflammatory chemokine, its function in the CNS appears better characterized as a homeostatic chemokine. The principal distinction of fractalkine and CXCL16, from all other chemokine molecules, lies in their extended structures, imparting membrane localization properties. For fractalkine, and quite possibly CXCL16, this provides a mechanism whereby chemokine receptor-dependent cell adhesion events can take place in the absence of mechanisms involving other classes of adhesion molecules. At a molecular and cellular level, issues relevant to the usage of membrane-attached versus shed forms of fractalkine are worthy of consideration. For instance, since cell– cell communication networks in the CNS are not subject to the same sheer forces that elements within the lumen of blood vessels normally encounter, the significance of expression and utilization of membraneattached fractalkine–CX3CR1 cell adhesion events in the CNS becomes debatable. In other words, these neural cells may only be utilizing fractalkine as a cell–cell signaling molecule although usage of this chemokine receptor pair in static adhesion events should not be discounted. On the flip side, in considering the function of fractalkine in peripheral systems, how critical is a CX3CR1 signaling event in biological processes dependent on fractalkine cell adhesion events? It seems certain that receptor signaling events are occurring concomitantly with cell adhesion, even if the latter process is not dependent on the former one. Properties of fractalkine shared with other members of the chemokine gene family include regulated expression by endothelial cells in a proinflammatory-

dependent manner, i.e. induced by IL-1, TNFa and LPS. Shed forms of fractalkine are chemotactic for a variety of leukocyte cell types including monocytes, T cells and NK cells. The expression of fractalkine is regulated, and typically up-regulated in a variety of inflammatory pathologies characterized by a TH1 bias. While the specific role of fractalkine in many of these diseases is not clearly defined, in rat glomerulonephritis and murine cardiac allograft rejection, a prominent role for this chemokine and CX3CR1 in disease initiation and progression seems clear. Furthermore, a role for CX3CR1 in coronary artery disease is also suggested. In this regard, the role of fractalkine in these pathologies clearly fits with classically described properties of a number of chemokine peptides, namely the recruitment of inflammatory cell populations. The constitutive expression of fractalkine and CX3CR1 in many diverse tissues certainly begs the question for a role of this chemokine system in normal physiology, processes that would preclude involvement of mechanisms normally associated with inflammatory pathologies. Yet, no significant phenotype of either fractalkine or CX3CR1 gene-disrupted mice are evident, outside of prolonging cardiac allograft survival. Given the dramatic phenotype of SDF-1 and CXCR4 (another prominent constitutively expressed chemokine ligand:receptor pair) knockout mice, i.e. dying perinatally with several pathological consequences (Nagasawa et al., 1996; Tachibana et al., 1998; Zou et al., 1998; Ma et al., 1998), it was certainly expected that fractalkine or CX3CR1 gene-deleted animals would have displayed more obvious phenotypes. For instance, the relatively high expression of fractalkine and CX3CR1 in the CNS might have lent predictions of abnormal brain pathologies in the gene-disrupted mice. These expected results would have obviously made it easier to glean insights into the function of fractalkine in the CNS. Thus, it now seems certain that this chemokine system is playing a more subtle role in CNS physiology. Perhaps a role for fractalkine and CX3CR1 in brain cognition will be revealed from future experimentation. In this light, it is worth considering the function of these genes in the context of the more central concept in dualism, namely the relationship of the mind and body so elegantly and systematically proposed by the French mathematician, philosopher and physiologist, Rene

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Descartes. While cognitive and/or behavioral analysis of experimental animals are often met with difficulty and near impossibility, perhaps a role for fractalkine in human cognition will be revealed from thoughtful self examinations. Given that functional differences among the human CX3CR1 variants exist, more careful comparisons of these individuals and their associated psychological behaviors may be necessary before a true understanding of the function of CNS expressed fractalkine is realized. Finally, since chemokines participate actively in the development of inflammatory diseases, they have attracted significant attention from the pharmaceutical industry and their receptors have become targets in the development of novel therapeutics. Logically, there is especially an intense effort on the elaboration of antagonist molecules, both as novel antiinflammatory drugs as well as agents useful in slowing the progression of HIV-induced AIDS. With certainty, a CX3CR1 antagonist will probably prove useful in various peripheral inflammatory diseases, and current experimental evidence indicates a clear use in controlling cardiac transplantation rejection. However, it will be necessary to take into account the particular features of fractalkine in the CNS. Since fractalkine controls astrocyte secretion of trophic factors, microglia activation and neuronal survival the development of a CX3CR1 agonist should also be considered.

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populations and in vivo production of fractalkine in human. Eur. J. Immunol. 30, 87–97. Foussat, A., Bouchet-Delbos, L., Berrebi, D. et al. (2001). Deregulation of the expression of the fractalkine/ fractalkine receptor complex in HIV-1-infected patients. Blood 98, 1678–1686. Fraticelli, P., Sironi, M., Bianchi, G. et al. (2001). Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses. J. Clin. Invest. 107, 1173–1181. Fujimoto, K., Imaizumi, T., Yoshida, H. et al. (2001). Interferon-gamma stimulates fractalkine expression in human bronchial epithelial cells and regulates mononuclear cell adherence. Am. J. Respir. Cell. Mol. Biol. 25, 233–238. Furuichi, K., Wada, T., Iwata, Y. et al. (2001). Upregulation of fractalkine in human crescentic glomerulonephritis. Nephron 87, 314–320. Gao, J.L. and Murphy, P.M. (1994). Human cytomegalovirus open reading frame US28 encodes a functional beta chemokine receptor. J. Biol. Chem. 269, 28539–28542. Garcia, G.E., Xia, Y., Chen, S. et al. (2000). NF-kappaBdependent fractalkine induction in rat aortic endothelial cells by IL-1beta, TNF-alpha, and LPS. J. Leukoc. Biol. 67, 577–584. Garton, K.J., Gough, P.J., Blobel, C.P. et al. (2001). TACE (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J. Biol. Chem. 276, 37993–38001. Goda, S., Imai, T., Yoshie, O. et al. (2000). CX3Cchemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms. J. Immunol. 164, 4313–4320. Greaves, D.R., Hakkinen, T., Lucas, A.D. et al. (2001). Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine and thymus- and activationregulated chemokine, are expressed in human atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 21, 923–929. Harrison, J.K., Barber, C.M. and Lynch, K.R. (1994). cDNA cloning of a G-protein coupled receptor expressed in rat spinal cord and brain related to chemokine receptors. Neurosci. Lett. 169, 85–89. Harrison, J.K., Jiang, Y., Chen, S. et al. (1998). Role for neuronally-derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc. Natl Acad. Sci. USA 95, 10896–10901. Harrison, J.K., Jiang, Y., Wees, E.A. et al. (1999). Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart. J. Leukoc. Biol. 66, 937–944. Harrison, J.K., Fong, A.M., Swain, P.A. et al. (2001). Mutational analysis of the fractalkine chemokine domain: basic amino acid residues differentially contribute to CX3CR1 binding, signaling, and cell adhesion. J. Biol. Chem. 276, 21632–21641. Haskell, C.A., Cleary, M.D. and Charo, I.F. (1999). Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction; rapid flow arrest of CX3CR1expressing cells is independent of G-protein activation. J. Biol. Chem. 274, 10053–10058. Haskell, C.A., Cleary, M.D. and Charo, I.F. (2000). Unique role of the chemokine domain of fractalkine in cell capture. Kinetics of receptor dissociation correlate with cell adhesion. J. Biol. Chem. 275, 34183–34189.

Haskell, C.A., Hancock, W.W., Salant, D.J. et al. (2001). Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection. J. Clin. Invest. 108, 679–688. Hiroyama, T., Iwama, A., Nakamura, Y. and Nakauchi, H. (2001). Fractalkine shares signal sequence with TARC: gene structures and expression profiles of two chemokine genes. Genomics 75, 3–5. Hoover, D.M., Mizoue, L.S., Handel, T.M. and Lubkowski, J. (2000). The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement. J. Biol. Chem. 275, 23187–23193. Imai, T., Hieshima, K., Haskell, C. et al. (1997). Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91, 521–530. Imaizumi, T., Matsumiya, T., Fujimoto, K. et al. (2000). Interferon-gamma stimulates the expression of CX3CL1/ fractalkine in cultured human endothelial cells. Tohoku J. Exp. Med. 192, 127–139. Inngjerdingen, M., Damaj, B. and Maghazachi, A. A. (2001). Expression and regulation of chemokine receptors in human natural killer cells. Blood 97, 367–375. Jiang, Y., Salafranca, M.N., Adhikari, S. et al. (1998). Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis. J. Neuroimmunol. 86, 1–12. Jung, S., Aliberti, J., Graemmel, P. et al. (2000). Analysis of fractalkine receptor CX3CR1 function by targeted deletion and green fluorescent protein reporter insertion. Mol. Cell. Biol. 20, 4106–4114. Kanazawa, N., Nakamura, T., Tashiro, K. et al. (1999). Fractalkine and macrophage-derived chemokine: T cellattracting chemokines expressed in T cell area dendritic cells. Eur. J. Immunol. 29, 1925–1932. Kansra, V., Groves, C., Gutierrez-Ramos, J.C. and Polakiewicz, R.D. (2001). Phosphatidylinositol 3-kinase-dependent extracellular calcium influx is essential for CX(3)CR1mediated activation of the mitogen-activated protein kinase cascade. J. Biol. Chem. 276, 31831–31838. Kennedy, K.J., Strieter, R.M., Kunkel, S.L. et al. (1998). Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1alpha and monocyte chemotactic protein-1. J. Neuroimmunol. 92, 98–108. Kledal, T.N., Rosenkilde, M.M., Coulin, F. et al. (1997). A broad-spectrum chemokine antagonist encoded by Kaposi’s sarcoma-associated herpesvirus. Science 277, 1656–1659. Kledal, T.N., Rosenkilde, M.M. and Schwartz, T.W. (1998). Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirusencoded broad-spectrum receptor US28. FEBS Lett 441, 209–214. Kreutzberg, G.W. (1996). Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Ludwig, A., Berkhout,T., Moores, K. et al. (2002). Fractalkine is expressed by smooth muscle cells in response to IFNgamma and TNF-alpha and is modulated by metalloproteinase activity. J. Immunol. 168, 604–612. Ma, Q., Jones, D., Borghesani, P.R. et al. (1998). Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl Acad. Sci. USA 95, 9448–9453.

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Yoshida, H., Imaizumi, T., Fujimoto, K. et al. (2001). Synergistic stimulation, by tumor necrosis factor-alpha and interferon-gamma, of fractalkine expression in human astrocytes. Neurosci Lett. 303, 132–136. Zou, Y.-R., Kottmann, A.H., Kuroda, M. et al. (1998). Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599. Zujovic, V., Benavides, J., Vige, X. et al. (2000). Fractalkine

modulates TNF-alpha secretion and neurotoxicity induced by microglial activation. Glia 29, 305–315. Zujovic, V., Schussler, N., Jourdain, D. et al. (2001). In vivo neutralization of endogenous brain fractalkine increases hippocampal TNFalpha and 8-isoprostane production induced by intracerebroventricular injection of LPS. J. Neuroimmunol 115, 135–143.

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Subsection G. The TGF Beta Family Members

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49 Transforming growth factor b Philip H. Howe Cleveland Clinic Foundation, Cleveland, OH, USA

We will discover the nature of our particular genius when we stop trying to conform to our own or other people’s models, learn to be ourselves, and allow our natural channel to open Shakti Gawain

INTRODUCTION Twenty years ago an activity secreted in conditioned medium of transformed cells was found to promote the ‘transformed’ phenotype of non-neoplastic reader cells and given the name transforming growth factor b (TGFb) (Moses et al., 1981; Roberts et al., 1981; Cheifetz et al., 1987). It was subsequently purified from human platelets (Assoian et al., 1983), and cloned and sequenced from a human placental cDNA library (Derynck et al., 1985). It soon became apparent that its name was not only misleading, in that it does not promote oncogenic transformation, but was restrictive in terms of the protein’s multifunctional biologic activity. Early studies with TGFb demonstrated that it was a potent inhibitor, rather than stimulator, of cellular proliferation in epithelial and certain tumor cell lines, an inducer of chondriogenic differentiation, an immunosuppressor, and an inhibitor of myogenic differentiation (Massagué, 1990, 1998). It also became apparent early on that TGFb is The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

prototypic of a superfamily of growth, differentiation and morphogenic factors that now comprises over 50 members. These include the activins and inhibins, bone morphogenetic proteins (BMPs), decapentaplegic product (DPP), Mullerian inhibiting substance (MIS) and other TGFbs. The widespread distribution of these factors from C. elegans to humans, as well as their strict evolutionary conservation, suggested early on that these factors were important regulators of cell growth and developmental processes (Massagué 1990, 1998). During the past decade, studies have revealed a signal transduction pathway, in which activation of receptor serine/threonine kinases at the cell surface leads to phosphorylation of specific intracellular substrates, the Smad proteins, which translocate into the nucleus (Massagué and Chen, 2000; Wrana, 2000; ten Dijke et al., 2000; Derynck et al., 1998). Once inside the nucleus Smad proteins interact with other DNA-binding proteins to regulate transcription of specific target genes thereby eliciting the many

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different effects of TGFb. More recent research has implicated a role for members of the MAPK signaling pathway, independently or in cross-talk with the Smads, as mediators of TGFb responses. Thus, light is being shed at a molecular level on the regulation and integration of diverse signaling pathways which underlie the multifunctionality of this protein. Previous reviews on TGFb have covered its numerous biologic responses including its role in mediating cellular proliferation, tumorgenicity and various other disease states (Massagué and Wotton, 2000; Massagué, 1998; Massagué et al., 2000; ten Dijke et al., 2000). Other chapters in this book will also review the TGFb-related factors, activins/inhibins and BMPs. Therefore, in this chapter, I will limit my discussion to TGFb. In particular, I will review its structure, its signal transduction pathway from receptor activation to regulation of gene expression, and its role in regulating apoptosis.

MOLECULAR STRUCTURE OF TGFbs TGFbs are typical of other secretory polypeptides in that they are synthesized as larger glycosylated preproproteins containing an amino-terminal signal sequence for endoplasmic reticulum translocation and secretory pathway targeting. In the case of TGFb1, (Plate 49.1A) (see Plate section) it is synthesized as a 390-amino-acid glycosylated pre-proprotein that contains a 29-amino-acid hydrophobic signal sequence which is cleaved, resulting in pro-TGFb1 (Derynck et al., 1985; Sharples et al., 1987). The carboxyl-terminal 112-amino-acid sequence, representing monomeric TGFb1, is cleaved from the amino-terminal propeptide at a dibasic (RXXR) cleavage site. Homodimerization of these 112-amino-acid subunits generates the biologically active 25-kDa dimeric polypeptide. It is not clear at which point during the processing, either synthesis or transit, dimerization of the mature subunits occurs (Gentry et al., 1987, 1988; Purchio et al., 1988; Brunner et al., 1988). The amino-terminal prodomains of each family member are poorly conserved yet cross-species conservation for a particular family member is less divergent (Derynck et al., 1986; Cate et al., 1986). The prodomains appear to be required for normal syn-

thesis and secretion and in the case of the TGFbs bind to the mature form of the protein forming a latent complex (Plate 49.1B; Gray and Mason, 1990; Hammonds et al., 1991). Unlike most other hormones and other members of the TGFb superfamily, the cleaved amino-terminal pro-TGFb1, also known as the latency-associated peptide (LAP), remains non-covalently associated with the mature TGFb1 after secretion to produce an inactive complex which is not recognized by betaglycan or the signaling receptors (Gentry and Nash, 1990; Kingsley, 1994). In most cells, LAP is covalently linked by disulfide bonds to an additional glycoprotein, latent TGFbbinding protein (LTBP), forming a large latent complex (Saharinen et al., 1999). LTBPs are not required for the latency of TGFb complex but are required for efficient secretion, correct folding and storage of TGFb in the extracellular matrix (Wakefield et al., 1988; Brown et al., 1990; Miyazono et al., 1991; Saharinen et al., 1999). Latency, however, is dependent on the presence of LAP, and modification of the cysteine residues required for LAP dimerization results in altered TGFb secretion. This suggests that the tertiary structure of LAP is important for the formation of the latent complex (Sha et al., 1991; Miller et al., 1992; McMahon et al., 1996). The molecular mechanism of activation of the TGFb–LAP is not fully understood. It is known that changes in ionic strength, acidification of cellconditioned media or acid extraction of tissue, are capable of disrupting the non-covalent interactions, thus releasing mature, biologically active TGFb1 (Lyons et al., 1988; Brown et al., 1990; Miyazono et al., 1990). Proteolysis has also been shown to activate latent TGFb. Specifically, plasmin, a serine protease, appears to cleave within the amino-terminal glycopeptide resulting in conformational changes which destabilize the latent complex and release mature TGFb1 (Lyons et al., 1988; Lyons and Moses, 1990). The role of plasmin as a physiological activator of latent TGFb1 was further corroborated by the demonstration that in vivo activation of TGFb during coculture of bovine endothelial cells and pericytes could be blocked by specific inhibitors of plasmin (Sato and Rifkin, 1989). However, plasminogen knockout mice do not phenocopy TGFb knockout mice, suggesting that plasmin is not the sole activator of TGFb in vivo. Exposure to reactive oxygen species and radiation

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treatment have also been implicated in TGFb activation (Barcellos-Hoff and Dix, 1996; Barcellos-Hoff et al., 1994). In addition, thrombospondin 1 (TSP-1) (Schultz-Cherry and Murphy-Ullrich, 1993; SchultzCherry et al., 1994a, 1994b) and the integrin aVb6 (Munger et al., 1998) have been implicated in the physiologic activation of latent TGFb. The matrix protein TSP-1 has been shown to bind LAP through distinct domains resulting in a rearrangement of LAP, a disruption of the electrostatic interactions between LAP and the active protein, and a conversion of the latent complex into a biologically active molecule (Schultz-Cherry et al., 1994b, 1995; Riberio et al., 1999b). Genetic evidence also supports a role for TSP1 in TGFb activation in vivo. TGFb1 and TSP-1 knockout mice have similar histologic abnormalities and treatment of TSP-1 null mice with a TSP-1 peptide, required for TGFb activation, results in reversion of the lung and pancreatic abnormalities being observed as well as detection of active TGFb in situ (Crawford et al., 1998). In addition, treatment of wild-type mice with a peptide that blocks TGFb activation results in phenotypic alterations similar to those seen in TGFb1 null animals (Crawford et al., 1998). Similarly, in vitro (Munger et al., 1998), as well as genetic evidence (Munger et al., 1999), implicates a role for integrin aVb6 in TGFb activation. LAP has been shown to bind to aVb6 through an integrin-binding RGD sequence and this interaction is postulated to localize latent TGFb to the surface of specific cells and result in TGFb activation (Munger et al., 1998). Mice which lack this integrin develop enhanced inflammation and are protected from developing pulmonary fibrosis, both suggestive of localized deficiencies of active TGFb as the basis of the aVb6/ phenotype (Munger et al., 1999). Most of the amino-acid sequence homology between the family members is in the mature, C-terminal region of the molecule. Sequence identity in this domain ranges from 25 to 90% between the various family members (Massagué, 1990). This region also contains the characteristic seven cysteines conserved in all members of the superfamily (Massagué 1990). Crystallography studies and NMR analysis of TGFb2 (Daopin et al., 1992; Schlunegger and Grütter, 1992), BMP-7 (Griffith et al., 1996), and TGFb1 (Archer et al., 1993; Hinck et al., 1996) have demonstrated a common structural motif shared by all family mem-

bers. Six of the seven invariant cysteines are closely grouped together through three intramolecular disulfide bonds known as the cystine knot. This knot locks the base of two pairs of antiparallel b-sheet strands together and is postulated for conferring the resistance of TGFb family members to heat and denaturation. The seventh cysteine in each monomer forms an intermolecular cystine bridge that links the two monomers into a dimer. Five TGFbs, termed TGFb1–5, have been identified to date (Massagué, 1990; Kingsley, 1994). These are derived from different species with TGFb4 possibly representing the chick homologue of TGFb1 (Burt and Paton, 1992) and TGFb5 identified as a cDNA in Xenopus (Kondaiah et al., 1990). Mammalian TGFb4 and 5 have not been identified. The degree of identity between the mature TGFb isoforms is well conserved (70 to 80%) and cross-species homology between the various TGFbs is extremely high (Derynck et al., 1986; Miller et al., 1989a, 1989b). There is greater than 97% identity between mammalian and avian TGFb1 and TGFb2 (Madisen et al., 1988; Jakowlew et al., 1988). The mammalian TGFb1 gene contains a seven-exon structure that is conserved in the other TGFb genes suggesting that the TGFbs arose through gene duplication from a common ancestor, albeit these genes are found on different chromosomes in humans and mouse (Derynck et al., 1987, 1988). Each isoform is controlled by unique gene regulatory promoter/ enhancer elements and their expression is strictly regulated in tissues (Roberts et al., 1991). These observations suggest that each isoform has distinct cellular functions although discriminating these biologic activities in vitro has been elusive (Massagué, 1990; Kingsley, 1994).

RECEPTORS The cellular actions of TGFb are mediated through binding to three distinct cell surface receptors. These receptors were originally revealed through affinity crosslinking studies and were initially characterized according to their molecular weights and termed type I (TbR-I; 53 kDa), type II (TbR-II; 80 kDa), and type III (TbR-III, also known as betaglycan or endoglin; 300 kDa) (Cheifetz et al., 1986, 1987; Segarini and Seyedin, 1988; Massagué, 1990).

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Type III receptors Betaglycan, the type III receptor, is a transmembrane glycoprotein with a large extracellular region that binds TGFb and a short cytoplasmic region without any clearly distinguishable signaling motif or enzymatic activity (Segarini and Seyedin, 1988; Andres et al., 1989; Massagué, 1998). It is a membrane proteoglycan composed of heparan and chondroitin sulfate chains and a 120-kDa core protein which binds all three isoforms of TGFb (Segarini and Seyedin, 1988; Andres et al., 1989). Betaglycan also binds the type II receptor (TbR-II)andispostulatedtoactin‘presenting’theTGFb ligand to TbR-II (Cheifetz et al., 1992; López-Casillas et al., 1993; Moustakas et al., 1993). This role of ‘presentor’ of ligand to the TbR-II signaling receptor is exemplified with TGFb2. TGFb2 signals through the TbR-I and TbR-II, as do TGFb1 and TGFb3, yet it has much lower binding affinity for TbR-II than do these isoforms (Segarini et al., 1987; Andres et al., 1991; Mitchell et al., 1992). TGFb2 is also weaker in eliciting biologic responses in cells that lack betaglycan than are TGFb1 and TGFb3 isoforms and transfection of betaglycan augments TGFb2 binding and activity in these cells (López-Cassilas et al., 1993; Sankar et al., 1995). Recently, it has been shown that betaglycan serves as a co-receptor for another TGFb superfamily member, inhibin A (Lewis et al., 2000). Betaglycan binds to inhibin A in complex with the activin type II receptor ActR-II, and it is postulated that this ternary complex mediates inhibin antagonism of activin activity by sequestration of ActR-II from the activin and activin type I receptor (ActR-I) complex. Furthermore, betaglycan is capable of conferring inhibin sensitivity to cells that are otherwise insensitive to this factor (Lewis et al., 2000). A more direct role for betaglycan in mediating TGFb signaling has recently been demonstrated (Blobe et al., 2001). Despite a lack of identifiable signaling motifs in its cytoplasmic tail, it has been shown that a betaglycan cytoplasmic deletion mutant is still able to bind TGFb, to bind TbR-II and to enhance TGFb binding to TbR-II but is unable to enhance TGFb2 signaling. These data not only confirm the role of betaglycan as co-receptors in modulating ligand–receptor interaction and sensitivity, but also demonstrate functional roles of the cytoplasmic domain of betaglycan in regulating TGFb signaling (Blobe et al., 2001).

Endoglin is a homodimeric cell surface glycoprotein composed of disulfide-linked subunits of 90–95 kDa (Gougos and Letarte, 1988, 1990; Gougos et al., 1992). It is expressed at high levels in vascular endothelium and at lower levels in pre-erythroblasts and leukemic cells of lymphoid and myeloid lineages. Its expression is induced upon activation of peripheral blood monocytes and transient expression is observed in heart development in endothelium during heart septation and valve formation (Qu et al., 1998). It has regions of strong sequence similarity with betaglycan, particularly in the extracellular domain (Moren et al., 1992). Endoglin binds TGFb1 and TGFb3, but unlike betaglycan, it does not bind TGFb2 (Cheifetz et al., 1992). Similarly to betaglycan, it has been shown to complex with TbR-I and TbR-II (Yamashita et al., 1994a; Zhang H. et al., 1996; Lastres et al., 1996; Pece et al. 1997). Endoglin does not bind TGFb1 and TGFb3 on its own but requires the coexpression of TbR-II. In addition, it binds activin A, BMP-2 and BMP-7 only in the presence of their respective ligand-binding receptors (Barbara et al., 1999). Recent results have demonstrated that endoglin is the binding partner for the ALK1 type I TGFb receptor whose corresponding type II receptor and ligand are unknown (Marchuk, 1998; Lux et al., 1999). Genetic evidence also suggest that endoglin and ALK1 may act in the same pathway and that endoglin may facilitate ligand binding to ALK1. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by epithelial vascular dysplasia and excessive hemorrhaging in the nasal and gastrointestinal mucosa. The two genes identified as targets for inactivating mutations in this disease encode for endoglin and ALK1 (McAllister et al., 1994; Johnson et al., 1996).

Signaling receptors: type I and type II TGFb and other members of the superfamily signal through serine/threonine kinases collectively referred to as the TGFb receptor family (Massagué, 1998). The exception is the glial cell-derived neurotrophic factor (GDNF), the most divergent member of the TGFb family, which signals through the tyrosine kinase receptor Ret (Massagué, 1996). The first member of this family isolated was Daf-1 an orphan receptor from C. elegans involved in dauer larva development (Georgi et al., 1990). Its identity as a TGFb receptor

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family member was not appreciated until the cloning of the activin receptor, now referred to as ActR-II (Mathews and Vale 1991). The strong sequence homology between Daf-1 and ActR-II not only established Daf-1 as a TGFb receptor family member but also led to the isolation and identification of many other members of this receptor family (Massagué 1996; Miyazono et al., 1994). Structural and functional characteristics allow this receptor family to be divided into two subfamilies termed type I and type II receptors (Plate 49.2A). Both are glycoproteins of approximately 55 kDa and 70 kDa, respectively, with core proteins of 500 to 570 amino acids (Mathews andVale, 1991; Attisano et al., 1992; Lin et al., 1992; ten Dijke et al., 1993; Ebner et al., 1993). The domain structure of each is similar in that they contain a short extracellular ligand-binding domain, a single transmembrane region and a long cytoplasmic region which consists almost entirely of a serine/threonine kinase domain. The ligand-binding domain is abundant in cysteine residues (10 or more) and an especially conserved motif of three cysteines near the transmembrane region, the cysteine cluster, is characteristic of all members (Wrana et al., 1994b). The cytoplasmic juxtamembrane region of each receptor has been shown to contain important regulatory phosphorylation sites. A serine in this region of TbR-II is a site for autophosphorylation and required for signaling activity (Luo and Lodish, 1997), and a serine in this region of TbR-I is the site for phosphorylation by TbR-II (Souchelnytskyi et al., 1996). The protein kinase domain of each receptor type conforms to the canonical serine/threonine kinase sequence, and each phosphorylates their respective substrates only on serine and threonine residues (Franzén et al., 1993). Several sequence features distinguish type I from type II receptors. The spacing of several of the cysteine residues in the extracellular domain of type I receptors is more highly conserved than in type II receptors. The kinase domains of the type I receptors are more related to each other (60–90% identity) than the kinase domains of type II subfamily members (30–40% identity) (ten Dijke et al., 1993; Matzuzaki et al., 1993; He et al., 1993; Ebner et al., 1993). In addition, type I receptors contain a highly conserved 30-amino-acid region located just N-terminal of the kinase domain referred to as the ‘GS’ domain because of a characteristic TTSGSGSG sequence (Wrana et al.,

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1994a). Ligand-induced phosphorylation of serine and threonine residues in this sequence by type II receptors is required for receptor activation and signaling (Wrana et al., 1994a; Wieser et al., 1995; Souchelnytskyi et al., 1996). C-terminal to the ‘GS’ domain is a conserved Leu–Pro motif found in all type I receptors that is required for binding of the immunophilin FKBP12 (Charng et al., 1996; Chen,Y.G., et al., 1997), postulated to be a negative regulator of receptor signaling (Wang et al., 1996; Chen, Y.G., et al., 1997; Yao et al., 2000). Type II receptors also contain a short C-terminal extension following the kinase domain which is rich in serine and threonine residues. Another important distinction between type I and type II receptors is the inability of type I receptors to bind TGFb in the absence of type II receptors, evidence which was provided using TGFb receptor mutant cell lines (Wrana et al., 1992; Ebner et al., 1993; ten Dijke et al., 1994). Conversely, the type II receptor can bind ligand independently of type I, yet it is insufficient for signal transduction. The latter two distinctions between type I and II receptors underlie the heteromeric receptor model for TGFb signaling and will be discussed in greater detail below (Wrana et al., 1992; Ebner et al., 1993). Plate 49.2B presents an overview of the two subfamilies of receptors and categorizes them with respect to their respective receptor and ligand binding partners (ten Dijke et al., 1996). One striking observation is that there are many more ligand family members (approximately 50) than there are receptor family members, thus multiple ligands share a given receptor. Also, there is overlap in specificity in that different type I and type II receptors can pair to bind different ligands. Activins and BMPs in vertebrates can each bind to several pairs of type I and type II receptors (ten Dijke et al., 1996). In general, type II receptors define their own specificity of ligand binding, while type I receptors have their ligand specificity determined by the co-expressed type II receptor (Attisano et al., 1993; Ebner et al., 1993). Also, in vertebrates, another categorization within the type I subfamily can be assigned based on the degree of sequence homology in the kinase domains and their respective signaling activity. One group includes TbR-I, ActR-I and IB, another includes BMPR-IA and IB, and the last includes Alk2 and Alk-1 whose ligand is unknown (Massagué, 1998).

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Receptor activation Studies with TGFb mutant mink lung epithelial cells, generated by chemical mutagenesis, and somatic cell hybrids between these mutant cell lines revealed early on that many of the cellular responses elicited by TGFb required both a functional type I and type II receptor (Boyd and Massagué, 1989; Laiho et al., 1990, 1991). These studies also demonstrated the cooperative nature of these receptors in TGFb signaling. It was observed that type I receptor could not bind ligand in the absence of type II, and type II, while able to bind ligand on its own, could not signal without type I. Additional evidence for cooperation between the two receptor types was demonstrated by their coimmunoprecipitation (Wrana et al., 1992). These observations led to the formulation of the heteromeric receptor model for TGFb signaling (Plate 49.3) (Wrana et al., 1992, 1994b). According to this concept, ligand binding to the type II receptor induces the formation of a heteromeric complex of type I and type II receptors. Formation of this complex is required for signaling. There is evidence that heterotetrameric receptor complexes form upon ligand binding and this is postulated to be due to the dimeric nature of the ligand itself which could contact a receptor subunit with each of its monomeric chains (Yamashita et al., 1994b). Ligand binding does not increase the phosphorylation or activity of the type II receptor which exists as a constitutively active and autophosphorylated homodimer (Chen and Derynck, 1994; Henis et al., 1994; Wrana et al., 1994b). Autophosphorylation occurs primarily on five serine residues; Ser-223, -226, -227 in the juxtamembrane domain, and Ser-549 and -551 in the C-terminal tail (Souchelnytskyi et al., 1996). This basal phosphorylation of the type II receptor is partially maintained in cells expressing a kinase-defective mutant of the receptor suggesting that other kinases may use the type II receptor as substrate (Wrana et al., 1994b; Chen and Weinberg, 1995). The interaction of the type II receptor with the type I receptor following ligand binding leads to rapid phosphorylation of the type I receptor (Wrana et al., 1994b). This phosphorylation is catalyzed by the type II receptor and occurs at serine (ser-187, -189, -191) and threonine (thr-186) sites in the TTSGSGSGLP sequence of the GS box (Plate 49.2A), and Ser-165 in the juxtamembrane region

(Wrana et al., 1994b; Souchelnytskyi et al., 1996). This transphosphorylation of the type I receptor by the type II receptor kinase results in activation of the type I receptor and initiates subsequent signaling of the receptor complex (Wrana et al., 1994b). Evidence for the role of the type I receptor as the effector of signaling is provided by the T204D type I receptor mutant (GS box mutant) which has increased kinase activity and acts as a constitutive activator of TGFb signaling, albeit with diminished signaling activity compared with the wild-type heteromeric complex (Weiser et al., 1995; Luo and Lodish, 1996).

Receptor modulators Regulators of TGFb receptor signaling have been identified. These include FKBP12 (Wang et al., 1994), Bambi (Onichtchouk et al., 1999), caveolin-1 (Razani et al., 2001) and Lefty (Ulloa and Tabibzadeh, 2001). FKBP12 is a ubiquitously expressed cytoplasmic protein that is a member of the immunophilin family. It serves as a receptor protein for the immunosuppressive drugs FK506 and rapamycin (Kay, 1996; Gothel and Marachiel, 1999). The FKBP12/FK506 complex inhibits calcineurin phosphatase activity and nuclear factor of activated T cell translocation, whereas the FKBP12/rapamycin complex inhibits TOR (target of rapamycin) family proteins (Liu et al., 1991; Abraham and Wiederrecht, 1996). In the absence of ligand, FKBP12 binds to the GS domain of TbR-I and blocks the phosphorylation of the activation sites by TbR-II thereby preventing receptor complex activation (Chen Y.G., et al., 1997). FKBP12 binding has also been shown to prevent receptor-mediated endocytosis, a process required for TGFb signaling (Yao et al., 2000). Ligand-induced receptor complex formation and transphosphorylation by TbR-II is postulated to release FKBP12 from TbR-I allowing receptor activation (Chen Y.G., et al., 1997; Huse et al., 1999). Bambi (BMP and activin membrane-bound inhibitor) is a transmembrane protein which is related to the TGFb family type I receptors but lacks an intracellular kinase domain. It has been shown to bind to most type I receptors and to TbR-II and inhibits BMP, activin and TGFb signaling by preventing the formation of receptor complexes. It is coexpressed with BMP-4 during ventralization in Xenopus, requires BMP signaling for its expression,

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and is thought to serve as a negative feedback loop of BMP signaling during this embryogenic process (Onichtchouk et al., 1999; Grotewold et al., 2001). Caveolae are cholesterol-rich membrane microdomains, that with their principal marker protein caveolin-1 (Cav-1), have been implicated in the compartmentalization and regulation of certain signal transduction pathways. It has been shown that Cav-1 co-localizes with TbR-I, TbR-II and Smad2 in caveolae and that Cav-1 directly interacts with TbR-I. Cav-1 is able to suppress Smad2 phosphorylation induced by TGFb, its association with Smad4, and its subsequent translocation to the nucleus and thereby dampen TGFb-mediated signaling (Razani et al., 2001). Lefty polypeptides are novel members of the TGFb superfamily and have been shown to be involved in the formation of embryonic lateral patterning (Meno et al., 1996, 1997; Kothapalli et al., 1997). Recent studies have shown that Lefty inhibits phosphorylation of Smad2 following activation of the TGFb receptor complex (Ulloa and Tabibzadeh, 2001). Lefty was also shown to inhibit downstream events including Smad4 heterodimerization and nuclear translocation, and activation of TGFb-induced reporter genes. The molecular mechanism of this inhibition by Lefty remains to be elucidated (Ulloa and Tabibzadeh, 2001; Ulloa et al., 2001).

INTRACELLULAR EFFECTORS Smad proteins The downstream events following receptor activation have begun to become elucidated with the discovery of the Smad proteins and the demonstration that they serve as direct substrates for the type I receptor kinase and mediate gene responses to the TGFb family. The first member of this Smad family identified, through a genetic screen, was the Drosophila Mad gene (mothers against decapentaplegic; Raftery et al., 1995). Its identification led to the discovery of three related genes in C. elegans termed Sma-2, -3 and -4, (Savage et al., 1996) and the subsequent identification of many vertebrate homologues, designated Smads from the contraction of the names Sma and Mad (Derynck and Feng, 1997; Massagué, 1998).

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The Smad family of proteins can be subdivided into three categories based on structural and functional characteristics (Plate 49.4A) (Massagué, 1998; ten Dijke et al., 2000; Wrana, 2000). The first category consists of the receptor-regulated Smads (R-Smads) which are direct substrates of type I receptor kinases. Smads 1, 5 and 8 are activated by BMP receptors type IA (BMPRIA/Alk3) or type IB (BMPR-IB/Alk6) (Hoodless et al., 1996; Liu et al., 1996), whereas Smads 2 and 3 are substrates and mediators of TGFb (TGFbR-II) and activin (ActR-IB) receptors (Chen Y., et al., 1996; Lagna et al., 1996; Zhang Y., et al., 1996). Mad, Sma-2, -3 and Daf-8 are also in this subfamily of Smad proteins and act as mediators of the Dpp and BMP-like receptors in Drosophila and C. elegans (Plate 49.4A; Savage et al., 1996; Newfeld et al., 1996). The second subfamily contains the common mediator Smads (Co-Smads) that participate in signaling by associating with the R-Smads (Zhang Y. et al., 1996; Lagna et al., 1996; Kretzschmar et al., 1997b). Two distinct Co-Smads have been identified in Xenopus (Smad4a and Smad4b/10) (Masuyama et al., 1999; Howell et al., 1999). In vertebrates, Smad4 was originally identified as the product of the tumor suppressor gene DPC-4 (deleted in pancreatic carcinoma locus 4) (Hahn et al., 1996). The functional interaction of Smad4 with the R-Smads is demonstrated in experiments in which a C-terminal dominant-negative Smad4 blocks not only TGFb responses but also BMP responses and blocks Smad1, 2 and 3 from entering the nucleus (Zhang Y., et al., 1996; Lagna et al., 1996). Medea and Sma-4 are Drosophila and C. elegans homologs of Smad4, respectively (Plate 49.4A), and are postulated to associate with R-Smads in these organisms and mediate nuclear translocation and signaling (Das et al., 1998; Savage et al., 1996). The third subfamily are the antagonistic Smads (anti- or inhibitory I-Smads) that inhibit the signaling activity of the R-Smad/Co-Smad complexes (Hill, 1999). Smad7 inhibits R-Smad phosphorylation by occupying type I receptors for TGFb, activin and BMP (Nakao et al., 1997; Hayashi et al., 1997). Recently, it has been shown to serve as a binding partner of the E3 ubiquitin–ligase complex that targets TGFb receptors for degradation (Kavsak et al., 2000; Ebisawa et al., 2001). Smad6 preferentially inhibits BMP signaling by competing with Smad4 for binding to Smad1 thus forming an inactive Smad1/ Smad6 complex (Imamura et al., 1997; Hata et al.,

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1998). Dad protein is a Smad6, 7 Drosophila homolog that inhibits Mad signaling (Tsuneizumi et al., 1997; Plate 49.4A). Structurally, the R-Smads and Co-Smads share two domains of high-sequence similarity at their respective N and C-termini termed the MH1 and MH2 domain, respectively (Heldin et al., 1997; Massagué, 1998) (Plate 49.4B). These Mad-homology domains are separated by a proline-rich linker region of variable length and function. The MH1 domain has DNA binding activity (except for the major splice form of Smad2 which contains an insert that prevents DNA binding) and associates with other transcription factors (Kim et al., 1997; Liu et al., 1997). The Smad3 and Smad4 MH1 domains have been shown to bind to the transcription factor ATF-2 (Sano et al., 1999), and the MH1 domain of Smad3 is required for binding c-Jun (Zhang et al., 1998) and TFE-3 (Hua et al., 1999). It is approximately 130 amino acids and is highly conserved in the R-Smads and Smad4 but not in the I-Smads. The MH1 domain interacts with the MH2 domain and this interaction is postulated to inhibit the transcription and biologic activities of MH2 (Liu et al., 1996; Baker and Harland, 1996). Receptor-mediated phosphorylation of the C-terminal sequence SSXS in the MH2 domain relieves inhibitory interaction of the two domains and is postulated to allow the TGFb-induced association of Smad2 and Smad4 MH2 domains and ultimately R-Smad activation and nuclear translocation (Hata et al., 1997). Thus MH1 and MH2 binding may regulate Smad basal state transcription activity. The MH2 domain of certain Smads is responsible for receptor interaction (Macias-Silva et al., 1996), transcription activation (Liu et al., 1997; Chen Y.G., et al., 1997) and is also important for homo- and hetero-oligomerization (Hata et al., 1997; Wu et al., 1997). It is also the domain that interacts with the Smad anchor for receptor activation (SARA) which is postulated to recruit and scaffold Smad2 to the receptor kinase (Tsukazaki et al., 1998; Wu et al., 2000) (discussed below). The MH2 domain is approximately 200 amino acids long and contains receptor phosphorylation sites (SSXS) at its C-terminus (Plate 49.4B). Structural investigation has revealed that the L3 loop and the a helix-1 in this domain mediate interaction with the L45 loop of the cytoplasmic Ser/Thr kinase domain of TbR-I (Chen Y.G. et al., 1998; Chen

and Massagué, 1999; Lo and Massagué, 1999). This interaction was also shown to specify Smad–receptor interaction in that exchanging several residues between two Smad subtypes is sufficient to switch signaling specificity between the TGFb and BMP pathways (Chen and Massagué, 1999). Other regions within this MH2 domain have been demonstrated to be required for recognition of other transcription factor interactions (Liu et al., 1997; Chen X. et al., 1997). The linker region between the MH1 and MH2 domains is less well conserved than the MH domains. Consensus phosphorylation sites for prolinedirected kinases are found in this region as well as the MAPK phosphorylation sites which are postulated to be inhibitory for Smad nuclear translocation (Kretzschmar et al., 1997a). The linker region of both the R-Smads and I-Smads also contain the conserved proline–tyrosine (PY) motif that bind with WW domains (Plate 49.4B). This interaction has been shown to mediate binding to the E3 ubiquitin–ligase Smurf1 that induces the ubiquitination and proteosome-mediated degradation of the BMPregulated Smads, Smad1 and 5, and thus antagonizes BMP signaling in Xenopus (Zhu et al., 1999; Lo and Massagué, 1999). The molecular pathway of TGFb signaling from the activated receptor complex at the cell membrane to the regulation of transcription in the nucleus is illustrated in Plate 49.3. TGFb-induced activation of the heteromeric receptor complex results in transphosphorylation, within the GS domain, and activation of TbR-I by TbR-II (Wrana et al., 1994b; Souchelnytskyi et al., 1996). This leads to transient recruitment of the R-Smads to the receptor complex and phosphorylation of Smads in the C-terminal SSXS motifs by TbR-I (Kretzschmar et al., 1997b; Macias-Silva et al., 1996). The recruitment of Smad 2 and 3 to the TGFb receptor complex is governed by the membrane-associated protein SARA (Smad anchor for receptor activation) (Tsukazaki et al., 1998; Wu et al., 2000). This occurs through a direct interaction between the Smadbinding domain (SBD) of SARA and the MH2 domain of non-phosphorylated Smad 2 and 3 (Wu et al., 2000). SARA contains a FYVE domain which interacts specifically with the phospholipid phosphatidylinositol 3-phosphate and localizes SARA to subcellular regions where the TGFb receptors are also localized. SARA can interact with TGFb receptors and functions

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to recruit and scaffold Smad proteins to their receptor kinase. SARA thus regulates the subcellular localization of the Smads and SARA mutants that cause mislocalization of the Smads also inhibiting TGFb signaling (Tsukazaki et al., 1998). Upon phosphorylation of the R-Smads by activated TbR-I, R-Smads and SARA dissociate from the receptor complex (Plate 49.3) (Tsukazaki et al., 1998). The phosphorylation of the R-Smads relieves the autoinhibitory MH1–MH2 association and allows the R-Smads to associate with Smad4, through their MH2 domains, in the cytosol (Hata et al., 1997). The precise stoichiometry of the Smad complexes remains to be elucidated. Earlier studies showed that R-Smads exist primarily as monomers in the non-activated state and upon receptor-mediated phosphorylation can form homo- and hetero-oligomers with each other, as well as hetero-oligomers with Smad4 (Kawabata et al., 1998). The prevailing view was that in vivo the Smad2 homomer appears to be a trimer, and the heteromeric complexes composed of Smad2 and Smad4 are heterotrimers composed of two and one, or one and two molecules each of Smad2 and Smad4, respectively (Kawabata et al., 1998). More recent studies, however, suggest that in the basal state Smad 2, 3, and 4 form separate and strikingly different complexes, either homomeric or multiple oligomeric states (Jayaraman and Massagué, 2000). Upon activation by receptor phosphorylation, Smads form a multitude of complexes ranging in size from Smad2/Smad4 heterodimers to assembly of large complexes of greater than 650 kDa (Jayaraman and Massagué, 2000). Following oligomerization, the R-Smad/Smad4 complex translocates into the nucleus where it associates with other DNA binding proteins to modulate transcription of specific target genes (Plate 49.3). It has been shown that the nuclear import of Smad2 is masked by SARA and that TGFb-dependent phosphorylation and subsequent release from SARA allows the nuclear localization sequence (NLS) of Smad2 to be recognized for import into the nucleus (Xu et al., 2000). Furthermore, it has been shown that receptor activation induces nuclear import of Smad3 in an importin b- and Ran-dependent manner (Xiao et al., 2000; Kurisaki et al., 2001). In the nucleus, Smads have been shown to act as transcription factors through direct binding to DNA and to regulate transcriptional responses through

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cooperativity with other transcription factors. This was first demonstrated with the Drosophila Mad protein which was shown to bind directly to a GC-rich sequence (Mad-binding element) in the promoter of the Dpp target gene vestigial (Kim et al., 1997). Smad3 and Smad4 were subsequently shown to bind DNA via their MH1 domain (Kim et al., 1997; Shi et al., 1998). While full-length Smad4 can bind DNA, it was shown that for Smad3 to bind DNA the auto-inhibitory MH1–MH2 interaction had to be relieved either through TbR-I phosphorylation or deletional removal of the MH2 domain of the Smad3 protein (Kawabata et al., 1998; Zawel et al., 1998; Dennler et al., 1998). DNA binding is mediated by an 11-amino-acid b-hairpin structure which protrudes from the MH1 domain and binds to the major groove (Shi et al., 1999). Smad2 is unable to bind DNA due to a 30-amino-acid sequence insert just N-terminal to the b-hairpin loop which may interfere with DNA recognition (Yagi et al., 1999). An alternatively spliced variant of Smad2, found in various cell types, that lacks exon 3 which encodes for the 30-amino-acid insert or artificial removal of the insert from wild-type Smad2, allows for direct interaction with DNA (Yagi et al., 1999). The amino-acid sequences of the b-hairpin loop are identical in all the R-Smads, and that of Smad4 is highly conserved, yet Smad1 and 5 (BMP/R-Smads) preferentially bind GC-rich sequences while the MH-1 domain of Smad3 preferentially binds CAGA sequences. Recently, it has been shown that while the b-hairpin loop is required for DNA binding, specificity in DNA binding and thus elicitation of specific biologic activity between the TGFb family members is provided by the a helix 2 (H2) sequence, N-terminal to the b-hairpin loop in the MH1 domain (Kusanagi et al., 2001). Mutational analysis and crystallographic studies with Smad3 and Smad4 have identified numerous DNA binding motifs in various TGFb and activin target genes. The consensus and optimal Smad-binding element (SBE) was determined to be AGAC, which has been estimated to be present at least once in the regulatory region of any average size gene (Shi et al., 1998; Zawel et al., 1998). Thus, the binding of Smads to DNA is of extremely low specificity and furthermore, the affinity of MH1 domain binding for SBE is very weak (~107 M; Shi et al., 1998). These two facts suggest that DNA binding alone is insufficient for Smaddependent transcriptional activation and that other

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cooperating factors are necessary for specific, highaffinity binding of a Smad complex to its target gene.

Smad-interacting transcription factors The ability of the Smads to activate transcriptional responses results from a functional interaction with other Smad-interacting factors. These Smad/DNAbinding partner interactions, more than the Smad complex alone, provide specificity to this signaling pathway in several different ways. These co-factors, in cooperation with the Smads, specify the promoter sequence to which this Smad/DNA-binding partner complex will bind. Also, since some of these co-factors are expressed in a tissue/cell-specific manner they specify and govern the context-dependent Smad gene responses. Finally, the multiple possible combinatorial interactions between the various R-Smads and the many DNA-binding co-factors provides pathway specificity for the various TGFb family members (Massagué and Wotton, 2000). The first identified interaction of Smad with a DNAbinding partner was demonstrated with the FAST (forkhead activin signal transducer) family of wingedhelix transcription factors which mediate activininduced regulation of the Mix.2 gene in Xenopus and the goosecoid gene in mouse (Chen X., et al., 1997; Liu et al., 1997; Labbé et al., 1998; Yeo et al., 1999). FAST factors bind constitutively to specific elements in the promoter of these genes, adjacent to SBE sites, but cannot activate transcription until a transcriptionally active complex, induced by activin or TGFb, is formed containing FAST, Smad2 or Smad3, and Smad4. In this scenario, Smad4 promotes the interaction of Smad2 and FAST-1 and provides a stabilizing function to the complex (Chen X., et al., 1997; Liu et al., 1997; Labbé et al., 1998; Yeo et al., 1999). Smads have subsequently been shown to physically or functionally interact directly with a rapidly expanding number of DNAbinding partners. Listed below are but a few examples; others are the subject of excellent reviews (ten Dijke et al., 2000; Massagué and Wotton, 2000). Unlike the FAST factors, most of these DNA binding partners possess transactivating activity independent of the Smads. Smad3 has been shown to interact with c-Jun and c-Fos (Zhang et al., 1998; Liberati et al., 1999), forming multimeric transcriptional complexes, which transactivate at TGFb-responsive AP-1 sites (Zhang

et al., 1998; Wong et al., 1999). The MH1 domain of Smad3 and Smad4 has been shown to bind ATF-2 and cooperatively activate CRE-containing elements (Sano et al., 1999; Hanafusa et al., 1999). Smad3 and Smad4 have been shown to cooperate with the basic helix–loop–helix protein TFE3 to induce TGFbinduced transcription of plasminogen activator inhibitor-1 (PAI-1) (Hua et al., 1998). Smads have also been shown to associate, either physically or functionally through other co-factors, with SP-1 in the p15 and p21 promoters (Datto et al., 1995) AML in the Ia promoter in the IgA gene (Hanai et al., 1999; Pardali et al., 2000), BF-1 in the PAI-1 and p15 promoter (Dou et al., 2000; Rodriguez et al., 2001), vitamin D receptor (VDR) in vitamin D response element-containing promoters (Yanagisawa et al., 1999), and STAT3 (Nakashima et al., 1999), through CBP/p300, in synergizing BMP- and leukemia inhibitory factor (LIF)induced glial fibrillary acidic protein (GFAP), and cooperate with NFjB in transcriptional regulation of the junB promoter (Lopéz-Rovira et al., 2000). Smad proteins have also been shown to activate transcription by inhibiting the actions of transcriptional repressors. Smad1, in response to BMP stimulation, has been shown to bind to and dislodge the transcriptional repressor Hoxc-8 from the osteopontin promoter and induce osteopontin transcription (Shi et al., 1999). More recently, TGFb treatment has been shown to prevent recruitment of the Myc co-repressor to the p15INK4B promoter through the Miz-1 protein (Myc-interacting zinc-finger protein-1). This relieves Myc-induced repression and enables transcriptional activation by a TGFb-induced Smad complex (Seoane et al., 2001). The transcriptional activity of Smads also depends on interactions with coactivators and corepressors within the complex. This transcriptional activity of the Smads was revealed through the use of GAL4-RSmad fusions and was shown to be mediated through the MH2 domain and required the presence of the Co-Smad, Smad4. More recent studies have demonstrated that the transcriptional activity of the R-Smads occurs through their ability to recruit the transcriptional co-activators CBP and p300 (Feng et al., 1998; Janknecht et al., 1998; Pouponnot et al., 1998; Topper et al., 1998). This interaction is mediated directly through the Smad MH2 domain and is dependent on type I receptor kinase activity. The interaction can be

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inhibited by the viral oncoprotein E1A, which binds directly to the Smads and competes for binding with CBP/p300. E1A thus blocks CBP/p300-Smad interactions and also inhibits TGFb signaling (Topper et al., 1998). CBP and p300 have intrinsic histone acetyl transferase (HAT) activity which through histone acetylation decreases chromatin condensation and facilitates transcription presumably by increasing Smad accessibility to the basal transcription complex. CBP and p300 also serve as co-activators for many other sequence-specific transcription factors by bringing these in proximity to the general transcription machinery. Thus, the observed cooperative interactions between Smads and other transcription factors mentioned above (i.e. AP1, ATF-2, VDR and STATS) may be mediated through CBP and p300 (Massagué and Wotton, 2000; Wrana, 2000). Smads can also recruit co-repressors to a transcriptional complex. TGIF, a co-repressor, binds directly to Smad2 and Smad3 via the MH2 domain in a TGFbinducible fashion, and represses Smad-induced transcription by recruiting histone deacetylases (HDACs) (Wotton et al., 1999). The binding of TGIF to Smads is competed by CBP/p300 binding, suggesting that transcriptional activity of the Smad complex is tightly regulated by the respective cellular levels of the TGIF repressor and CBP/p300 activator (Wotton et al., 1999). Ski and SnoN have been identified as Smad2 and Smad3 co-repressors (Luo et al., 1999; Sun et al., 1999a, 1999b; Akiyoshi et al., 1999; Stroschein et al., 1999). These proteins bind Smads and similarly to TGIF, recruit HDAC to a Smad transcriptional complex via the adaptor N-CoR. Similarly to TGIF, these proteins compete with CBP/p300 for Smad binding, but unlike TGIF whose binding to Smads is induced by TGFb, Ski and SnoN bind Smads under basal conditions and TGFb induces their dissociation in a timedependent manner (Sun et al., 1999b; Stroschein et al., 1999). TGFb induces both the expression of Ski/SnoN and their dissociation from the Smad complex with accompanying proteosomal degradation. Thus, Ski/SnoN are postulated to be part of a TGFbinduced negative feedback loop regulating Smad transcriptional activity (Stroschein et al., 1999). Other inhibitors of Smad transcriptional activation include SIP1 (Smad interacting protein 1) (Verschueren et al., 1999) and Evi-1 (Kurokawa et al., 1998). Both are nuclear, zinc-finger proteins that interact with Smads

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and attenuate TGFb-responsive reporters and growth inhibition. A novel Smad nuclear interacting protein, SNIP1, has recently been described as a repressor of TGFb signaling (Kim et al., 2000). The C-terminus of SNIP1 interacts with Smad1 and Smad2 while its N-terminus has binding sites for both Smad4 and CBP/p300. Similarly to the co-repressor TGIF, SNIP1 binding to Smad4 is mutually exclusive of CBP/p300 binding. Therefore, a similar competitive mechanism has been proposed whereby the expression level of SNIP1 in a cell determines whether Smad4 interacts with the transcriptional co-activator CBP/p300 or the transcriptional co-repressor SNIP1 (Kim et al., 2000).

Negative regulators of Smad signaling Ligand-induced signaling is a highly coordinated and specific process requiring not only a temporal and spatial activation but also an inactivation or termination of signaling. Negative regulatory mechanisms exist throughout the TGFb signaling pathway; not only at the ligand and receptor level but also at the Smad level to switch off activated Smad function. One such mechanism involves the targeted degradation of activated R-Smads through the ubiquitin–proteosome pathway (Plate 49.3). Ubiquitination of proteins is mediated through E3 ligases, which physically interact and recruit substrate protein to the ubiquitinconjugating machinery (Herschko and Ciechanover, 1998; Ciechanover et al., 2000). Smurf1, a member of the HECT family of E3 ubiquitin–ligases, was originally identified as interacting with Smad1 and inducing its ubiquitination and degradation and inhibiting the BMP signal-mediating pattern formation in Xenopus (Zhu et al., 1999). The interaction was shown to be mediated through the PY motif in the linker region of Smad1 and the WW domain of Smurf1 (Zhu et al., 1999). A separate E3 ligase, Smurf2, has been identified and shown to be specific for TGFb activated Smads (Lo and Massagué, 1999; Lin et al., 2000; Zhang et al., 2001). TGFb, through Smurf2, was shown to target Smad2 for ubiquitination and degradation. Receptor-mediated phosphorylation of Smad2 is required for its ubiquitination and degradation to the extent that it mediates Smad2 nuclear accumulation. Smad mutant constructs that accumulate constitutively in the nucleus, in the absence of

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receptor-mediated phosphorylation, are constitutively ubiquitinated (Lo and Massagué, 1999). More recent studies have shown that both Smurf1 and Smurf2 can mediate degradation of TGFb receptors through the I-Smad, Smad7 (Kavsak et al., 2000; Ebisawa et al., 2001). In this scenario, Smurf2 associates constitutively with Smad7 and this binding induces export of the Smad7/Smurf2 complex from the nucleus and recruitment to the TGFb receptor complex where it causes degradation of receptors and Smad7 by the ubiquitin and lysosomal pathways (Kavsak et al., 2000). Smurf1 has also been shown to use Smad7 as an adaptor in its targeting and degradation of TGFb receptors (Ebisawa et al., 2001). Thus, this E3 family of ligases can mediate negative regulation of TGFb signaling by two distinct mechanisms (1) by interaction and degradation of R-Smads; and (2) through interactions with Smad7 they target TGFb receptor complexes for degradation. Another negative regulatory mechanism exerted at the Smad level is mediated through the inhibitory Smads, Smad6 and Smad7. Smad6 is a specific inhibitor of BMP signaling in Xenopus embryos and mammalian cells (Ishisaki et al., 1999; Hata et al., 1998; Imamura et al., 1997), while Smad7 can inhibit TGFb signaling (Souchelnytski et al., 1998; Ishisaki et al., 1999). Smad7 has been suggested to inhibit TGFb signaling by inhibiting the phosphorylation of R-Smads by TbR-I (Imamura et al., 1997; Nakao et al., 1997; Hayashi et al., 1997), and more recently, as described above, by binding and targeting Smurfs to TGFb receptor complexes for proteosomal-mediated degradation (Kavsak et al., 2000; Ebisawa et al., 2001). Smad7 expression is also up-regulated by TGFb stimulation, suggesting that it is involved in negative feedback regulation of TGFb signaling (Nakao et al., 1997; Ishisaki et al., 1998). Induction of Smad7 has also been shown to be mediated by interferon c (Ulloa et al., 1999), TNFa through NFjB (Bitzer et al., 2000) and by CD40 in B lymphocytes (Patil et al., 2000), suggesting that Smad7 may be involved in other signaling pathways and mediate cross-talk with TGFb signaling.

Non-transcription factor Smadinteracting proteins Smad proteins interact with not only nuclear-binding partners but have recently been shown to bind to

cytoplasmic and membrane-associated proteins which play a role in Smad function. As described above, SARA is a cytoplasmic/membrane protein which has been implicated in Smad subcellular localization and facilitates signaling by bringing the Smad substrate to the activated receptor complex (Tsukazaki et al., 1998; Wu et al., 2000). Another FYVE domain protein termed Hgs (Hrs) has also been shown to be involved in intracellular localization and functional activity of Smads (Miura et al., 2000). Under non-stimulated conditions, Hgs binds Smad2, in the cytosol, through its C-terminal domain. Upon ligand activation Hgs cooperates with SARA to recruit and present Smad2 to the receptor and stimulate activin-mediated signaling (Miura et al., 2000). Microtubule (MT) binding to Smads has also been implicated in TGFb signaling (Dong et al., 2000). Under non-stimulated conditions, Smads bind to b-tubulin, a major component of MTs, and activation by TGFb triggers dissociation from b-tubulin and the MTs. Dissociation promotes Smad2 and Smad3 phosphorylation, and nuclear translocation with ensuing activation of transcription. Destabilization of the MT network with nocodazole or colchicine disrupts the Smad/tubulin complex and increases TGFb-induced Smad2 phosphorylation on the C-terminal SSXS motif and activated Smad-dependent transcription (Dong et al., 2000). Recently, another cytoskeletal component, filamin, has been shown to bind Smads and this association has been implicated in TGFb signaling (Sasaki and Sugamura, 2001). Filamin is an actinbinding protein that acts as a scaffold for intracellular proteins involved in signal transduction. Its association with Smad5 was revealed through a two-hybrid screen and subsequently confirmed, as well as shown to bind to Smad2, when overexpressed in mammalian cells. It was further demonstrated that in filamindeficient cells, Smad2 phosphorylation, nuclear translocation and Smad2-dependent transcriptional responses were impaired but could be restored following transient transfection of full-length filamin (Sasaki A. et al., 2001). It is not clear how MTs and filamin regulate Smad function. It is possible that they serve as scaffolding proteins, akin to SARA, in controlling the localization of Smad near cell surface receptors. Alternatively, these cytoskeletal elements could be involved in conformational presentation of the Smad to the receptor kinase.

THE CYTOKINES AND CHEMOKINES

OTHER RECEPTOR - ASSOCIATED PROTEINS

Other proteins have recently been identified and postulated to serve as Smad adaptors or scaffolds. Disabled-2 is a previously identified signaling molecule of unknown function which contains an N-terminal phosphotyrosine binding domain (PTB) and a C-terminal proline-rich domain (PRD), both characteristic of signaling adaptors (Hocevar et al., 2001). Dab2 was shown to restore TGFb signaling to a TGFb-signaling defective cell line. Dab2 was shown to bind to Smad2 and Smad3, and restored Smad2 phosphorylation, nuclear translocation and Smadmediated transcriptional activation. The molecular mechanism by which Dab2 mediates TGFb signaling remains unknown. Perhaps, similarly to SARA, MTs and filamin, Dab2 is another Smad-interacting protein that regulates Smad activity through subcellular localization (Hocevar et al., 2001). This is supported by the recent demonstration that Dab2 binds to the clathrin adaptor molecule AP-2 and co-localizes in clathrin-coated, early endocytic vesicles (Morris and Cooper 2001). Interestingly, these early endocytic vesicles are the same intracellular compartment where the TGFb receptor resides and where signaling is postulated to initiate (Doré et al., 1998). Another recently identified Smad3-interacting protein is Axin, a negative regulator of the Wnt signaling pathway (Furuhashi et al., 2001). Axin binds to Smad3 through its C-terminal domain and co-localizes with Smad3 in the cytosol in the absence of ligand stimulation. Upon receptor activation, Smad3 is highly phosphorylated in the presence of Axin, and dissociates from TbR-I and Axin. Axin was further shown to promote the transcriptional transactivation of Smad-dependent reporter constructs, and an axin mutant construct abrogated this response (Furuhashi et al., 2001).

OTHER RECEPTORASSOCIATED PROTEINS In addition to the Smads, other proteins that interact with TGFb receptors have been identified. These include FKBP-12 (Wang et al., 1994; Yao et al., 2000), TRIP-1 (Chen R.H., et al., 1995; Choy and Derynck, 1998), the a chain of farnesyl transferase (Kawabata et al., 1995), clusterin (Reddy et al., 1996), STRAP (Datta et al., 1998; Datta and Moses, 2000), B-a subunit of protein phosphatase 2A (PP2A; Griswold-

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Prenner et al., 1998), SARA (Tsukazaki et al., 1998; Wu et al., 2000), TRAP-1 (Charng et al., 1998; Wurther et al., 2001), sorting nexin 6 (Parks et al., 2001) and XIAP (Yamaguchi et al., 1999; Reffey et al., 2001). FKBP-12 and SARA have been discussed earlier in this chapter. Functional roles for the others in TGFb signaling need to be more clearly defined; however, recent data with several of these further supports their roles in the TGFb pathway. STRAP (serine threonine kinase receptor-associated protein) is a WD40 repeat protein which was originally shown to bind to both TbR-I and TbR-II and inhibit TGFb signaling (Datta et al., 1998). Recently, STRAP has been shown to stabilize the association of Smad7 with the activated receptor and synergize with Smad7 to prevent Smad2 and Smad3 access to the receptor (Datta and Moses, 2000). TRAP-1 (TbR-I associated protein-1) was originally shown to recognize activated TbR-I and that a C-terminal portion when overexpressed in cells could inhibit TGFb-mediated effects (Charng et al., 1998). It has recently been shown that TRAP-1 acts as a chaperone for Smad4 helping to recruit Smad4 to the vicinity of the receptor complex and facilitating its transfer to the activated R-Smads (Wurther et al., 2001). Sorting nexin 6 (SNX6), belongs to a family of proteins, the sortin nexins (SNX), which have been implicated in receptor trafficking, specifically members of the receptor tyrosine kinases, including receptors for EGF, PDGF and insulin. SNX6 has recently been shown to interact with members of the type I and type II TGFb receptors and is postulated to play a role in selection and transport of these molecules within the intracellular vesicular transport pathways (Parks et al., 2001). XIAP (X chromosome-linked inhibitor of apoptosis protein) was recently isolated in a two-hybrid screen as binding to the TAB1 protein (Yamaguchi et al., 1999). TAB1 is an upstream activator of TAK1 (TGFbactivated kinase), a MAP kinase kinase kinase (MEKKK) homolog (Yamaguchi et al., 1995), which has been implicated in the TGFb pathway and also participates in BMP signaling during mesoderm induction and patterning in early Xenopus embryos (Shibuya et al., 1998). In this study it was also demonstrated that XIAP associated not only with TAB1 but also with BMP receptors in mammalian cells. XIAP mRNA injection into dorsal blastomeres was shown to enhance the ventralization of embryos and a

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truncated form of XIAP, lacking the TAB1-binding domain, blocked the expression of ventral mesoderm induced by a constitutively active BMP type I receptor (Yamaguchi et al., 1999).

MAPK SIGNALING PATHWAY It has long been known that Ras acts as a modulator of TGFb signaling. Early studies focusing primarily on the antimitogenic effects of TGFb demonstrated that Ras transformation of lung, liver, intestinal and mammary epithelial cells results in a loss of TGFbmediated growth arrest (Schwarz et al., 1988; Houck et al., 1989;Valverius et al., 1989; Longstreet et al., 1992; Filmus et al., 1992; Filmus and Kerbel, 1993). A more direct antagonistic effect of Ras on TGFb-mediated growth arrest was demonstrated in studies where microinjection of oncogenic forms of H-Ras into TGFb-arrested lung epithelial cells was shown to overcome TGFb-growth inhibition and drive cell cycle progression into S phase (Howe et al., 1993). While these studies suggested an antagonism between the TGFb and Ras pathways, other studies demonstrated cell-specific synergistic effects of these two pathways in mediating growth arrest (Mulder and Morris, 1992; Yan et al., 1994; Hartsough et al., 1996). Similarly, both antagonistic and synergistic effects of these two pathways on other TGFb-mediated developmental and biologic processes have been observed. TGFb can potently overcome the mitogenic effects of EGF, a Ras-activating factor, in epithelial cells (Massagué, 1990; Howe et al., 1991; Alexandrow and Moses 1995a, 1995b). EGF is also antagonistic to the BMP pathway mediating osteogenic differentiation (Bernier and Goltzman, 1992). BMPs and FGF, another Ras-activating factor, mediate antagonistic effects during limb bud development (Niswander and Martin, 1993), during interdigital membrane apoptosis in digit formation (Ganan et al., 1996), and in tooth development (Neubüser et al., 1997). Synergistic effects between the Ras and TGFb or activin pathway have been observed during anchorage-independent growth of normal rat kidney (NRK) fibroblasts (Roberts et al., 1983) and during embryonic development (Bhushan et al., 1994; LaBonne and Whitman, 1994; Whitman, 1998). TGFb and FGF act synergistically during heart formation (Lough et al., 1996), myo-

genesis (Stern et al., 1997), and chondriogenesis (Frenz et al., 1994). TGFb also collaborates with oncogenic Ras in mediating the metastatic and invasive phenotype in epithelial cells (Oft et al., 1996, 1998). Further synergy between TGFb and another Rasactivator, hepatocyte growth factor (HGF), has been demonstrated in up-regulation of plasminogen activator inhibitor-1 (PAI-1) (Keski-Oja et al., 1988) and during scattering of epithelial cells by HGF or EGF (Stolz and Michalopoulos, 1997). Thus, depending on the cellular and biological context, the interaction between the Ras and TGFb signaling pathways can be antagonistic or cooperative. The molecular mechanisms that underlie Ras and TGFb pathway interactions have begun to emerge and reveal that cross-talk between these two pathways occurs at several levels. First, it was shown early on that H-Ras overexpression in intestinal epithelial cells leads to an altered level of TGFb receptors with a decrease in the expression of TbR-II (Filmus et al., 1992; Zhao and Buick, 1995). The interplay between Ras and TGFb signaling was also demonstrated at the cyclin-dependent kinase level (CDKs), where it was shown that Ras signaling stimulates the activation of the cdks whereas TGFb down-regulates cdk activity through the induction of cdk inhibitors (Howe et al., 1991; Longstreet et al., 1992; Hartsough et al., 1996; Koff et al., 1993; Ewen et al., 1993; Hannon and Beach, 1994; Reynisdóttir et al., 1995). Another level of interaction between these two pathways demonstrated that TGFb directly modified the GTP-bound state of Ras (Mulder and Morris, 1992; Howe et al., 1993) and one of its downstream targets, ERK-1, a member of the MAPK signaling pathway (Mulder and Morris, 1992; Howe et al., 1993; Hartsough and Mulder, 1995). These initial studies on ERK-1 were underscored by the observation that many TGFb target genes were regulated by transcription factors which served as nuclear substrates for the MAPK pathway. Subsequent identification of the Smad proteins and the demonstration that they interact, directly and/or functionally, with many of these same transcription factors to regulate TGFb gene expression, prompted further investigation into the role of the MAPK pathway in TGFb signaling. Emerging evidence indicates that this pathway plays a prominent role in TGFb signaling. It can integrate with the Smad pathway to mediate TGFb cellular responses or can elicit them

THE CYTOKINES AND CHEMOKINES

MAPK SIGNALING PATHWAY

directly, in a Smad-independent manner. The interplay or cross-talk between the Smad and MAPK pathways is multifaceted as well, occurring both in the cytosol and nucleus, and resulting in either synergistic or antagonistic effects on TGFb signaling, thus reflecting the modulatory effects of Ras on TGFb cellular responses.

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BMP type I receptor to the TAB1/TAK1 pathway (Yamaguchi et al., 1999) and the TGFb type I receptor to JNK and NFjB activation (Reffey et al., 2001). More recently, Hgs/Hrs, Smad and SARA-interacting FYVEfinger protein (described above), have been shown to associate with TAK1 and p21-activated kinase 1 (PAK1) and regulate transactivation of the SRE and CRE through JNK (Sasaki and Sugamura, 2001).

TGFb regulation of MAPK pathway TGFb and many of its family members have now been shown to activate the three terminal kinases of the MAPK signaling pathway in a cell type-specific manner. ERK activation by TGFb has been shown in colon carcinoma, intestinal, mink lung epithelial and fibroblasts (Yan et al., 1994; Hartsough and Mulder, 1995; Mucsi et al., 1996; Frey and Mulder, 1997). The stressactivated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) (Atfi et al., 1997; Frey and Mulder, 1997; Hocevar et al., 1999; Engel et al., 1999) and the p38 mitogen-activated protein kinase (p38) (Hannigan et al., 1998; Adachi-Yamada et al., 1999; Sano et al., 1999; Iwasaki et al., 1999; Hanafusa et al., 1999) have also been shown to be regulated by TGFb in a variety of cell types. In addition, upstream activators of these terminal kinases have also been shown to be activated by TGFb, including MKK4 (Hocevar et al., 1999), the hematopoietic progenitor kinase-1 (HPK1) (Wang et al., 1997; Zhou et al., 1999), and the MAPK kinase kinase (MAPKKK) homolog, the TGFb-activated kinase, TAK1 (Yamaguchi et al., 1995). The TGFb responses regulated by this kinase cascade are varied ranging from reporter construct transactivation to regulation of cellular proliferation and apoptosis. The kinetics of the responses also vary, ranging from rapid activation observed within minutes to more sustained activation on the order of 12 or more hours. The molecular link between the TGFb receptors and activation of these pathways is currently unknown. The most receptor proximal activators of this pathway to have been implicated in TGFb signaling are the non-Ras, GTPases RhoA, RhoB and Rac (Musci et al., 1996; Atfi et al., 1997; Engel et al., 1998, 1999; Choi et al., 1999). It has also been suggested that HPK1 in association with TAK binding protein 1 (TAB1) couples the TGFb receptor to the TAK1 and JNK1 pathways (Zhou et al., 1999). The X-linked inhibitor of apoptosis (XIAP) has been shown to couple the

Nuclear cross-talk between Smads and MAPK Targets for the activated MAPKs are commonly transcription factors such as CREB, Elk-1, ATF-2 and c-Jun (Karin, 1995; Treisman, 1996). Homo or heterooligomers of many of these transcription factors have been shown to bind and regulate transcriptional responses from specific promoter elements such as CRE, TRE and AP-1 sites (Karin, 1995; Treisman, 1996). The early observation that many TGFb-regulated gene promoters contain such AP-1, TRE and CRE sites suggested that the MAPK pathway may mediate TGFb responses. Also, the fact that in many TGFb-regulated genes, Smad-binding elements (SBEs) are often juxtaposed to AP-1, CRE and TRE response elements suggested that MAPK and Smad signaling might converge at these promoter sites to regulate TGFb-mediated gene expression. Both of these notions have now been demonstrated. TGFb has been shown to transactivate through AP-1 and CRE sites through the phosphorylation of c-Jun and ATF-2 by JNK and p38, respectively (Hocevar et al., 1999; Sano et al., 1999; Hanafusa et al., 1999). The Smad proteins have also been shown to interact physically with components of these complexes and regulate transactivation of TGFb reporter constructs. Smads have been shown to directly bind to c-Jun and c-Fos and regulate transactivation of AP-1 sites (Zhang et al., 1998; Liberati et al., 1999; Wong et al., 1999; Verrecchia et al., 2001a). Direct binding of Smads with ATF-2 has also been demonstrated to regulate ATF-2 transactivation (Sano et al., 1999; Hanafusa et al., 1999). Recently, two studies have demonstrated that this binding and cooperation between MAPK components and the Smads may also regulate endogenous TGFb gene responses. In rat hepatocytes, EGF and TGFb co-treatment was shown to induce association of Smad3 with c-Jun and mediate transactivation of Smad3 (Péron et al., 2001), and

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in a chondrogenic differentiation system, the ATDC5 cell line, Smad2, ERK1/2 and p38 were shown to cooperate in mediating TGFb-induced transcriptional activation of aggrecan gene expression, a marker gene characteristic of the chondrocyte phenotype (Watanabe et al., 2001). The interaction between the MAPK and Smad signaling pathways in the nucleus results not only in transcriptional transactivation but also in transcriptional repression. Early studies demonstrated that c-Jun expression, induced by TNFa, was inhibitory for TGFb induction of the a2 type I collagen promoter (Chung et al., 1996). A molecular basis for this antagonism has recently been proposed whereby promoterspecific elements determine whether the Smad/c-Jun interaction results in transactivation or transrepression (Dennler et al., 2000; Verecchia et al., 2001b). It was shown that Smad3 synergizes with c-Jun in AP-1 site-mediated transcription but c-Jun represses Smad3 transactivation from CAGAC sites (SBE sites). Smad3 and c-Jun do not associate when bound to DNA (Wong et al., 1999; Verecchia et al., 2001b). It is postulated that simultaneous DNA binding of Smad and AP-1 may not be compatible with the threedimensional structure of these complexes when bound to DNA (Shi et al., 1998). Thus Smad3/Jun interactions result in promoter-specific transcriptional activation or repression, independent of DNA binding (Verrecchia et al., 2001a, 2001b). Further support for the role of the MAPK pathway in transcriptional repression of Smad3 transcriptional activity was provided by results showing that constitutively active or dominant-negative mutants of JNK, respectively, repress or activate a Smad3-driven reporter construct (Dennler et al., 2000). Several models have been proposed to explain how MAPK/cJun modulate Smad3 transcriptional activity. In one model, off-DNA interactions between Smad3 and Jun result in the reduction of Smad3/DNA interactions and decreased transcriptional activity from Smadspecific promoters (Verrecchia et al., 2001a). However, others have shown that c-Jun does not inhibit Smad3 binding to DNA in vitro (Dennler et al., 2000), instead it acts as a suppressor of Smad transactivation by recruiting the transcriptional co-repressor TGIF (discussed above) (Pessah et al., 2001). Activation of the JNK pathway was shown to increase both the interaction between c-Jun/TGIF and Smad2/TGIF.

c-Jun was shown to stabilize the interaction between Smad2/TGIF and thus interfere with assembly of Smad2 with the transcriptional co-activator p300 in response to TGFb (Pessah et al., 2001). It has also been shown recently that activation of the Ras/MAPK pathway by EGF leads to the phosphorylation and stabilization of the TGIF co-repressor, increasing its steady-state levels and thus promoting formation of TGFb Smad/TGIF corepressor complexes (Lo et al., 2001).

Cytoplasmic cross-talk between Smads and MAPK The interplay between the Ras/MAPK and Smad pathways occurs not only at the level of transcriptional regulation in the nucleus but has also been demonstrated to occur in the cytoplasm. Further, as in the nucleus, cytoplasmic cross-talk between these two pathways can result in either antagonistic or synergistic responses. Early studies demonstrated that activation of mitogen growth factor signaling through the EGF and HGF receptor protein tyrosine kinases (RTKs) results in the phosphorylation of the BMP mediator Smad1 on serine residues in four PXSP motifs in its linker domain (Kretzschmar et al., 1997b). This phosphorylation, distinct from BMPR-Istimulated phosphorylation of the C-terminal SSVS sequence, was shown to be catalyzed by the ERK MAPK pathway based upon the use of the selective MEK1 inhibitor, PD98059. EGF and HGF-mediated phosphorylation of Smad1 was shown to block its nuclear accumulation and transcriptional transactivating activity but not its association with Smad4 (Kretzschmar et al., 1997a). Subsequent studies showed similar antagonistic effects of Ras activation on TGFb-mediated Smad2 and Smad3 transactivation (Kretzschmar et al., 1999). Ras activation, and to a lesser extent EGF receptor activation, was shown to mediate the ERK-dependent phosphorylation of Smad2 and Smad3 in their linker domains. This phosphorylation resulted in inhibition of Smad2 and Smad3 nuclear translocation and transactivating activity (Kretzschmar et al., 1999). More recent studies, however, have demonstrated a synergistic interplay between the MAPK and Smad pathways. It was shown that EGF and HGF, through a MEK1-catalyzed phosphorylation on the SSXS motif of Smad2, results

THE CYTOKINES AND CHEMOKINES

SMAD - INDEPENDENT SIGNALING

in its nuclear accumulation and transactivating activity (deCaestecker et al., 1998). Also, in endothelial cells, it has been demonstrated that a constitutively active form of MEKK1 mediates the phosphorylation of Smad2, independent of the SSXS motif, and results in enhanced Smad2/Smad4 interaction, nuclear localization of Smad2 and Smad4, and transcriptional transactivation (Brown et al., 1999). Synergistic interaction between TGFb-mediated JNK activation and Smad3 has also been shown. TGFb was shown to rapidly activate JNK in a Smad-independent manner, resulting in Smad3 phosphorylation outside its SSXS motif (Engel et al., 1999). This JNK-mediated phosphorylation of Smad3 enhanced its phosphorylation by TbR-I and its nuclear accumulation, and potentiated its transcriptional activation (Engel et al., 1999). Thus, in both the nucleus and cytoplasm, the interplay between the Ras/MAPK and Smad pathways provides a molecular basis for the antagonistic and synergistic effects of these two pathways on TGFbmediated developmental and biological processes. Dependent upon the combinatorial association of Smads with other transcriptional regulators, the cisregulatory elements present in TGFb target genes, and the specific kinase(s) mediating cytoplasmic Smad phosphorylation, the interplay can lead to transcriptional transactivation or repression. In both the cytoplasm and nucleus, the cross-talk between these two pathways may serve to integrate signaling elicited by multiple stimuli into a coordinated cellular response. For example, the ability of oncogenic forms of Ras to override the antiproliferative effects of TGFb, the opposing effects of EGF and HGF on TGFb or BMPmediated cellular responses, and the ability of TGFb to growth inhibit and override the proliferative effects of EGF in epithelial cells may all be attributable to the interplay between the MAPK and Smad pathways. Interestingly, the cross-talk can also serve as a feedback control mechanism to regulate multiple signaling pathways. In the case of TGFb, it can first induce activation of both Smad and MAPK/ c-Jun which modulate transcriptional regulation of multiple genes, including c-Jun itself. c-Jun accumulation or modification, through TGFb-induced JNK activation, subsequently leads to repression of Smad transcriptional activity and attenuation of TGFb signaling (Plate 49.5).

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SMAD-INDEPENDENT SIGNALING While most TGFb signaling results from the coordinated integration of the Smad and MAPK pathways, recent results suggest that certain TGFb responses are Smad independent and rely solely on TGFb activation of the MAPK pathway. It has been shown that TGFbinduced fibronectin (FN) expression is mediated through the JNK pathway, independent of Smad pathway activation. TGFb-mediated FN expression was demonstrated in cell lines which do not express endogenous Smad4 but was abrogated by dominantnegative forms of JNK or its upstream activator MKK4 (Hocevar et al., 1999). As described above, the rapid activation of JNK by TGFb was shown to be Smad independent. Early JNK activation by TGFb was achieved in the presence of a dominant-negative mutant form of Smad3, as well as in Smad4-deficient cells (Engel et al., 1999). More recent studies have shown that the antiapoptotic properties of XIAP in suppressing Bax-induced apoptosis are independent of Smad4 (Reffey et al., 2001). XIAP was shown to associate and co-localize with several type I receptor family members and potentiate TGFb-mediated activation of JNK and transcription from a TGFbresponsive reporter. XIAP-induced activation of JNK and reporter transactivation could both be blocked by a dominant-negative (DN) form of Smad4; however, XIAP inhibition of BAX-induced apoptosis was insensitive to DN Smad4 (Reffey et al., 2001). While these studies failed to demonstrate a direct antiapoptotic role of XIAP in TGFb-mediated apoptosis, they do suggest that XIAP-induced signaling is Smad4 dependent, whereas its antiapoptotic properties are Smad4 independent (Reffey et al., 2001). Other growth factors, much less pleiotropic than TGFb, transduce their signal through receptor complexes which couple to a large array of signaling pathways. Furthermore, most TGFb studies, for mostly technical reasons, have reduced signal transduction for this multifunctional protein to an ability to transactivate reporter constructs containing minimalistic promoter sequences. As such, many of the complexities and nuances of the TGFb signaling may not yet be fully appreciated. Therefore, while the Smad and MAPK pathways have emerged as major mediators of TGFb signaling, it is not unreasonable to expect that

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other pathways mediating some of TGFb’s cellular responses will soon be discovered.

TGFb REGULATION OF APOPTOSIS Normal development and maintenance of multicellular organisms requires the homeostatic balance between cellular proliferation and programmed cell death, apoptosis. Abnormalities in apoptotic control, similarly to disregulated cellular proliferation, can contribute to a variety of diseases, including cancer, degenerative disorders, and autoimmunity (Strasser et al., 2000). It is not surprising that a multifunctional protein such as TGFb, as well as its related family members, play crucial roles in the maintenance of this homeostatic balance. In addition to its ability to either potently inhibit or stimulate cellular proliferation in a cell type-specific manner, TGFb exerts both proapoptotic and antiapoptotic effects depending on the cell type or cellular context. In this section I will briefly discuss the antagonistic effects of TGFb on apoptosis in certain cellular contexts followed by a review of TGFb’s role in mediating apoptosis in three different cell types, prostate epithelium, hepatocytes and B lymphocytes. This is not intended to be a comprehensive review of the different apoptotic pathways but rather a short overview of those pathways activated by TGFb in these three apoptotic model systems.

Antiapoptotic effects of TGFb Although in many cellular systems TGFb is a potent inducer of apoptosis (discussed below), antiapoptotic or prosurvival actions of TGFb have also been reported. TGFb has been shown to protect keratinocytes from DNA fragmentation and cell death following loss of adhesion (cell substratum and/or cell–cell contact; Sachsenmeier et al., 1996). In human leukemic HL-60 cells, TGFb has been shown to inhibit the induction of cell density-dependent apoptosis (Saeki et al., 1997) and ceramide-induced apoptosis (Kuo et al., 1997). TGFb inhibits apoptosis in cells of neuronal origin (Prehn et al., 1994; Ren et al., 1997), and has also been shown to inhibit Fas-mediated apoptosis in human T cells (Cerwenka et al., 1996; Chen R.H., et al., 1998), rheumatoid synovial cells

(Kawakami et al., 1996), and in murine bone marrow progenitor cells (Dybedal et al., 1997). Several molecular mechanisms have been proposed as mediating some of the antiapoptotic or protective effects of TGFb. In neuronal cells induced to apoptose by excessive activation of glutamate receptors accompanied by calcium overloading, TGFb inhibits apoptosis by up-regulation of the antiapoptotic regulator Bcl-2 (Prehn et al., 1994). In HL-60 human leukemic cells, TGFb abrogates the activation of caspase 3 (CPP32/Yama) and cleavage of poly(ADPribose) polymerase (PARP) resulting in an attenuation of ceramide-induced apoptosis (Kuo et al., 1997). TGFb was shown to inhibit the ceramide-induced down-regulation of Bcl-2, maintaining constant Bcl-2 levels, thereby inhibiting caspase 3 activation (Kuo et al., 1997). In the human monoblastic myelogenous leukemia cell line, U937, TGFb increases cellular viability through an inhibition of apoptotic cell death without affecting cell proliferation and differentiation (Lee et al., 1999). It was further shown that TGFb inhibition of apoptosis was accompanied by a significant transcriptional up-regulation of the pro-survival factor Bcl-XL (Lee et al., 1999). More recently, several reports have implicated components of the MAPK pathway in the protective effects of TGFb in murine macrophages (Chin et al., 1999) and human lung carcinoma cells (Huang et al., 2000). TGFb inhibits serum withdrawal-induced apoptosis in the murine macrophage cell line, Raw 264.7, with an accompanying increase in ERK activation. TGFb does not rescue serum deprivation-induced apoptosis in Raw 264.7, cells transfected with a dominant-negative mutant ERK or treated with the MEK1 inhibitor, PD098059 (Chin et al., 1999). In the human lung carcinoma cell line, A549, TGFb suppresses serum deprivationinduced cell death through differential effects on c-Jun and JNK activities (Huang et al., 2000). TGFb was shown to inhibit the abrogation of JNK activity associated with serum withdrawal and induced the expression of c-Jun, required for cell survival in these cells. Thus, the antiapoptotic effects of TGFb appear to be mediated through components of the apoptotic machinery such as Bcl-2 and Bcl-XL, and/or elements of the MAPK pathway. It remains to be determined, however, whether TGFb independently activates and/or elicits cross-talk between these two pathways in inducing its antiapoptotic effects.

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TGFb-mediated apoptosis in prostate epithelium The prostatic epithelium undergoes extensive apoptosis following androgen withdrawal by castration. TGFb has been shown to play an important role in mediating this castration-induced apoptosis in both androgen-dependent normal and malignant prostate epithelial cells (Kyprianou and Isaacs, 1989; Martikainen et al., 1990; Tu et al., 1996). TGFb ligands (Kyprianou and Isaacs, 1989; Landström et al., 1996), receptors (Kim et al., 1996; Landström et al., 1996) and Smads (Brodin et al., 1999) are all induced following androgen ablation; however, the mechanisms through which TGFb induces apoptosis in prostate epithelium are just beginning to emerge. As previously shown in mammary epithelium (Nass et al., 1996) and in B lymphocytes (Chaouchi et al., 1995; Saltzman et al., 1998), TGFb-induced apoptosis in the rat prostatic epithelial cell line NRP-154 is accompanied by a suppression of Bcl-XL expression (LarischBloch et al., 2000; Chipuk et al., 2001). TGFb down-regulates mRNA and protein levels of the prosurvival Bcl-XL in a time-dependent manner with maximal effects observed in 12–16 hours (Chipuk et al., 2001). Mutant prostate epithelial cells that overexpress Bcl-XL (Larisch-Bloch et al., 2000) or stable transfectants over-expressing Bcl-XL (Chipuk et al., 2001) were shown to be resistant to TGFb-induced apoptosis. Furthermore, TGFb was shown to induce mitochondrial cytochrome c release with subsequent activation of caspases 3 and 9 and cleavage of the caspase 3 substrate, PARP; overexpression of Bcl-XL was shown to block these effects (Chipuk et al., 2001). In another prostatic cell line, LNCaP, TGFb-induced apoptosis was also accompanied by a downregulation of Bcl-2 and an increased expression and activation of caspase 1 (Guo and Kyprianou, 1999). Interestingly, while Bcl-XL was shown to block TGFbmediated cytochrome c release and apoptosis, it was unable to inhibit TGFb-mediated fibronectin expression, cell cycle arrest, and TGFb-dependent transcriptional responses (Larisch-Bloch et al., 2000; Chipuk et al., 2001). These observations suggest that the apoptotic signaling pathway of TGFb is distinct or involves additional molecules than those required for mediating TGFb-dependent transcriptional and growth arrest responses. Recently, this notion has

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been validated by the isolation of a novel mitochondrial septin-like protein, ARTS, which mediates TGFb-induced apoptosis of NRP-154 rat prostatic cells (Larisch et al., 2000). ARTS localizes to the mitochondria in cells but translocates to the nucleus upon TGFb treatment. ARTS overexpression sensitizes Cos cells to TGFb-mediated apoptosis and TGFbmediated caspase 3 activation, and anti-sense ARTS blocks TGFb-mediated apoptosis in NRP-154 cells (Larisch et al., 2000). While it is unclear how the TGFb signal couples to regulation of ARTS and/or Bcl-2 and Bcl-XL, a role for Smad proteins has also been shown in TGFb-induced apoptosis in prostate epithelium. In the Dunning R3327-PAP prostate tumor model, androgen deprivation-induced apoptosis correlates with an upregulation in the immunohistochemical expression of the Smad proteins, including Smad2, Smad3, Smad4, Smad6, Smad7, and an increase in Smad2 phosphorylation (Brodin et al., 1999). Smad3 has also been shown to directly interact, through its MH2 domain, with the androgen–receptor (AR) and prevent transcriptional transactivation of androgen-responsive promoters (Hayes et al., 2001). Thus, it is proposed that TGFb promotes apoptosis in the prostate by directly targeting and down-regulating, through the Smad proteins, the androgen-signaling pathway required for prostate epithelial cell growth. Further implicating a role for Smads in the regulation of TGFb-mediated cell death in the prostate is the demonstration that Smad7 overexpression induces apoptosis in the human prostatic carcinoma cell line (PC-3U) (Landström et al., 2000). In these cells, TGFb treatment or ectopic expression of Smad7 induces apoptosis, and inhibition of Smad7 expression by antisense mRNA or antisense oligonucleotides inhibits TGFb-induced apoptosis (Landström et al., 2000). While the mechanism by which Smad7 induces apoptosis in the prostate is unclear, it has recently been shown, in a mink lung epithelial cell model inducibly overexpressing Smad7 and in MDCK and Cos cells transiently overexpressing Smad7, that JNK activation is required for Smad7-induced apoptosis (Mazars et al., 2001). A dominant negative form of MKK4, an upstream activator of JNK, was able to inhibit Smad7-induced activation of JNK and apoptosis. Interestingly, the dominant negative form of MKK4 had no effect on Smad7-mediated inhibition of

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TGFb-mediated signaling, suggesting that the inhibitory function of Smad7 is independent of its ability to activate JNK (Mazars et al., 2001).

TGFb-mediated apoptosis in hepatocytes TGFb is an important regulator of liver homeostasis. Intravenous injection of TGFb has been shown to induce liver atrophy and apoptotic cell death in the normal and regressing liver (Oberhammer et al., 1991, 1992, 1993). TGFb also induces apoptosis in primary hepatocytes (Oberhammer et al., 1992; Sánchez et al., 1996; Cain et al., 1996) and hepatoma cell lines (Lin et al., 1992; Fukuda et al., 1993; Chuang et al., 1994; Chen and Chang, 1997). It was determined early on that the caspase family of cysteine proteases plays an important role in mediating TGFb-induced cell death in hepatocytes. In primary rat hepatocytes (Cain et al., 1996; Inayat-Hussain et al., 1997) and in hepatoma cell lines (Chen and Chang, 1997; Choi et al., 1998; Hung et al., 1998) caspase activation has been shown to be required for TGFb-mediated apoptosis. Treatment of cells with the pan-caspase inhibitor ZVAD FMK or CrmA blocks caspase 3 activation and TGFb-induced apoptosis (Cain et al., 1996; InayatHussain et al., 1997; Chen and Chang, 1997; Choi et al., 1998; Hung et al., 1998). More recent results, however, demonstrate that TGFb induces apoptosis by activating multiple caspases (Choi et al., 1998; Shima et al., 1999; Freathy et al., 2000). The emerging model (Plate 49.6) is that TGFb induces the release of cytochrome c from mitochondria (Freathy et al., 2000; Fabregat et al., 2000; Cain and Freathy, 2001; Herrera et al., 2001). The exact mechanism by which TGFb induces cytochrome c release remains to be elucidated. The role of Bcl-2 and Bcl-XL, which inhibit cytochrome c release, in TGFbmediated apoptosis is ambiguous. In several hepatocyte cell types, Bcl-XL levels are down-regulated by TGFb (Yamamoto et al., 1998; Shima et al., 1999; Fabregat et al., 2000), whereas in Hep-3B and FaO hepatocytes, TGFb has little, if any, effect on Bcl-XL (Ribeiro et al., 1999a; Freathy et al., 2000). Other studies suggest that reactive oxygen species (ROS), induced by TGFb, mediate the release of cytochrome c from mitochondria by decreasing the mitochondrial transmembrane potential (Sánchez et al., 1996;

Rodrigues et al., 1999; Herrera et al., 2001). A consequence of this decrease in mitochondrial transmembrane potential is postulated to be the opening of the membrane permeability transition (MPT) pore and the release of cytochrome c. A recent study has shown that ROS production induced by TGFb in fetal hepatocytes precedes its effects on Bcl-XL, the loss of mitochondrial transmembrane potential and the release of cytochrome c (Herrera et al., 2001). Cytochrome c released into the cytoplasm has been shown to associate with the apoptotic protease activating factor-1 (Apaf-1) and dATP, in an apoptosomelike structure, and catalyze the processing of caspase-9, thereby initiating the caspase cascade resulting in caspase 3 activation (Zou et al., 1997; Li et al., 1997). It has been shown recently that TGFb, through cytochrome c release, promotes the oligomerization of Apaf-1 into a 700 kDa apoptosome complex which recruits and processes caspase 3 and 7 (Freathy et al., 2000; Cain and Freathy 2001). The apoptotic effects of TGFb in hepatocytes can be inhibited by treatment of cells with EGF (Fabregat et al., 1996; Fasciati and Maier, 1997) and insulin (Chuang et al., 1994; Chen and Chang, 1997). Two recent studies suggest that these survival factors mediate their anti-apoptotic effects through activation of the phosphoinositide-3 kinase (PI-3K)dependent pathway and not through the MAPK pathway (Chen R.H., et al., 1998; Fabregat et al., 2000). The PI-3K inhibitor LY294002 and not the MEK inhibitor PD98059, or stable overexpression of an activated PI-3K or its downstream effector AKT, blocked the antiapoptotic effects of insulin on TGFb-mediated apoptosis (Chen R.H., et al., 1998). Furthermore, it was shown that the PI-3K pathway mediates the survival effects of EGF and insulin on TGFb-induced apoptosis by acting upstream from mitochondrial changes, such as Bcl-XL down-regulation, cytochrome c release and activation of caspase 3 (Chen R.H., et al., 1998; Fabregat et al., 2000). Interestingly, activation of PI-3K was specific in its inhibitory effects on TGFbmediated apoptotic events, in that it had no effects on Smad hetero-oligomerization or nuclear translocation (Chen R.H., et al., 1998). Another molecule shown to promote cell survival and antiapoptotic effects in hepatocytes is the NFjB/Rel family of factors (Arsura et al., 1997; Bellas et al., 1997). Further evidence for a role of NFjB in

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normal liver homeostasis is provided by RelA/ mice which undergo embryonic death at 15–16 days of gestation accompanying extensive hepatocyte apoptosis (Beg et al., 1995). In two untransformed murine hepatocyte cell lines, AML-12 and NMH, TGFb treatment caused an increased NFjB binding that was followed by a dramatic decrease in NFjB levels that preceded apoptosis. Overexpression of c-Rel abrogated TGFbinduced apoptosis. TGFb was further shown to increase IjBa expression due to hypophosphorylation and increased IjBa protein stability, thus correlating with NFjB down-regulation and apoptosis (Arsura et al., 1997). The signaling pathway that couples TGFb receptors to modulation of NFjB levels and IjBa phosphorylation, presumably through IjBa kinase, is currently unknown.

TGFb-mediated apoptosis in B lymphocytes Elimination or clonal deletion of self-reactive B cells during development is mediated through apoptosis and is essential for maintenance of immune tolerance. Failure to eliminate immature B cells results in lymphoproliferation and the development of autoimmune diseases and cancer (Lebman and Edmiston, 1999). TGFb is critical for the maintenance of normal immunological homeostasis (Christ et al., 1994; Letterio and Roberts, 1998) and lymphocyte proliferation (Beckwith et al., 1995; Diebold et al., 1995; Arsura et al., 1996; Brown et al., 1998, 1999). TGFb-deficient mice exhibit extensive lymphocytic hyperproliferation and systemic lupus erythematosus (SLE) like autoantibodies (Shull et al., 1992; Diebold et al., 1995; Dang et al., 1995; Letterio et al., 1996). The phenotypic consequences of TGFb ablation are similar to Sjogren’s sydrome, a human organ-specific autoimmune disease (Dang et al., 1995). In addition, 5% of patients with Sjogrens syndrome develop malignant B-cell lymphoma and an even higher percentage develop pseudolymphoma (Talil and Bunim, 1964). Thus the ability of TGFb to induce apoptosis in immature B lymphocytes may be a major mechanism controlling lymphocyte growth and subsequently the immune response. The mouse immature B cell line, WEHI 231, has been used as an in vitro model system to study B-cell tolerance (Warner et al., 1992; Fischer et al., 1994;

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Gottschlak and Quintáns, 1995; Wu et al., 1996) and apoptosis (Warner et al., 1992; Chaouchi et al., 1995; Beckwith et al., 1995; Wu et al., 1996; MacDonald et al., 1996; Brown T.L., et al., 1998, 1999; Patil et al., 2000), and TGFb has been shown to induce apoptosis in these cells (Warner et al., 1992; Wu et al., 1996; Arsura et al., 1996; Brown T.L., et al., 1998, 1999; Patil et al., 2000). The mechanism of TGFb-induced apoptosis in these cells involves TGFb-mediated targeting of components of the NFjB/Rel survival pathway (Wu et al., 1996, 1997; Arsura et al., 1996; Patil et al., 2000) and the caspase cascade (Brown et al., 1998, 1999; Brás et al., 1999). TGFb treatment of WEHI 231 cells has been shown to down-regulate NFjB/Rel, through its transcriptional induction of IjBa (Arsura et al., 1996), and leads to the transcriptional repression of the c-myc protooncogene (Lee et al., 1995a, 1995b; Wu et al., 1996). Overexpression of c-Rel or c-myc (Arsura et al., 1996; Wu et al., 1997) or stabilization of c-myc, ironically with antisense oligonucleotides to c-myc (Fischer et al., 1994) blocks TGFb-mediated apoptosis in these cells. Subsequent studies have shown that the p27Kip1 cyclin-dependent kinase inhibitor is induced in a c-myc-dependent fashion, and this induction correlates with apoptosis. Induction of an antisense c-myc vector was shown to induce p27Kip1 and lead to apoptosis (Wu et al., 1999). A signal for abrogating or rescuing WEHI 231 cells from TGFb-mediated growth arrest and apoptosis can be generated by concomitant signaling through CD40 (Arsura et al., 1996; Patil et al., 2000). CD40 is a transmembrane glycoprotein which is predominantly expressed by B cells (Stamenkovic et al., 1989) and plays a critical role in promoting cell survival, immunoglobulin class switching and memory cell formation (Kehry, 1996). CD40 has been shown to maintain NFjB/Rel and c-myc levels, and block induction of p27Kip1 levels in the presence of TGFb (Arsura et al., 1996; Schauer et al., 1996, 1998; Wu et al., 1999). Recent studies suggest that in B lymphocytes, CD40 survival is mediated through a PI-3 kinase/Akt-dependent pathway (Fruman et al., 1999; Curnock and Knox, 1998) which targets and activates NFjB (Romashkova and Makarov, 1999; Ozes et al., 1999) leading to up-regulation of NFjB (Barkett and Gilmore, 1999; Schwabe et al., 2001) and abrogation of B-lymphocyte apoptosis. CD40 has also been shown

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to induce, through an NFjB-dependent pathway, Smad7 levels which are inhibitory for TGFb-mediated apoptosis in WEHI 231 cells. Ectopic overexpression of Smad7 in WEHI 231 B lymphocytes (Patil et al., 2000) or in the mouse B-cell line HS-72 (Ishisaki et al., 1998) blocks TGFb- and activin A-induced apoptosis, respectively. Thus a model for TGFb-mediated apoptosis in B lymphocytes is emerging whereby the decline in NFjB/Rel-binding activity leads to a drop in c-myc levels and a concomitant induction of p27Kip1 resulting in an activation of apoptosis. The survival pathway induced by CD40 activation maintains NFjB/Rel activity, through a PI-3 kinase/Akt-dependent pathway, leading to the induction of the TGFb signaling inhibitor Smad7, ultimately resulting in abrogation of TGFb-mediated cell death (Plate 49.6). The caspase cysteine proteases have also been implicated in TGFb-mediated apoptosis in B lymphocytes (Brown et al., 1998, 1999; Schrantz et al., 1999; Inman and Allday, 2000). In WEHI 231 cells, specific caspase inhibitors had no effect but the broadspectrum caspase inhibitor, BD-fmk, blocked TGFbinduced cell death (Brown et al., 1998). BD-fmk was also able to block TGFb-mediated cleavage of aIIspectrin, a cytoskeletal protein involved in structural integrity and organization whose cleavage is postulated to play a role in apoptosis (Brown et al., 1999). In the human Burkitt’s lymphoma B-cell line BL41, TGFb-mediated nuclear fragmentation is blocked by the caspase 3 inhibitor DEVD-fmk. DEVD-fmk was shown to block TGFb-mediated expression of the active p17 subunit of caspase 3, and the cleavage of poly(ADP-ribose) polymerase, PARP, a caspase 3 substrate (Schrantz et al., 1999). In addition, TGFb treatment of BL41 cells promoted the expression of both the dephosphorylated and truncated forms of the growth suppressor protein, Rb, and this cleavage of Rb was also sensitive to the caspase 3 inhibitor. Thus it is postulated that TGFb-mediated apoptosis is dependent on caspase 3 activation and involves caspase-dependent cleavage of Rb (Schrantz et al., 1999). Also, in BL41 cells, TGFb-mediated apoptosis has been shown to involve activation of caspase 8 in a death receptor-independent fashion (Inman and Allday, 2000). In this study, levels of the Bcl-2 family members were not affected by TGFb but PARP cleavage was observed (Inman and Allday, 2000).

In summary, the mechanisms by which TGFb mediates apoptosis are beginning to become unraveled. As a rule, it does not appear as if TGFb activates a novel or distinct pathway that mediates cell death but rather targets and modulates components of the existing apoptotic and survival machinery within a cell. In the prostate, a novel protein termed ARTS is required but for the most part TGFb seems to utilize the apoptotic machinery that mediates cell death in response to other apoptotic inducers and factors. What remains to be determined is the mechanism by which the TGFb receptors couple to these downstream apoptotic pathways and the potential crosstalk and modulation among these in mediating TGFb cell death. Also, to be determined is the role of the TGFb-activated Smad and MAPK signaling pathways in mediating apoptosis. A recent study (Perlman et al., 2001) provides some initial clues as to how TGFb receptors couple to downstream apoptotic signaling pathways. Daxx, a protein associated with the Fas receptor that mediates activation of JNK and apoptosis induced by Fas, was shown to associate with the type II TGFb receptor. A dominant-negative inhibitor of Daxx blocks TGFb apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGFbinduced apoptosis in mouse hepatocytes. In addition, Daxx was shown to be involved in TGFb activation of JNK. These exciting results provide the first biochemical connection between TGFb receptors and the apoptotic machinery, and lay the ground for further experiments aimed at these coupling mechanisms.

ACKNOWLEDGEMENTS I would like to thank Drs Hocevar, Patil and Wildey for critical review and discussion. Work in the laboratory was supported by NIH grants CA 55536 and CA80095.

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Sano, Y., Harada, J., Tashiro, S. et al. (1999). ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-beta signaling. J. Biol. Chem. 274, 8949–8957. Sasaki, A., Masuda, Y., Ohta, Y. et al. (2001). Filamin associates with Smads and regulates transforming growth factor-beta signaling. J. Biol. Chem. 276, 17871–17877. Sasaki, Y. and Sugamura, K. (2001). Involvement of Hgs/Hrs in signaling for cytokine-mediated c-fos induction through interaction with TAK1 and Pak1. J. Biol. Chem. 276, 29943–29952. Sato, Y. and Rifkin, D.B. (1989). Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J. Cell Biol. 109, 309–315. Savage, C., Das, P., Finelli, A.L. et al. (1996). Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc. Natl Acad. Sci. USA 93, 790–794. Schauer, S.L., Wang, Z., Sonenshein, G.E. and Rothstein, T.L. (1996). Maintenance of nuclear factor-kappa B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of I kappa B proteins. J. Immunol. 157, 81–86. Schauer, S.L., Bellas, R.E. and Sonenshein, G.E. (1998). Dominant signals leading to inhibitor kB protein degradation mediate CD40 ligand rescue of WEHI 231 immature B cells from receptor-mediated apoptosis. J. Immunol. 160, 4398–4405. Schlunegger, M.P. and Grütter, M.G. (1992). An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2. Nature 358, 430–434. Schrantz, N., Blanchard, D.A., Auffredou, M-T. et al. (1999). Role of caspases and possible involvement of retinoblastoma protein during TGFb-mediated apoptosis of human B lymphocytes. Oncogene 18, 3511–3519. Schultz-Cherry, S. and Murphy-Ullrich, J.E. (1993). Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J. Cell Biol. 122, 923–932. Schultz-Cherry, S., Ribeiro, S.M.F., Gentry, L. and MurphyUllrich, J.E. (1994a). Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system. J. Biol. Chem. 269, 26775–26782. Schultz-Cherry, S., Lawler, J. and Murphy-Ullrich, J.E. (1994b). The type 1 repeats of thrombospondin 1 activate latent transforming growth factor-beta. J. Biol. Chem. 269, 26783–26788. Schultz-Cherry, S., Chen, H., Mosher, D.F. et al. (1995). Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J. Biol. Chem. 270, 7304–7310. Schwabe, R.F., Schnabl, B., Kweon, Y.O. and Brenner, D.A. (2001). CD40 activates NF-kappa B and c-Jun N-terminal kinase and enhances chemokine secretion on activated human hepatic stellate cells. J. Immunol. 166, 6812–6819. Schwarz, L.C., Gingras, M.C., Goldberg, G. et al. (1988). Loss of growth factor dependence and conversion of transforming growth factor-beta 1 inhibition to stimulation in

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50 The activin/inhibin family David J. Phillips Monash University, Melbourne, Australia

A multiple personality is in a certain sense normal George H. Mead

INTRODUCTION Unlike the relatively short history of most cytokines and growth factors, the origins of the activin/inhibin family can be sourced to a Science paper published back in 1932 by D. Roy McCullagh. He proposed the existence of a ‘water-soluble testicular factor, which is characterized by its action on the pituitary gland, [and] has been called “inhibin”’ (McCullagh, 1932). After a hiatus of around 50 years, inhibin was purified and sequenced by several groups (Ling et al., 1985; Miyamoto et al., 1985; Robertson et al., 1985), activin followed in 1986 (Vale et al., 1986), and the activinbinding protein, follistatin, in the following year (Esch et al., 1987; Robertson et al., 1987). At that time the overwhelming view based on available evidence was that these factors, as proposed by McCullagh, were reproductive hormones released by the gonad to regulate follicle-stimulating hormone in the pituitary gland. The subsequent isolation and purification of ‘erythroid differentiation factor’ (Eto et al., 1987), The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

‘mesoderm-inducing factor’ (Smith et al., 1990), ‘nerve survival molecule’ (Schubert et al., 1990) and ‘plasmacytoma growth inhibitor’ (Brosh et al., 1995), all shown to have sequence identity with activin, highlighted functions beyond those originally proposed. More recently, major advances have been made to elucidate these broader roles and these have led to the placement of activin, in particular, firmly in the category of a multifunctional cytokine with application to a number of cellular and tissue systems. Based on structural features, activin and inhibin have been assigned to the transforming growth factor b (TGFb) superfamily. Other members of the family, TGFb itself and the bone morphogenetic proteins (BMP), are considered in adjacent chapters. Inhibin is not believed to possess the characteristics of a cytokine, although recent evidence is suggestive of a role in tumour suppression (Risbridger et al., 2001). It will only be covered superficially herein. The majority of this chapter will focus on the biology of activin, and its binding protein, follistatin. The activin signalling

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pathway shares significant homology with those of the TGFb and BMP groups. As this has been dealt with comprehensively in the TGFb chapter by Dr Howe, discussion of this area will be limited to providing a reference point for functions of activin shared with other family members compared with more divergent roles. In the remaining sections, the regulation and actions of activin in various biological systems pertinent to its role as a cytokine will be examined, specifically in apoptosis and proliferation, tissue remodelling and in the immune system. Other aspects of activin biology such as mesoderm induction and effects on the reproductive system are beyond the scope of this chapter and the reader is referred to pertinent reviews of these areas (Woodruff, 1998; McDowell and Gurdon, 1999). It should also be stated at the outset that in fulfilling the criteria of a cytokine, activin and its receptors are expressed by many cell types in the body. This is not to infer, however, that the regulation by activin of specific processes is impossible, rather that its role needs to be integrated with those of other cytokines and growth factors.

GENETIC AND MOLECULAR CHARACTERIZATION The activins, like other members of the TGFb superfamily, are dimeric proteins formed following posttranslational processing of a number of distinct gene products. Genes for five activin subunits have been described, designated bA to bE. The chromosomal localization and homologies are given in Table 50.1. The inhibin a subunit is another member of the TGFb family that has less structural homology to the activin subunits, yet is able to form dimers with the activin bA and bB subunits to form dimeric inhibin A and inhibin B, respectively. Dimerization with other activin b subunits to form inhibins C–E is unlikely, at least on the basis of coexpression studies with the a and bC subunit (Mellor et al., 2000). Inhibin and activin subunits are synthesized as full-length precursor forms that undergo proteolytic cleavage intracellularly and also in serum (Robertson, 1991). The inhibin a subunit is formed as a pro-aN-aC entity, with the aC portion being the mature form. The activin b subunits have a

TABLE 50.1 Chromosomal locations and sequence homology for activin ligands, receptors and binding proteins Protein

Chromosomal % location Homology

Activin bA Activin bB Activin bC Activin bD Activin bE Inhibin a Activin receptor type IA Activin receptor type IB Activin receptor type IIA Activin receptor type IIB Follistatin Follistatin-related protein

7p14.1 2q14.2 12q14.1 _b 12q14.1 2q35 2q24.1 12q13.13 2q23.1 3p22.2 5q11.2 19p13.3

100a 64 51 56 52 21

100c 43.2c

a

Compared with the activin bA subunit precursor coding region. b Activin bD is not known for the human. c Compared with the follistatin coding region.

proregion which is cleaved to derive the mature subunit form. It should be stressed that unlike TGFb, there is no evidence to suggest that inhibin or activin subunits are held as an inactive complex in a similar mechanism to that of latency-associated protein (LAP) and TGFb (Munger et al., 1997). As alluded to above, a number of different activin forms are known based on the formation of heterodimeric or homodimeric partnerings. Activin A, the dimer of bA subunits, is the most well studied and the original actions for which activins were identified are based on its isolation and properties. The extent to which the various activins can substitute for each other is an interesting biological conundrum. Knocking out the activin bA subunit results in animals that survive to the early neonatal period, but who die soon after due to a cleft palate defect that prevents suckling (Matzuk et al., 1995b). The cleft palate defect is reminiscent of knockout phenotypes from other related family members, TGFb2 and b3 (Proetzel et al., 1995; Sanford et al., 1997), but clearly is not rescued in the activin bA null mutation. In contrast, the activin bB subunit knockout has a milder phenotype, with abnormal eyelid fusion and mammary gland defects (Schrewe et al., 1994; Vassalli et al., 1994), and the double knockout (bA/bB) has no further abnormalities (Matzuk et al., 1995b). This has led to the concept that

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the bB subunit, and therefore activin B, has a lower biological potency compared with forms containing bA subunits. In vitro data support this contention (Corrigan et al., 1991; Nakamura et al., 1992). More recently, an elegant study by Matzuk and colleagues (Brown et al., 2000) has added further support by generating mice in which the coding region for activin bA is replaced by the bB subunit coding region. In these mice the cleft palate defect in the activin bA knockout mice is rescued but other abnormalities in hair and lesions in gonadal function and accessory sexual organs were apparent. Thus, activin B can substitute in some biological roles but in others is unable to act as an equivalent ligand. Interestingly, the heterodimer, activin AB, is believed to have a similar potency to activin A, but does not circulate in the bloodstream in detectable quantities and is only measurable in ovarian follicular fluid (Evans et al., 1997). The above considerations are not intended to dismiss the activin bB subunit as irrelevant, for the following reasons; (1) the knockout animal has a clearly defined and abnormal phenotype; (2) there are a number of examples of differential expression of the activin bA and bB subunit, such as during development (Roberts and Barth, 1994) and during wound healing (Hübner et al., 1996); (3) examples exist, such as duct formation in the mammary gland, where the activin bA and bB subunits appear to have opposing actions (Liu, Q.Y. et al., 1996; Robinson and Hennighausen, 1997); (4) activin is known to be a potent paracrine regulator of FSH in the pituitary gland, with the bB subunit being the dominant form (the bA subunit is almost undetectable in pituitary cells, Corrigan et al., 1991); and (5) changes in the expression levels of the activin subunits are not concordant in vivo, such as following airway inflammation in mice (Rosendahl et al., 2001). The biological function of the activin bC and bE subunits is not well understood. In some paradigms such as liver regeneration or inflammation, mRNA for these subunits are perturbed (e.g. Esquela et al., 1997; O’Bryan et al., 2000), but gene knockouts of either subunit, or the double knockout, have no obvious abnormalities (Lau et al., 2000). The bD subunit is only known in Xenopus and is expressed during early development with mesoderm-inducing properties (Oda et al., 1995); a mammalian homologue has yet to be described. Deletion of the inhibin a subunit leads to the devel-

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opment of cachexia and tumour development in the gonads and adrenal glands (Matzuk et al., 1992, 1994). These observations are discussed briefly in the section on cancer and are extensively covered in a recent review (Risbridger et al., 2001); the role of inhibin A and B in reproductive function is also well defined and the subject of comprehensive coverage in other fora (Anderson and Sharpe, 2000; Knight and Glister, 2001; Muttukrishna, 2001).

BINDING PROTEINS, RECEPTORS AND SIGNALLING PATHWAYS Binding proteins No discussion of the biology of activin is complete without factoring in a number of extracellular binding proteins that impinge upon its availability and access to receptors and intracellular signalling pathways. At least half a dozen binding partners with differing affinities for activin and potentially different roles are known (Phillips, 2000). Of these, follistatin has receive the most attention for its ability to neutralize activin’s action in a number of biological contexts; the binding affinity of follistatin is in a similar range to that of the activin receptor subunits and is nearly irreversible. Follistatin was the third regulator of folliclestimulating hormone (FSH) isolated in the 1980s (Esch et al., 1987; Robertson et al., 1987) – inhibin and activin were the earlier isolates. It was only following further characterization that it was established that follistatin bound to activin and that its ‘modus operandi’ to inhibit FSH secretion was actually its ability to block the stimulatory effects of activin (Nakamura et al., 1990). Follistatin is the prototype member of a growing family of proteins which share one or more follistatin domains (Phillips and de Kretser, 1998) and includes proteins such as SPARC (Brekken and Sage, 2000) and follistatin-related protein (FSRP) (Schneyer et al., 2001). The follistatin domain is a canonical sequence with conserved cysteine residues, an amino b hairpin, two antiparallel a helices and three b sheets (Phillips and de Kretser, 1998). While family members share tertiary homology, this does not infer that the follistatin domain per se is required for activin binding.

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Mapping studies of follistatin indicate that the aminoterminal region and the first follistatin domain (of three) is important for activin binding (Inouye et al., 1991b; Sidis et al., 2001) whereas with FSRP the region containing the second follistatin domain (of two) and the carboxy-terminal sequence interact with activin (Tsuchida et al., 2000). Follistatin exists in a number of variant forms as a result of alternative splicing, proteolytic cleavage and glycosylation. Follistatin-315 (315 amino acids) is the form of follistatin predominant in the circulation, although it may be proteolytically cleaved at the carboxy-terminus to derive a 300/303-amino-acid form that is prevalent in ovarian follicular fluid (Esch et al., 1987; Inouye et al., 1991a; Sugino et al., 1993). Follistatin-288 is an alternatively spliced form lacking exon 6 of the gene. It is also found in the circulation, but in low abundance, and is primarily associated with cell surfaces due to its affinity for heparan sulphate proteoglycans (Sugino et al., 1993; Hashimoto et al., 1997). The

heparin-binding region is located in a region of the molecule (amino acids 72–86; Inouye et al., 1992; Sumitomo et al., 1995) that may be masked by the carboxy-terminal 15 amino acids in the full-length isoform, thereby explaining why follistatin-315 has minimal affinity for cell surfaces. While both forms bind activin with similar but not identical affinity (Hashimoto et al., 2000), follistatin-315 may act as a circulating sink for activin, whereas the follistatin-288 form facilitates a local degradation pathway. The formation of an activin–follistatin complex in association with heparan sulphate proteoglycans triggers the complex to be internalized and targeting to the lysosomal pathway for degradation (Figure 50.1; Hashimoto et al., 1997). The importance of follistatin to cellular function is highlighted by the neonatal lethality of follistatin knockout mice (Matzuk et al., 1995c); pups are small, pale and cyanotic and die within 24 hours due to breathing abnormalities induced by retarded inter-

FIGURE 50.1 Four mechanisms by which activin signalling is regulated by extracellular binding proteins or receptor mimetics: (A) follistatin binds to activin and prevents receptor binding, with alternative fates depending on whether binding is to follistatin-315 (extracellular form) or follistatin-288 (cell surface form); (B) a complex of inhibin and inhibin-binding protein (InhBP) associates with the type I receptor and prevents dimerization with the type II receptor; (C) a complex of inhibin and betaglycan associates with the type II receptor and prevents dimerization with the type I receptor; (D) the pseudoreceptor, BAMBI, associates with activin and the type II receptor, but lacks the phosphorylation domain required for Smad binding and activation. THE CYTOKINES AND CHEMOKINES

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costal and diaphragmatic muscle mass. Additional abnormalities are found in the ribs and lumbar vertebrae, incisors, whiskers, skin and secondary palate. While this phenotype is consistent with an unopposed action of activin in several of these organs, it is clear that some aspects of follistatin biology perturbed in the knockout model reflect other binding partners. Indeed, follistatin has been demonstrated to bind and neutralize other TGFb family members, most notably several of the BMPs (Yamashita et al., 1995; Fainsod et al., 1997; Iemura et al., 1998; Yamamoto et al., 2000a), growth/differentiation factor-11 (GDF-11) (Gamer et al., 1999), and more recently myostatin (Lee and McPherron, 2001). The binding of follistatin to myostatin, a potent regulator of muscle mass, is most likely reflected by the irregularities in bodysize and muscle development observed in the follistatin knockout phenotype. The recently defined involvement of FSRP in binding to activin (Tsuchida et al., 2000; Maguer-Satta et al., 2001; Tortoriello et al., 2001) also raises the issue of this protein in defining activin-mediated cellular signalling. An important difference in the biology of these proteins is that FSRP appears to be largely localized to the nucleus of cells (Tortoriello et al., 2001) and may function as an autocrine regulator of activin function. Some credence can be ascribed to this given the recently characterized nuclear localization signal found in the proregion of the activin bA subunit (Bläuer et al., 1999).

Receptors In common with other members of the TGFb superfamily, activins signal through transmembrane serine–threonine kinases, consisting of a ligandbinding extracellular domain, a short transmembrane domain and a large cytoplasmic domain involved in communicating with intracellular signalling partners (Piek et al., 1999). Activin binds to a constitutively activated type II receptor, after which a type I receptor is recruited to the complex and is phosphorylated. The mature ligand–receptor complex is believed to consist of an activin dimer coupled with two type I and two type II kinase subunits. For this reason, inhibin, containing one b subunit, is under some circumstances able to antagonize activin signalling by binding to receptor subunits but failing to activate

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downstream signalling through the Smad pathway (Xu et al., 1995; Lebrun and Vale, 1997; Martens et al., 1997). More recent studies implicate both betaglycan (Lewis et al., 2000) and inhibin-binding protein (Chapman and Woodruff, 2001) as receptor-binding co-factors mediating inhibin antagonism of activin signalling. The existence of other inhibin-binding proteins is likely (Hertan et al., 1999; Harrison et al., 2001), and the above do not rule out additional mechanisms for inhibin action exclusive of activin type I receptors or Smad signalling. The proposed mechanism for inhibin-binding protein is reminiscent of the recently described pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor) (Onichtchouk et al., 1999), which lacks an intracellular kinase domain and forms inactive complexes with TGFb superfamily type II receptors. Various combinations of type I and II receptors are known to be integrated into activin signalling. (Nomenclature for the activin receptors is interchangeable. In this chapter, the preferred format is ActRIA (ActRIALK2), ActRIB (ALK4), ActRIIA (ActRII) and ActRIIB.) The preferred type I receptor is ActRIB although activin binds to ActRIA to a lesser extent (Attisano et al., 1993). Further lines of evidence that support this concept are (1) that kinase-deficient ActRIB blocked activin responses in K562 cells whereas kinase-deficient ActRIA had little effect (Tsuchida et al., 1995), and (2) overexpression of ActRIB facilitated activin-induced apopotosis in HS-72 cells whereas ActRIA overexpression suppressed this effect (Hashimoto et al., 1998). Specificity is suggested by the observation that Smad7 preferentially inhibits ActRIA-mediated activin responses compared with ActRIB-mediated responses (Kitamura et al., 2000). In terms of type II receptors, two receptor subunits are utilized – ActRIIA and ActRIIB – both subunits have alternative receptor transcripts which have differing affinities for activin ligands (Attisano et al., 1992; Xu et al., 1994). The importance of the various receptor combinations is highlighted by the different phenotypes of the activin type II receptor knockouts (Matzuk et al., 1995a; Oh and Li, 1997); furthermore, the type I receptors have distinct localization patterns during embryonic development (Verschueren et al., 1995). The various activin receptor subunits are known to be promiscuous in binding other TGFb family members (Table 50.2). This is

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TABLE 50.2 Activin receptor subunits and known ligands Receptor subunit

Ligands

ActRIA (ALK2)

Activin Müllerian inhibitory substancea BMP7b Activin BMP3c Activin Inhibin BMP3c BMP7d GDF5e Activin Inhibin BMP7d Myostatinf

ActRIB (ALK4) ActRIIA

ActRIIB

Accessory proteins

Inhibin binding protein Betaglycan

Betaglycan

a

Clarke et al., 2001; bLiu, et al., 1995; cDaluiski et al., 2001; dYamashita et al., 1995; eNishitoh et al., 1996; fLee and McPherron, 2001.

clearly pertinent in defining ligand specificity, or its lack, through the assembly of various receptor combinations, but also in terms of competition for receptor subunits by subsets of TGFb superfamily members.

Intracellular signalling components Once receptor activation has occurred, activins utilize the Smad signalling system common to the TGFb superfamily. Our current understanding of this system is outlined clearly in the accompanying chapter on TGFb and in other comprehensive reviews (e.g. Massagué, 2000), and therefore the following discussion is confined to outlining the key points and features that are unique (or otherwise) to activin. Activin intracellular signalling is closely aligned with that of TGFb. Activin utilizes Smad 2 and 3 in combination with the co-Smad4; Smad7 is the presumptive inhibitory Smad in the activin pathway (Figure 50.2; Ishisaki et al., 1998; 1999). Consequently, it should not be surprising that in many contexts activin and TGFb share similar functions. What is surprising is that activin and TGFb have a number of disparate functions. One point at which these ligands exert different effects may be in the ontogeny and distribution of receptors, or indeed in the local availability of TGFb or activins. An example is in F5.5.fl erythroleukemic cells, which respond to activin but not TGFb; this cell line has the requisite expression of type

I and II activin receptors, but is deficient in TGFbRII (Machida et al., 2000). A similar argument for subtle differences in the expression patterns of activin and TGFb receptors may explain why gene deletions of TGFb2, TGFb3 and activin bA subunit all have superficially similar cleft palate defects (Matzuk et al., 1995b; Proetzel et al., 1995; Sanford et al., 1997), yet func-

FIGURE 50.2 The key aspects of the activin signalling system. Each box represents points at which regulation occurs and where alternative components interact, such as competition and sharing with the TGFb and BMP signalling pathways. See text for further description of the various steps and for a more detailed coverage refer to Chapter 49. Adapted from Massagué et al. (2000).

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tional rescue through shared Smad activation does not occur. Another point at which specificity may occur could be through a Smad-independent signalling mechanism or via receptor-specific accessory proteins. While convincing functional evidence for these concepts has yet to be elucidated, the recent description of activin receptor interacting protein 1 (ARIP1) (Shoji et al., 2000) provides a potential first hint of such a process. ARIP1 is a PDZ protein that interacts specifically with activin type II receptors but not with TGFb or BMP type II receptors in mammalian two-hybrid systems. It also associates with Smad3 and so may act as a chaperone for Smad3–activin receptor interactions. The precise function of this new intracellular signalling factor and whether other such receptor-specific entities exist is clearly an area of intense interest.

Interactions with other signalling pathways An important consideration in describing a role for activin in various cellular systems is the points of interaction with other cytokine and growth factor signalling pathways. These interactions have been largely based on a generalized schematic of interaction of transcription factors or intracellular signalling systems with Smad proteins. As such, they are unable to discern between modulation of TGFb versus activin pathways. Examples include phosphorylation of Smad2 by MEK1, a downstream regulator in the MAP kinase pathway (de Caestecker et al., 1998), and interactions with Smad3 and Smad7 by NFB and interferon -activated factors (Bitzer et al., 2000; López-Rovira et al., 2000). Other approaches have utilized more functional settings, with the most powerful showing synergistic or antagonistic effects of activin or follistatin with other growth factors or cytokines. Among the most profound include those related to the induction of activin by basic fibroblast growth factor (bFGF) as a neuroprotective factor (Tretter et al., 2000), branching morphogenesis interactions with hepatocyte growth factor (HGF) (Maeshima et al., 2000), and the synergistic action of follistatin in enhancing angiogenesis driven by bFGF (Kozian et al., 1997). Clearly, it is important to establish the relevance of these observations to other cell systems and processes, and whether they are activin-specific or a

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more general response resulting from inputs at the level of Smad signalling. It is also relevant to consider emerging data that activin and TGFb cross-regulate their own signalling pathways. For instance, TGFb can induce the gene expression of activin and its binding proteins follistatin and FSRP (Hübner and Werner, 1996; Zhang, Y.Q. et al., 1997; Bartholin et al., 2001; Maguer-Satta et al., 2001), and modulation of activin signalling by follistatin blockade or dominant-negative activin receptors stimulates TGFb synthesis (Kogure et al., 2000; Ichikawa et al., 2001).

ROLES IN PROLIFERATION AND APOPTOSIS Activin has an important action in maintaining the homeostatic balance between cell proliferation and cell cycle arrest and apoptosis. Several groups have purified factors with powerful apoptotic action, that upon sequencing, proved to be activin A (Brosh et al., 1995; Nishihara et al., 1995). However, activin is also known to be proliferative to a number of other cell lineages, such as certain subtypes of epithelial cells (Di Simone et al., 1996; Welt et al., 1997; Choi et al., 2001) and fibroblast-like cells (Hedger et al., 1989; Kojima et al., 1993; Sakurai et al., 1994; Ohga et al., 2000). Other studies raise the issue that the proliferative response may be biphasic, with activin being stimulatory in the lower nanomolar range, and inhibitory at higher concentrations (Shikone et al., 1994; Kotajima et al., 1995; Di Simone et al., 1998). Regardless of the proliferative or apoptotic effects induced by activin, an important consideration is the local cellular balance of activin and follistatin dictating how a cell will respond to activin signals (Di Simone et al., 1996; Delbaere et al., 1999; McPherson et al., 1999).

Apoptosis A specific apoptotic effect of activin was first noted in various myeloid and hybridoma cell lines (Nishihara et al., 1993). Subsequently, this phenomenon has been extended to include a number of other cell lineages, including prostatic epithelial carcinoma cell lines (Dalkin et al., 1996; Wang et al., 1996; McPherson

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et al., 1997), human breast cancer cells (Cocolakis et al., 2001) and possibly lymphocytes (Hedger et al., 1989; Hedger and Clarke, 1993), among others. Furthermore, in vivo administration of recombinant activin A threw up the surprising finding of massive induction of apoptosis in hepatocytes surrounding the central vein (Schwall et al., 1993; Hully et al., 1994). Follow-up studies of proliferation and apoptosis in cultured hepatocytes or hepatoblastoma cells (Yasuda et al., 1993; Zhang et al., 1996; Russell et al., 1999; Chen et al., 2000) have established activin and follistatin as potent regulators of liver homeostasis. The biological relevance of these findings has been encapsulated in models of liver regeneration using partial hepatectomy; in animals administered follistatin, hepatocyte proliferation and liver regeneration times are accelerated (Kogure et al., 1995, 1998). One of the consistent features of activin-induced apoptosis is a shift in cells towards G0/G1 arrest (Sternberg et al., 1995; Yamato et al., 1997; Ishisaki et al., 1998; Koseki et al., 1998), a point at which cells are most vulnerable to undergo apoptosis via the p53 pathway. This is often one of the first phenotypic features in cells undergoing activin-induced differentiation (Seishima et al., 1999; Davis et al., 2000; Sonoyama et al., 2000; Fukuchi et al., 2001), consistent with the notion that succession of proliferation and G0/G1 arrest is common to both commitment pathways and is one of the first points at which cell fate is decided following exposure to activin. Certainly, preliminary evidence implicates Mcl-1, a member of the Bcl-2 family, as a common mediator in the processes that dictate whether myeloid cells undergo differentiation or apoptosis (Fukuchi et al., 2001). Furthermore, in mouse B cells made resistant to apoptosis by overexpression of Bcl-2, activin-induced G0/G1 arrest still occurs (Yamato et al., 1997), indicating that this effect is an obligatory first step in the activin response of many cells, regardless of whether further apoptotic stimuli are received. In common with TGFb, activin induces apoptotic activation through the p53/retinoblastoma nexus. An increase in p53 expression itself is apparent in LNCaP prostatic epithelial cells exposed to activin (Ying et al., 1997; 1999; Lin and Ying, 1999), but not in T47D breast cancer cells (Cocolakis et al., 2001). Furthermore, modulation of a number of points in the cyclin and cyclin-dependent kinase (CDK) pathways are appar-

ent. Activin inhibits the production of cyclin D2 (Yamato et al., 1997) and CDK 4 (Zauberman et al., 1997), and increases the production of a number of CDK inhibitors such as p21CIP1/WAF1 and possibly p15INK4B and p16INK4A (Yamato et al., 1997; Zauberman et al., 1997; Ishisaki et al., 1998, 1999; Lin and Ying, 1999; Cocolakis et al., 2001). These dynamic changes combine to suppress cell cycle progression through enhancing the hypophosphorylated state of the retinoblastoma protein, as has been shown for a number of activin-treated cell types (Figure 50.3A; Yamato et al., 1997; Zauberman et al., 1997; Ishisaki et al., 1999). Other aspects of the cell death machinery that have been explored are potential interactions of activin

FIGURE 50.3 The role of activin in apoptotic pathways, with pointers representing steps at which activin affects apoptosis: (A) cell cycle arrest, (B) caspase activation through the Bcl-2 cascade, (C) the Fas/Fas ligand system. Adapted from Yang et al. (1997), Lundberg and Weinberg (1999) and Adams and Cory (2001).

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with the Bcl-2 family. Overexpression of Bcl-2 or Mcl-1 protects cells from activin-induced apoptosis (Koseki et al., 1995; Fukuchi et al., 2001). Furthermore, treatment of cells with activin modulates the balance of pro- and antiapoptotic family members, although changes in Bcl-2, Bcl-XS, Bcl-XL, Mcl-1 and Bax may vary with cell type (Koseki et al., 1995, 1998; Zhang, Z. et al., 1997; Fukuchi et al., 2001). The net result is reflected in an increase in caspase-9 and -3 production following activin exposure (Figure 50.3B; Fukuchi et al., 2001). Interest has also focused on the mechanism by which Fas ligand signals through the FADD and Jun N-terminal kinase (JNK) pathways to induce caspase activation and apoptosis. The recent indentification of Daxx (Yang et al., 1997), a novel Fas-binding protein, provides a mechanism linking Fas with JNK activation and subsequent apoptosis. Furthermore, the demonstration that Daxx interacts with both TGFb and activin type II receptors (Perlman et al., 2001) provides another mechanism by which activin induces apoptosis in responsive cell types (Figure 50.3C). This pathway may also underlie the induction of p38 map kinase in activin-treated T47D breast cancer cells (Cocolakis et al., 2001), as TGFb-induced p38 map kinase occurs independent of FADD signalling (Schrantz et al., 2001).

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expression of activin, inhibin or follistatin or indeed activin receptor mRNA related to the development of malignancy (see Risbridger et al., 2001). Other aspects of activin action that are relevant to tumour biology are potential stromal–tumour cell interactions (Shoham et al., 2001a), and a role for activin in inhibiting, and follistatin enhancing, angiogenesis through paracrine and autocrine endothelial cell interactions (Kozian et al., 1997; Breit et al., 2000).

ROLES IN THE IMMUNE SYSTEM Activin has been previously isolated as a potent mesoderm-inducing factor in embryonic development (Smith et al., 1990); the complex interplay between activin, follistatin and BMP-like ligands is beyond the scope of this chapter and the reader is referred to several recent reviews (e.g. King and Brown, 1999; McDowell and Gurdon, 1999). Nevertheless, in the following section the concept of activin and its role in the differentiation and commitment of cell phenotype and how this relates to the processes of branching morphogenesis, fibrosis and wound healing is discussed.

Differentiation Cancer Activin and inhibin have recently received attention for putative roles in tumorigenesis. This has been the subject of a recent and comprehensive review (Risbridger et al., 2001), and so only a brief overview of the salient points are given. Evidence from mouse knockout studies indicate that the inhibin a subunit is tumour-suppressive (Matzuk et al., 1992; 1994; Cipriano et al., 2001); where activin A is overexpressed, animals become cachetic, but the development of tumours in these mice is not related to signalling through the ActRIIA receptor (Coerver et al., 1996). This does not rule out, however, activin signalling through ActRIIB to modulate tumour development. As discussed above, in a number of cancer cell lines activin inhibits proliferation, whereas in some ovarian epithelial cancer cell lines, activin stimulates proliferation. Furthermore, numerous studies have documented perturbations in serum levels, tissue

Differentiation is a vital regulatory process in the ontogenesis, continued function and pathology of a number of tissue systems. The role of activin in this process is cell type-specific and also dependent on local concentrations of this factor. For instance, exposure of Xenopus embryos to different activin concentrations leads to different mesoderm lineages (Asashima et al., 2000): at concentrations of 0.3–1 ng ml1 ventral mesoderm such as blood-like cells, coelomic epithelium and mesenchyme are favoured, muscle develops preferentially between 5 and 10 ng ml1, whereas the most dorsal mesoderm, notochord, develops with concentrations of 50–100 ng ml1. Broad agreement with this schema has been found in human embryonic stem cell cultures, where exposure to activin A at 20 ng ml1 preferentially induced muscle at the expense of other mesodermal lineages (Schuldiner et al., 2000). The role of activin in differentiation processes

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needs to be contrasted with its effects on terminally differentiated cells, such as apoptotic effects discussed in the previous section. Furthermore, most of the data discussed below are concerned with effects on cell lineages that derive well after activin’s role as a mesoderm inducer and in body patterning. In general, activin favours differentiation towards more lineage-specific cells and terminal differentiation; examples include astrocytes (Satoh et al., 2000), retina (Belecky-Adams et al., 1999; Davis et al., 2000), pancreatic b-cells (Demeterco et al., 2000), intestinal epithelial cells (Sonoyama et al., 2000), osteoclasts (Fuller et al., 2000; Murase et al., 2001) and macrophage cells (Yamada et al., 1992; Nüsing et al., 1995). In these situations, activin may regulate the withdrawal of cells from further cell cycles to commit to differentiation. Interestingly, in vitro evidence suggests that in some cases this commitment to differentiation may be accompanied by an inhibition of proliferation (Seishima et al., 1999; Davis et al., 2000; Sonoyama et al., 2000; Fukuchi et al., 2001) whereas as in other cell types, no changes in proliferation are observed (Nüsing et al., 1995; Demeterco et al., 2000; Satoh et al., 2000). It should also be noted that at least one case exists where activin functions as a gate to regulate differentiation: while favouring the induction of cardiac myogenesis and differentiation (Johansson and Wiles, 1995; Yatskievych et al., 1997; Ladd et al., 1998; Moore et al., 1998), it appears to inhibit skeletal muscle differentiation by blocking the transformation of myoblasts into myocytes (Link and Nishi, 1997, 1998; Shiozuka et al., 1997). The mechanisms that account for the favouring of particular lineages are largely unexplored, although the recent finding that another TGFb superfamily member, myostatin (Grobet et al., 1997; McPherron et al., 1997; Lee and McPherron, 2001), not only binds to follistatin, but also signals through the activin type II receptor is highly relevant. Myostatin induces hyperplasia of skeletal muscles, suggesting that a complex interplay exists between activin, follistatin, myostatin and perhaps other structurally related family members to regulate myogenic differentiation and homeostasis.

Branching morphogenesis Glandular organs undergo a distinctive budding and branching process involving epithelial–mesenchymal

interactions, known as branching morphogenesis. Activins and follistatin have been implicated in the regulation of branching morphogenesis in all organs examined so far, including kidney, prostate gland, mammary gland, lung, pancreas and salivary gland (Ball and Risbridger, 2001). Specificity in the response is dependent on the localization of particular activin subunits, their actions in regulating duct formation, and receptor populations within different cell types. For instance, in the mammary gland, the activin bB subunit is necessary for normal mammary function; mice lacking the bB subunit gene are unable to support their offspring due to abnormal mammary gland development (Vassalli et al., 1994). A more comprehensive analysis of the phenotype indicated delayed ductal elongation and alveolar development and a lack of differentiated alveolar epithelial cells (Robinson and Hennighausen, 1997). Tissue transplantation studies verified that stromally derived bB subunit was necessary for normal development of the mammary gland, as transplantion of bB null glands into wildtype recipients recapitulated the mutant phenotype, whereas wild-type glands transplanted into knockout females developed normally. While the activin bB subunit appears to favour branching morphogenesis (at least in the mammary gland), the bA subunit inhibits duct formation in mammary gland cells (Liu et al., 1996) and in other branching organs. The prostate gland shows a complex interaction between activin and follistatin in branching morphogenesis, but in this paradigm the activin bA subunit appears to be the dominant activin subunit at work (Cancilla et al., 2001). Activin A strongly inhibits branching and overall glandular size, with particular features including an increase in the size of epithelial tips and the surrounding mesenchymal caps. The activin bA subunit is strongly localized to these sites indicating a profound paracrine action in these cell populations. In contrast, the activin bb subunit is localized in a diffuse pattern within the prostatic mesenchyme and stroma, but is relatively unchanged in those areas of the gland undergoing branching morphogenesis. Consistent with these effects are the observations that antisense deoxyoligonucleotides against Smad 2, 3 and 4 stimulates branching morphogenesis in mouse lung cultures (Zhao et al., 1998). While this model cannot distinguish between activin- versus TGFb-mediated effects,

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these two growth factors are likely to have differing roles in the ductal regulation as suggested by subtle but distinctive branching patterns induced by these ligands (Ritvos et al., 1995; Cancilla et al., 2001). The mechanism by which activin A regulates branching morphogenesis is not well understood, although it clearly involves aspects of the proliferative and differentiative pathways. In the kidney, activin A appears to modulate branching morphogenesis downstream of HGF (Maeshima et al., 2000). Activin alone has no effect on the proliferation or branch formation of aggregates of MDCK cells, which form tubule-like structures in culture. HGF induces significant branching and is able to negatively regulate both activin bA subunit mRNA and activin A protein, but does not appear to influence follistatin mRNA levels. Conversely, activin A is able to block HGF-stimulated tubulogenesis in these kidney cell cultures. A similar point of interaction between activin and HGF appears to exist in the salivary gland (Furue et al., 1999), suggesting that this may be a mechanism common to all organs where mesenchymal–epithelial cell interactions induce branching morphogenesis. The mechanism through which the activin bB subunit enhances duct formation in the mammary gland is not clear.

Fibrosis Fibrosis is another pathogenic process in which activin regulates tissue remodelling, with actions additional and complementary to the extensive role determined for TGFb. Activin bA subunit mRNA and protein has been localized to fibrotic lesions in a number of pathologies such as pulmonary fibrosis (Matsuse et al., 1995; 1996), vascular restenotic (Pawlowski et al., 1997) and arteriosclerotic lesions (Inoue et al., 1994; Engelse et al., 1999), proliferative vitreoretinal diseases (Yamamoto et al., 2000b) and hepatic fibrosis (De Bleser et al., 1997; Sugiyama et al., 1998; Date et al., 2000; Kobayashi et al., 2000). The Smad signalling pathway is known to stimulate profibrotic genes such as type VII collagen (Vindevoghel et al., 1998) and a2 (I) collagen (Dooley et al., 2000; Zhang et al., 2000; Poncelet and Schnaper, 2001; Reisdorf et al., 2001). This mediation functions through Smad3 and not Smad2, consistent with the lack of DNA-binding ability exhibited by Smad2 (Shi et al., 1998; Yagi et al., 1999). While these effects on

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collagen synthesis have been largely attributed to TGFb-stimulated Smad signalling, activin effects on these genes are likely. Evidence for profibrotic actions of activin include mitogenic activities in fibroblasts (Hedger et al., 1989; Sakurai et al., 1994; Ohga et al., 1996; Kotsuji et al., 2000), enhancing collagen gel contraction (Ohga et al., 2000), fibronectin production in hepatic stellate cells (Date et al., 2000) and smooth muscle cell migration (Riedy et al., 1999). Furthermore, activin may act synergistically with TGFb to induce differentiation of fibroblasts into myofibroblasts (Ohga et al., 1996, 2000), and in the stimulation of collagen genes (Sugiyama et al., 1998). Once fully differentiated, myofibroblast-like cells appear to lose their response to further stimulation via the Smad signalling pathway (Dooley et al., 2000, 2001a, 2001b; Reisdorf et al., 2001).

Wound healing Aspects of the activin bA subunit and follistatin knockouts point to a role in epidermis formation and tissue remodelling. The activin bA null animals lack whiskers due to a reduction in the growth and/or differentiation of the whisker follicle (Matzuk et al., 1995b). Follistatin knockout animals have taut, shiny skin resulting from hyperkeratosis and also have defective whisker development (Matzuk et al., 1995c; Jhaveri et al., 1998); furthermore overexpression of follistatin induces a dishevelled coat appearance (Guo et al., 1998). More recent investigations have focused on the wound healing response. Activin bA subunit mRNA is induced rapidly and strongly in the granulation tissue of wounded skin, whereas the bB subunit is induced transiently in the hyperproliferative epithelial layer at the wound edge (Hübner et al., 1996). Activin A appears to be a potent autocrine growth factor for keratinocytes, as shown by hyperkeratosis in the follistatin knockout mouse (Matzuk et al., 1995c), although in vitro evidence examining activin effects on the proliferation of keratinocytes is conflicting (Seishima et al., 1996; 1999; Shimizu et al., 1998). Both activin and TGFb induce the Mad1 transcription factor in keratinocytes (Werner et al., 2001), consistent with a proliferative/differentiative role. Furthermore, when a mouse model was developed where activin bA subunit was overexpressed behind a keratin 14

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promoter, transgenic mice were significantly smaller in size and had smaller ears and tails consistent with activin’s role in epidermis formation and keratinocyte proliferation (Munz et al., 1999). After skin injury, transgenic mice had noticeable increases in the amount of granulation tissue and an enhanced wound healing response. In contrast, overexpression of follistatin behind the same promoter results in animals with larger ears and tails and an irregular coat (Wankell et al., 2001b), the latter a similar phenotype to a previous overexpressor model (Guo et al., 1998). Significantly, follistatin overexpressors had reduced connective tissue and epidermal layers in the skin, and following wound healing, reduced granulation tissue formation and retarded wound healing (Wankell et al., 2001b). More recently, FSRP has also been localized to the dermis and epidermis (Wankell et al., 2001a), but with expression patterns and profiles during wound healing distinct from that of follistatin. The phenotype in the above models where activin– follistatin ligand concentrations are manipulated is apparently contradictory to the finding that Smad3 knockout mice have enhanced wound healing (Ashcroft et al., 1999). This scenario would suggest that activin induces its stimulatory effects on wound healing through Smad2, and the Smad3 knockout phenotype is derived through the loss of TGFbmediated processes. Activin and its role in wound healing of the skin is also relevant to neural models of injury. Activin bA subunit mRNA is up-regulated in response to neuronal injury (Lai et al., 1996, 1997; Tretter et al., 1996). Earlier reports that activin enhanced neuronal survival (Schubert et al., 1990; Krieglstein et al., 1995; Iwahori et al., 1997) have been substantiated in models of acute injury where exogenous activin A administration has been shown to be neuroprotective (Hughes et al., 1999; Wu et al., 1999; Tretter et al., 2000). In a model of kainic acid-induced injury, activation of activin in neuronal populations was induced through bFGF (Tretter et al., 2000), suggesting that activin is a potent neuroprotective factor downstream of the bFGF response. It should also be pointed out that activin may not be protective in all cellular systems, as in a renal model of ischaemia, follistatin was beneficial in reducing ischaemic damage to tubules and reduced apoptosis; conversely activin administration aggravated apoptosis of tubular cells

(Maeshima et al., 2001). The differential response in the kidney compared with skin or brain most probably relates to the apoptotic versus proliferative actions of activin in these respective tissues.

ROLES IN THE IMMUNE SYSTEM Haematopoiesis Linkages between activin and the regulation of immune cells have been inferred for some years, particularly given the early observations that activin was able to induce differentiation of erythroleukemic Friend cells in culture (Eto et al., 1987). A number of follow-up studies expanded on this function by examining the effects of activin on haematopoietic precursor lineages. Activin enhances the formation of both early stage (erythroid burst-forming units, BFU-E) and late stage (erythroid colony-forming units, CFU-E) progenitors, but this action is only operant in the presence of erythropoietin (Yu et al., 1987; Broxmeyer et al., 1988; Mizuguchi et al., 1993; Shiozaki et al., 1998). Furthermore, the effect is largely indirect as depletion of T lymphocytes and/or monocytes from bone marrow or peripheral blood-derived cultures ablates the ability of activin to stimulate differentiation of BFU-E or CFU-E progenitors (Broxmeyer et al., 1988; Yu et al., 1989; Shao et al., 1992b). A role in enhancing multipotential progenitors (granulocyte, erythrocyte, macrophage and megakaryocyte, CFU-GEMM) has also been reported (Broxmeyer et al., 1988), which may also extend down the megakaryocyte lineage with activin dose dependently inducing differentiation of L8057 murine megakaryoblastic leukemia cells (Nishimura et al., 1991). In terms of myeloid lineages, reports in the literature are somewhat conflicting. Activin A has been shown to have little effect on colony or cluster formation of granulocyte–macrophage progenitor cells (CFU-GM) (Broxmeyer et al., 1988; Nakao et al., 1991), whereas in another study activin inhibited the interleukin (IL)-3 responsive proliferation of CFU-GM colonies (Mizuguchi et al., 1993). Furthermore, it has been demonstrated to induce differentiation of the promyelocytic cell line, HL-60, into a monocyte/ macrophage phenotype (Yamada et al., 1992), and to

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induce differentiation characteristics in J774.1 murine macrophage cells (Nüsing et al., 1995). Clearly, further analysis is needed to establish the precise role, if any, of activin in myelopoiesis. A noted function of activin is in the regulation of lymphocyte populations. Activin is strongly inhibitory for both B (Brosh et al., 1995; Sternberg et al., 1995) and T lymphocytes (Hedger et al., 1989; Hedger and Clarke, 1993; Tompkins et al., 1998). Activin A is produced in significant amounts by bone marrow stromal cells (Brosh et al., 1995) and is proposed to act as a local lineage-specific regulator of lymphocytes within this microenvironment (Zipori and BardaSaad, 2001). The localization and status of activin in the thymus to regulate thymocyte differentiation and function has not been studied to date. Is activin a significant regulator of haematopoiesis in vivo ? Administration of recombinant human activin A to mice induced a small but significant increase in haematocrit, with proportional increases in the numbers of red blood cells and haemoglobin (Schwall et al., 1989). In a more comprehensive follow-up study, activin A administration increased the incorporation of iron by erythrocytes, stimulated the release of reticulocytes and enhanced the proportion of bone marrow and splenic CFU-GM, BFU-E and CFU-GEMM progenitors in S phase (Broxmeyer et al., 1991). Furthermore, follistatin administration decreased CFU-E progenitors in bone marrow and spleen and BFU-E progenitors in the spleen (Shiozaki

et al., 1992), lending credence to the concept that activin can be regarded as being a physiologically relevant mediator of haematopoietic lineages. Inhibin has opposite effects to those of activin in these systems (Yu et al., 1987; Broxmeyer et al., 1988), but these are almost certainly mediated through its ability to antagonize activation of the activin signalling pathway. Interestingly, preliminary evidence suggests that FSRP, rather than follistatin, may be the more relevant activin-binding protein in haematopoietic lineages (Maguer-Satta et al., 2001).

Proinflammatory actions In sharing significant signalling componentry with TGFb, it is not surprising that activin has actions that can be classified as being both pro- and antiinflammatory. This duality in phenotype has been extensively discussed for TGFb elsewhere (Letterio and Roberts, 1998), with the consensus being that TGFb is largely anti-inflammatory but with some notable proinflammatory actions. While the body of data on activin is far less than its more well-known cousin, what is emerging are some clear points of difference between the two cytokines (Table 50.3). As discussed earlier, until the fundamental makeup of the activin and TGFb downstream target genes are known, we cannot dissect out the mechanistic divergence in the two proteins and what effects this may have on inflammatory actions.

TABLE 50.3 Actions of activin A, compared with those of TGFb (see Letterio and Roberts, 1998) relating to inflammatory function. These have been loosely grouped into pro- and anti-inflammatory settings Action

TGFb

Activin A

Pro-inflammatory Secreted by monocytes Monocyte chemotaxis Plasminogen activation Cytokine release from monocytes Integrin receptor and collagenase expression Pro-inflammatory cytokines by macrophages Interferon production by T lymphocytes

Yes Enhances Yes Enhances Enhances Reduces Yes

Yesa Enhancesb No(?) Enhancesc Enhancesd Enhancese No effectb

Anti-inflammatory Reactive oxygen and nitrogen intermediates MIP-1a production by macrophages Acute phase protein production

Reduces Reduces Enhances

Enhancese No effectf Reducesg

a Erämaa et al., 1992, Shao et al., 1992a; b Petraglia et al., 1991; c Yamashita et al., 1993; d Ogawa et al., 2000; e Nüsing and Barsig, 1999; f Maltman et al., 1996; g Russell et al., 1999.

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One emerging role for activin is in the recruitment and activation of monocyte/macrophages. As well as responses from extracellular sources, an autostimulatory mechanism is suggested by the rapid synthesis and release of activin A from activated monocytes and macrophages (Erämaa et al., 1992; Shao et al., 1992a; Matsuse et al., 1995; Nishihara et al., 1995; Ogawa et al., 2000). As mentioned above, activin appears to enhance the differentiation of monocyte lineages towards a macrophage-like phenotype (Yamada et al., 1992; Nüsing et al., 1995). Furthermore, activin is chemotactic for peripheral blood-derived monocytes (Petraglia et al., 1991) and stimulates macrophages to produce matrix metalloproteinase-2, which may assist in migration and infiltration through the basement membrane (Ogawa et al., 2000). In terms of monocyte–macrophage-derived mediators, activin stimulation of bone marrow-derived macrophages or peripheral blood monocytes induces a rapid and profound increase in tumour necrosis factor a (TNFa), IL-1b and IL-6 protein production (Yamashita et al., 1993; Nüsing and Barsig, 1999). Other parameters measured from macrophage cultures also indicate that activin induces prostanoid (prostaglandin E2 and thromboxane B2) activation through an increase in cyclo-oxygenase 2 (COX-2) mRNA, and increases inducible nitric oxide synthase mRNA. One report in human monocytic cell lines THP-1 and U-937 suggested that activin may be anti-inflammatory by increasing IL-1 receptor antagonist levels through a posttranscriptional effect and inhibiting the production of mature IL-1b protein (Ohguchi et al., 1998). The veracity of this mechanism and whether it relates to endogenously derived or in situ monocyte/ macrophage populations is an important issue to resolve.

Anti-inflammatory actions While proinflammatory actions centre around the stimulation of monocyte/macrophage function, activin is also known to exhibit some anti-inflammatory properties (see Table 50.3). The well-established property to inhibit proliferation and induce apoptosis in lymphocytic cell types (Hedger et al., 1989; Hedger and Clarke, 1993; Brosh et al., 1995; Sternberg et al., 1995) may relate more to the lineage and cell typespecific regulation of haematopoiesis than a strict

anti-inflammatory action. Certainly in the bone marrow environment, the relatively high concentration of activin A is proposed to favour erythroid precursors at the expense of myeloid lineages (Zipori and Barda-Saad, 2001). Another well-studied effect of activin is to block a number of aspects of IL-6 function. Activin is able to antagonize IL-6 stimulated proliferation of B cells and block IL-6 stimulated acute phase protein production in HepG2 cells (Brosh et al., 1995; Yu et al., 1998; Russell et al., 1999; Hedger et al., 2000). The mechanism by which activin antagonizes IL-6 action is not completely understood, although it is known not to affect IL-6 receptor binding or the formation of Stat dimers in the Jak/Stat signalling cascade (Brosh et al., 1995). A recent examination of the interaction of activin in IL-6 stimulated production of the acute phase protein, haptoglobin, indicates that several of the Smad proteins, specifically Smad 3, 4 and 7 are capable of interacting with C/EPBb and STAT3 complexes, thus affecting the transcriptional activation of the haptoglobin promoter (Zauberman et al., 2001). This is entirely consistent with the previous report that activin either in the presence or absence of IL-6 can affect haptoglobin production (Russell et al., 1999). Of interest will be whether other actions of activin to block IL-6 signalling are also mediated through inhibition of C/EPBb and STAT3 interactions or other mechanisms are at work. Certainly, under some circumstances activin can enhance IL-6 action, such as in the proliferation of rat thymocyte cultures (Hedger et al., 2000). It should also be pointed out that the interaction of activin with IL-6 is likely to extend to other gp130 family members, as suggested by activin antagonism of IL-11-mediated proliferation of B9 cells (Brosh et al., 1995). Other cytokine actions may also be affected by activin, such as IL-1b. A net reduction in IL-1 bioactivity was observed in THP-1 and U-937 monocytic cell lines treated with activin A (Ohguchi et al., 1998). This effect was mediated by an increase in the production in IL-1 receptor antagonist and a decrease in the conversion of pro-IL1b to the mature form. A similar mechanism acting through IL-1 receptor antagonist may also underlie the finding that activin inhibited the IL-1b mediated proliferation of mouse thymocyte cultures (Hedger et al., 2000). Another preliminary report suggests that in prostatic epithelial cells, activin may block the production of the anti-

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inflammatory cytokine, IL-10 (Wang et al., 1999), but further studies are required to elucidate its relevance in other cell systems.

Synthesis and release following inflammatory activation In addition to effects to regulate pro- and antiinflammatory cytokines, activin A synthesis and release is mediated by proinflammatory events. Initial studies focused on monocytes and macrophages and showed that upon activation by agents such as lipopolysaccharide (LPS), synthesis of activin bA subunit and subsequent activin A release occurred (Erämaa et al., 1992; Shao et al., 1992a; Matsuse et al., 1995; Nishihara et al., 1995). Subsequently, other cell types, including bone marrow stromal cells (Shao et al., 1992a; 1998; Yamashita et al., 1992), vascular endothelial cells (Brauman et al., 2000), fibroblasts, keratinocytes (Hübner and Werner, 1996) and placental cells (Keelan et al., 1998; 2000; Mohan et al., 2001) have been shown to exhibit this response. Furthermore, follistatin mRNA and protein are also regulated by inflammatory stimulus under in vitro conditions. This stimulus appears to be cell-specific, as follistatin production is activated by LPS or other cytokines in vascular endothelial cells (Michel et al., 1996; Brauman et al., 2000), but no follistatin response is evident in placental cells subjected to equivalent stimuli (Keelan et al., 2000; Mohan et al., 2001). Confirmation of these in vitro responses has recently been made using various in vivo settings. In a sheep model, follistatin release in the peripheral circulation is elevated from around 6 hours through to 24–48 hours using a variety of inflammatory stimuli such as surgical trauma, injection of live yeast, LPS or IL-1b (Klein et al., 1993; 1996; Phillips et al., 1996; 1998). Following the availability of assays suitable for measuring activin in sheep samples, these findings have been extended to include activin. Blood concentrations of activin A increase rapidly within 1 hour of LPS challenge and follow a similar temporal profile to TNFb (Figure 50.4, Jones et al., 2000). Several points are relevant to this profile: that activin A release is relatively early in the release of inflammatory cytokines following immune activation; that this rapid response suggests release of protein rather than a synthetic mechanism; and that its release precedes that of its

FIGURE 50.4 Cytokine and temperature profiles in adult sheep treated with lipopolysaccharide (LPS). Each bar represents the period over which an elevated response for each parameter is detected (data from Jones et al., 2000). neutralizing protein, follistatin, and therefore it is likely to be bioactive and able to target appropriate cells. Further work is required to establish the cellular sources of this release, but based on the preceding discussion, activated monocytes/macrophages, bone marrow stromal cells and endothelial cells are likely candidates. Additionally, the functional relevance of this release of activin A has yet to be established, and is an important consideration in dissecting out the relative contributions of activin to pro- and antiinflammatory actions.

Relevance to clinical inflammatory syndromes Despite emerging data on activin (and follistatin) and putative roles in immune processes, there are relatively few studies addressing their relevance in clinical studies. Serum follistatin levels are elevated in patients with sepsis but there is no correlation with leukocyte counts or patient outcome (Michel et al., 1998). In contrast, meningitic patients have serum follistatin levels in the normal range but cerebrospinal concentrations are elevated (Michel et al., 2000). Information on activin concentrations in either of these syndromes is lacking apart from a small set of patients who had contracted food poisoning and where activin was in the normal range several days postinfection (Phillips et al., 2001). Informative samples in the early period following infection, where activin A might be expected to be elevated, were not available. Activin A has also been implicated in localized inflammatory syndromes such as inflammatory bowel disease, arthropathies and wound healing. In both ulcerative colitis and Crohn’s disease, activin bA

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subunit mRNA is undetectable in unaffected bowel areas, but shows a graded increase in expression in inflamed tissue that is highly correlated with IL-1b (Hübner et al., 1997). The mRNA is largely localized to the mucosal and submucosal layers, specifically in fibroblasts and infiltrating leukocytes. In inflammatory arthropathies such as gout and rheumatoid arthritis, activin A concentrations in synovial fluid are increased compared with non-inflammatory osteoarthritis (Yu et al., 1998). Immunohistochemical localization indicates preferential expression in the developing pannus (hypertrophic synovial membranes), specifically in fibroblast-like synovial cells and endothelial and smooth muscle cells of infiltrating blood capillaries (Gribi et al., 2001). As discussed previously, elevated expression of activin has been documented in wound healing models. In this paradigm both the activin bA and bB subunits have enhanced expression, but they show differing temporal and expression distributions (Hübner et al., 1996). Further work is required to establish if the action of activin in wound healing is related to enhanced dermal fibrosis or may also involve processes to improve the resolution of the local inflammatory response. Another example where multiple activin subunits are up-regulated is in airway inflammation. Previously, activin A detected by immunohistochemistry and as bioactive protein was demonstrated in alveolar macrophages after treatment of mice with bleomycin (Matsuse et al., 1995). More recently, increased activin receptors, phosphorylation and nuclear translocation of Smad2 and increased expression of all activin mammalian subunits (bA, bB, bC and bE) was observed in airway inflammation (Rosendahl et al., 2001). Interestingly, overexpression of the inhibitory Smad7 in T cells leads to an increase in airway inflammation (Nakao et al., 2000), which should reduce the inputs through both TGFb and activin signalling pathways. Several explanations can be envisioned, including a T cell-specific effect, a reduction in anti-inflammatory action of TGFb that is independent of effects of activin, or that increases in activin subunit expression and receptors are a consequence of, rather than mediating, airway inflammation. While the majority of reports document enhanced expression and/or production of activin in inflammatory syndromes, a recent report has suggested that

activin A expression is reduced in nasal polyp tissue (Shoham et al., 2001b). A characteristic feature of these pathologies is the massive infiltration of a number of haematopoietic cell types including eosinophils, mast cells, neutrophils and lymphocytes (Kramer and Rasp, 1999). A reasonable hypothesis outlined in this study is that the reduced expression of activin is related to the accummulation of lymphocytes, particularly given that activin is apoptotic to these cell types (Hedger et al., 1989; Hedger and Clarke, 1993; Brosh et al., 1995; Sternberg et al., 1995).

SUMMARY AND FUTURE RESEARCH AREAS The key points that need to be addressed in future research into activin and its biology are first, to provide a clinically relevant setting that enhances what is known of the basic biology, and second, to delineate the undoubtedly complex interplay between activin signalling and other intersecting systems. For the former, a number of heritable mutations have been determined that relate to ligands, receptors or signalling molecules belonging to the TGFb superfamily (Massagué et al., 2000). While mutations relating to the activin subfamily are not represented in the current list, the fundamental role of activin in many cell systems suggests that candidate disorders will eventually be elucidated. Furthermore, several studies relevant to clinical medicine, such as the beneficial effects of activin in wound healing (Munz et al., 1999) and neuroprotection (Tretter et al., 2000), are important steps in defining diagnostic and therapeutic opportunities using these molecules. The use of follistatin to block activin action, with potential clinical benefit, has also begun to be explored (Kogure et al., 1995; Maeshima et al., 2001). A conundrum in the biology of the TGFb superfamily is how specificity of action is maintained. The use of different sets of receptors and Smads between the activin/TGFb group and the BMP group allows one to envisage how these two subfamilies can be defined as separate on a functional basis. However, it becomes more perplexing to determine how activin and TGFb, which share the same Smads, could exist as distinct entities instead of just pseudonyms. Some discussion of how this could work has been given in the previous

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sections, but this remains a significant puzzle both on a mechanistic level and also in terms of deriving specific therapeutic strategies based around TGFb and/or activin. In summary, the quote that was selected to accompany this chapter was chosen to highlight how far the activin field has come in recent years; specifically, that we have now moved away from the concept that activin is a one-dimensional reproductive hormone with strictly defined activities. Outlined have been emerging roles in apoptosis and differentiation, tissue remodelling and the immune system. This is not an inclusive list. Why activin has developed into a multifunctional cytokine and growth factor is not yet clear. How this is achieved is beginning to become apparent and clearly relates to the many interchangeable components of the signalling cascade that activin, TGFb and BMP utilize. This has been succinctly put by others (van Grunsven et al., 2001): ‘The ability of Smads to regulate gene expression in a contextdependent manner through interaction with a variety of transcription factors provides the molecular basis for the wide range of biological effects that TGFb ligands can exert in an animal’.

ACKNOWLEDGEMENTS The author thanks Bernadette Scott, Mark Hedger and my students for careful review of the manuscript, and Sabine Werner and Dov Zipori for making pre-proof manuscripts available. The author’s research is supported by a Program Grant from the National Health and Medical Research Council of Australia and by the March of Dimes Birth Defects Foundation.

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51 Bone morphogenetic proteins A. Hari Reddi University of California, Davis School of Medicine, Sacramento, CA, USA

If you are able to state a problem, any problem, then the problem can be solved. Edwin S. Land

INTRODUCTION Among the many tissues in the body, bone has considerable potential for repair and regeneration. In fact, bone grafts with associated hematopoietic cells are routinely used to stimulate bone repair in nonunions of fractures of long bones. What is the molecular basis of the actions of bone grafts? Although demineralized matrix has been known for over a century, a critical discovery is the induction of new bone formation by demineralized, lyophilized segments of bone (Urist, 1965). The induction of new bone by demineralized extracellular matrix is a sequential developmental cascade (Reddi and Huggins, 1972; Reddi and Anderson, 1976; Reddi 1981). The key steps in this developmental cascade are chemotaxis of progenitor stem cells, mitosis of progenitor cells and then differentiation first into cartilage and then replacement of cartilage by bone, a process commonly known as endochondral ossification. Implantation of the demineralized matrix results in interaction and binding of The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

plasma fibronectin to implanted collagenous matrix (Weiss and Reddi, 1980). Peptides of fibronectin are chemotactic for mesenchymal cells. Chemotaxis is the directed migration of cells towards the source of a chemical gradient of chemoattractants. The mesenchymal cells attach to the collagenous matrix and proliferate and the daughter cells emerged as chondroblasts on day 5. With continued progression, chondrocytes were abundant on day 7. The chondrocytes hypertrophy on days 8–9 and the hypertrophic cartilage matrix mineralized on day 9. Concomitant angiogenesis and vascular invasion resulted in new bone formation on days 10–11. The appearance of osteoblasts was marked by increased alkaline phosphatase initially, and later, osteocalcin (also known as bone gla protein). With continued remodeling, hematopoietic marrow differentiation was evident in the ossicle. This entire sequence is a recapitulation of the stages of the embryonic limb bud development and differentiation. (Reddi and Anderson, 1976). Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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The bioassay for bone induction is based on in vivo implantation. The demineralized bone matrix is in the solid state. Dissociative extractants, such as 4 M guanidine hydrochloride or 8 M urea containing 1% NaCl and 1% sodium dodecyl were used to solubilize proteins. The soluble proteins were reconstituted with insoluble collagenous matrix and bioassayed. Neither the extract nor the matrix alone were osteoinductive; however, together they induced new bone formation. Thus there is a collaboration between a soluble signal and the insoluble matrix. This key advance (Sampath and Reddi, 1981, 1983) was an important step in the eventual purification of BMP. The BMPs are dimeric and the disulfide bonds are critical for activity (Wozney et al., 1988; Luyten et al., 1989; Celeste et al., 1990).

BMPS: ISOLATION AND STRUCTURE The fact that demineralized bone matrix devoid of any cells induced de novo cartilage and bone differentiation pointed to the extracellular collagenous matrix and associated proteins as the inducer of bone. Since the extracellular matrix is insoluble, various dissociative extractants were used; 4 M guanidine hydrochloride, 8 M urea containing 1 M sodium chloride and 1% sodium dodecyl sulfate buffered to pH 7.4. Approximately 2 to 3% of the proteins were solubilized and bioassays in vivo revealed that extract alone or matrix alone was devoid of the bone induction activity. However, when the extract was combined with the inactive matrix, the osteoinductivity was restored. This critical reconstitution experiment permitted further purification and characterization of the bone morphogenetic proteins (Sampath and Reddi, 1983; Wozney et al., 1988; Luyten et al., 1989). Further, the reconstitution experiments demonstrated that the soluble extract and insoluble extracellular matrix substratum collaborate to initiate new cartilage and bone formation. Initial rough estimates revealed about 1 lg of purified osteogenic protein was present in 1 kg of bone (Luyten et al., 1989). In view of this, bovine bone was used for further work. However, demineralized bovine or human matrix was not osteoinductive in rats. This is potentially due to either species specifically of bone morphogenetic proteins

or immunogenicity of the matrix. To further delve into this question, the demineralized bovine and human matrices were extracted in 4.0 M guanidine and the extracts were fractionated by molecular sieve chromatography. It is noteworthy that fractions containing proteins less than 50 kDa were osteoinductive in rats when combined with inactive matrix prepared from rats; however, when inactive human or bovine matrix was implanted with active fractions, the induction of bone was blocked. Also, soluble fractions exceeding 100 kDa obtained by chromatography when added to the bioactive fractions, the bone formation was inhibited. Thus, these experiments demonstrated bone morphogenetic signals are not species-specific; however, both the xenogeneic matrix and soluble components over 100 kDa were, perhaps, immunogenic and/or contained inhibitors or binding proteins to BMP (Sampath and Reddi, 1983). Further work with over a ton of bovine bone allowed the complete purification of osteogenin, a bone morphogenetic protein (Luyten et al., 1989). Amino acid sequences of tryptic peptides permitted the eventual cloning and expression of bone morphogenetic proteins (BMPs). The incisive work of Wozney and colleagues (1989) cloned BMP 2, BMP 2B (now called BMP 4) and BMP 3 (osteogenin). Recombinant BMP 2B can be produced by engineering a hybrid BMP 2A/2B molecule (Hammonds et al., 1991). Osteogenic protein-1 (OP-1) was cloned by Ozkaynak et al., (1990, 1992). OP-1 is also called BMP 7 by others. The BMP family is related to TGFb, and related activins and inhibins implicated in mesoderm induction and follicle stimulating hormone release and Mullerian duct inhibitory substance (MIS) involved in degeneration of Mullerian duct male embryos. Each BMP monomer contains seven cysteine residues of which six are involved in intrachain disulfides, with one demonstrated in interchain disulfide. The BMP family members are initially synthesized as a precursor of about 400 amino acids and are processed to a mature molecule of about 110 amino acids (Constam and Robertson, 1999). The BMPs are dimeric molecules with a single critical interchain disulfide bond. Disruption of this bond by reduction in dithiothreitol or mercaptoethanol results in loss of biological activity (Luyten et al., 1989). Each BMP monomer has six cysteine residues involved in three intrachain disulfides. The pro-

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domains of BMPs are processed by furin-like proprotein convertases (Constam and Robertson, 1999). The three-dimensional structure of BMP 7 is now available (Griffith et al., 1996). The alignment of amino acids among the 15 members of the BMP family, BMP 2 to BMP 15, reveals the characteristic motifs. The determination of protein sequence led to the eventual molecular cloning of BMPs. BMP 1 is a metalloprotease related Drosophila Tolloid (Tld). Purified procollagen Cproteinase involved in the cleavage of the carboxyterminal precursor from procollagen, a biosynthetic precursor of collagens identical to BMP-1. Tolloid in Xenopus has been demonstrated to play a role in proteolytic cleavage of a BMP antagonist chordin. Thus, tolloid may play a critical role in activation of inactive BMPs during morphogenesis of diverse tissue during embryogenesis and pattern formation and during regeneration of tissues in adults. The incisive investigation of Wozney and colleagues (1988), cloned BMP 2, BMP 2B (now called BMP 4), and BMP 3 (also called osteogenin). There are over 30 members in the BMP family, although in mammals there appears to be about 15 BMPs (Table 51.1). Ozkaynak and colleagues (1990) cloned osteogenic

protein 1 (also known as BMP 7). The genes for BMPs revealed that they are induced by BMP treatment indicating that there is auto-regulation. Two novel genes related to BMPs were isolated from a cDNA library obtained from calf articular cartilage, and called cartilage-derived morphogenetic proteins 1 and 2 (Chang et al., 1994). These were independently isolated by Lee and colleagues (Storm et al., 1994), and called growth/differentiation factor, GDF 5 and GDF 6. BMP 11 is expressed in developing limb and spinal cord (Gamer et al., 1999). BMP 4 stimulated the expression of TGFb1, indicating that members of the BMPs may cross-regulate each other (Cunningham et al., 1992).

PLEIOTROPY OF BMPs BMPs are pleiotropic morphogens. Pleiotropy is the property of a single gene or protein to act in a multiplicity of ways. A single BMP, such as recombinant human BMP 4 is chemotactic for human monocytes at femtomolar levels (Cunningham et al., 1992), mitogenic to cells and is bone inductive in vivo (Reddi, 1997). BMPs are stimulatory to alkaline

TABLE 51.1 The BMP family in humans and chromosome location BMP subfamily BMP 2/4 BMP 3 OP-1/BMP7

Others CDMP

Generic name BMP-2A BMP-2B Osteogenin Growth/differentiation factor-10 (GDF-10) BMP-5 Vegetal related-1 (Vgr-1) Osteogenic protein-1 (OP-1) Osteogenic protein-2 (OP-2) Osteogenic protein-3 (OP-3) BMP-9 BMP-10 Growth/differentiation factor-11 (GDF-11) Cartilage-derived morphogenetic protein-1 (CDMP-1) Growth/differentiation factor-5 (GDF-5) Cartilage-derived morphogenetic protein-2 (CDMP-2) Growth/differentiation factor-6 (GDF-6) Cartilage-derived morphogenetic protein-3 (CDMP-3) Growth/differentiation factor-7 (GDF-7) BMP-15

BMP designation

Chromosome location

BMP-2 BMP-4 BMP-3 BMP-3B BMP-5 BMP-6 BMP-7 BMP-8 BMP-8B BMP-9 BMP-10 BMP-11

20 14 4 10 6 6 20 — —

BMP-14

20

BMP-13

—

BMP-12 BMP-15

— —

BMP-1 is not a BMP family member with seven canonical cysteines. It is a procollagen-C proteinase related to Drosophila Tolloid and may play a role in modulating BMP actions by proteolysis of BMP antagonists/binding proteins, such as noggin, chordin, and gremlin. THE CYTOKINES AND CHEMOKINES

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phosphatase activity and collagen synthesis in osteoblasts (Vukicevic et al., 1989; T.L. Chen et al., 1991). BMPs 2 and 3 stimulated alkaline phosphatase activity in mouse osteoblast cell line MC3T3-E1 (Vukicevic et al., 1990; Takuwa et al., 1991; Yamaguchi et al., 1991). BMPs stimulate chondrogenic phenotype in vivo (P. Chen et al., 1991). BMPs have been implicated in a wide variety of epithelial–mesenchymal interactions during development and in regeneration (Yamaguchi et al., 1991; Vainio et al., 1993; Nakashima et al., 1994; Vukicevic et al., 1994; ). BMPs are highly conserved in nature from nematodes, such as C. elegans, insects to humans (Padgett et al., 1993; Reddi, 1997). BMPs bind to extracellular matrix components such as collagen I, collagen IV and heparin (Paralkar et al., 1990, 1991).

between BMP receptor activated turning on of genes and their turning off by Smads 6 and 7 by inhibiting type I BMP receptor kinase. BMPs regulate cell cycle progression, mitosis and differentiation of progenitor stem cells in marrow into bone. The ability of BMPs to interact with BMP receptors and activation of BMP-response genes is determined by the bioavailability of BMPs. Recent work has identified BMP-specific antagonists such as noggin, chordin and members of the DAN family such as gremlin. These antagonists have the same affinity to BMPs and the BMP receptors. Thus, these antagonists could be used therapeutically in clinical conditions with pathologically excessive bone formation. It is noteworthy that these antagonists may play a role in fibrous nonunions of fractures. It is also well known local irradiation inhibits BMP-induced bone formation.

BMP RECEPTORS AND SMADs

PATHOPHYSIOLOGY

The biological actions of BMPs are transduced via specific BMP receptors. BMP receptors are of two types, I and II, and are serine/threonine protein kinases. These kinases are enzymes that phosphorylate substrate Smads and activate them. The terms Smad is derived by the fusion of terms Sma, a gene family in the nematode worm Caenorhabditis elegans, and Mad, a gene in fruit fly Drosphila. Mothers against decapentaplegic (Mad) gene was identified as a factor critical in actions of decapentaplegic (dpp) a homologue of human BMP2/4 genes. The Smad genes are conserved from worms, flies to mice and men. The Smads are cytoplasmic substrates for cell membraneassociated BMP type I receptor. BMP type II receptor phosphorylates type I receptor. There are eight different Smads. Smads 1, 5 and 8 are substrates for BMP receptors. Smads 2 and 3 are substrates for TGFb and activin receptors. Phosphorylation of either Smad 1, 5 or 8 activates them to interact with common partner Smad 4 and this heteromeric complex enters the nucleus to activate and turn on BMP-responsive genes (Plate 51.1) (see Plate section). There are two inhibitory Smads 6 and 7 which are normally resident in nucleus which act as a relay to inhibit and turn off BMP receptor I mediated phosphorylation of Smad 1, 5 and 8. Thus there is an intricate homeostatic control

It is now well documented that BMPs play a critical role in embryonic pattern formation and limb morphogenesis (Reddi, 1998a, 1998b). In general, during fracture healing of long bones, BMPs are re-expressed in the callus (Nakase et al., 1994; Bostrom et al., 1996). In certain human diseases, such as fibrodysplasia ossificans progressiva (also known as myositis ossificans progressiva), BMPs have been implicated. In these patients, there is inexplicable formation of new endochondral bone in muscle (Shafritz et al., 1996). It is also possible that heterotopic bone formation, following hip surgery, is also triggered by BMPs. A variety of ectopic ossifications in aorta, kidney and placenta potentially implicate BMPs in diverse tissues. This is not surprising given the fact members of the BMP family play a critical role in cardiac, renal and eye morphogenesis. In addition BMP 4 is expressed in certain osteosarcomas in mice and men (Takaoka et al., 1993).

CLINICAL APPLICATIONS BMPs induce de novo bone formation in heterotopic and orthotopic sites in rats, rabbits, dogs and primates, including baboon (Reddi, 1998b; Riedel and Valentin-Opran, 1999; Cook, 1999). Recombinant

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ACKNOWLEDGMENTS

BMP 2 is effective in the rabbit ulnar non-union model and in sheep (Gerhart et al., 1993; Bostrom et al., 1996). BMPs are local cytokines functioning at the site of implantation, although they may also function in a systemic endocrine manner. Although bone grafts can stimulate new bone formation at the sites of non-union, it is handicapped due to limitation of autograft harvest sites, such as iliac bone and further complications due to donor site morbidity (Younger and Chapman, 1989). Although fractures are treated by both physical and chemical approaches, including electrical fields and ultrasound, the long-term solutions must mimic the biological and mechanical microenvironment of the fracture (McKibbin, 1978; Lanyon et al., 1982; Yasko et al., 1992; Einhorn, 1995; Marsh, 1998). Human recombinant BMP 2 is being developed as a therapeutic product by Genetics Institute, Inc. An absorbable collagen sponge was found to be efficacious in augmentation of maxillary sinus floor and extensive clinical trials in orthopedic trauma are underway (Riedel and Valentin-Opran, 1999). Human osteogenic protein 1 (OP-1), also known as BMP 7 in conjunction with bovine type I insoluble collagen particles prepared from bone (Sampath and Reddi, 1983) is currently in clinical trials in tibial non-unions in both Europe and the United States (Cook, 1999) sponsored by Stryker-Biotech. The imminent completion of the clinical trials may aid the healing of recalcitrant non-unions. However, the progress in this area is limited by the availability of optimal carriers. Collagen appears to be an ideal carrier, although research should continue on synthetic substrates with defined release kinetics (Ma et al., 1990; Hollinger et al., 1996). It is noteworthy that the geometry of the delivery system is critical for bone induction by BMPs (Ripamonti et al., 1992). The accelerating pace of advances in the role of morphogens in the newly emerging science of tissue engineering augers well for clinical applications of BMPs. Tissue engineering is the science of manufacture of spare parts for human body based on morphogenetic cytokines, responding stem cells and extracellular matrix scaffolds (Reddi, 1998b). In fact, a prototype model has established the proof of concept for tissue transformation in vivo with applications in surgery (Khouri et al., 1991). The evolution of tissue engineering strategies comes at a most

opportune time when there is a chronic shortage of donor tissue for transplantation. The advances in BMPs have provided the prototype bone tissue fabricated on the basis of cytokines (Khouri et al., 1991). In conclusion, BMPs are at the center of the problem of the new biology of tissue engineering of other tissues, including heart and kidney.

SUMMARY This chapter describes the role of bone matrix as a repository of growth and morphogenetic factors, including bone morphogenetic proteins. Demineralized bone matrix is the initial starting biomaterial for isolation, purification and molecular cloning and expression of bone morphogenetic proteins (BMPs). There are 15 members of the BMP family in humans and they have wide-ranging actions on tissues beyond bone. The action of BMPs is mediated by receptors for BMPs. The type I and II BMP receptors bind BMPs and the activated receptors phosphorylate intracellular substrates, called Smads. The term Smad is a fusion of two genes Sma in worms and a human homologue of a Drosophila gene Mad (mothers against decapentaplegic). BMP receptors phosphorylate Smads 1, 5 and 8 in cytoplasm. Upon phosphorylation they enter the nucleus in partnership with a common partner Smad 4 and turn on the gene expression. The gene expression cascade mediates BMP responses such as chemotaxis, mitosis and differentiation of cartilage first and replacement by bone. The bioavailability of BMPs for receptor binding is influenced by BMP-binding proteins such as noggin and chordin, and interactions with extracellular matrix components, such as type IV collagen and heparan sulfate. BMPs may have potential clinical utility in fracture healing, spine fusion, reconstructive surgery, oral surgery and plastic surgery.

ACKNOWLEDGMENTS I wish to thank Rita Rowlands for truly outstanding help with the bibliography and preparation of the manuscript. This work is supported by the Lawrence J. Ellison Chair in Musculoskeletal Molecular Biology.

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REFERENCES Bostrom, M., Lane, J.M., Tomin, E. et al. (1996). Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin. Orthop. Rel. Res. 327, 272–282. Celeste, A.J., Iannazzi, J.A., Taylor, R.C. et al. (1990). Identification of transforming growth factor b family members present in bone-inductive protein purified from bovine bone. Proc. Natl Acad. Sci. USA 87, 9843–9847. Chang, S.C., Hoang, B., Thomas, J.T. et al. (1994). Cartilagederived morphogenetic proteins. J. Biol. Chem. 269, 28227–28234. Chen, P., Carrington, J.L., Hammonds, R.G., and Reddi, A.H. (1991). Stimulation of chondrogenesis in limb bud mesodermal cells by recombinant human BMP-2B and modulation by TGF-bl and TGF-b2. Exp. Cell. Res. 195, 509–515. Chen, T.L., Bates, R.L., Dudley, A. and Hammonds, G. (1991). Bone morphogenetic protein-2B stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-b1. J. Bone Min. Res. 6, 1387–1393. Constam, D.B. and Robertson, E.J. (1999). Regulation of bone morphogenetic protein activity by pro domains and pro protein convertases. J. Cell. Biol. 144, 131–149. Cook, S.D. (1999). Preclinical and clinical evaluation of osteogenic protein-1 (BMP 7) in bony sites. Orthopaedics 22, 669–671. Cunningham, N.S., Paralkar, V. and Reddi, A.H. (1992). Osteogenin and recombinant bone morphogenetic protein-2B are chemotactic for human monocytes and stimulate transforming growth factor-B1 mRNA expression. Proc. Natl Acad. Sci. USA 89, 11740–11744. Einhorn, T.A. (1995). Current concepts review enhancement of fracture-healing. J. Bone Joint Surg. 77, 940–952. Gamer, L.W., Wolfman, N.M., Celeste, A.J. et al. (1999). A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in xenopus embryos. Dev. Biol. 208, 222–232. Gerhart, T.N., Kirker-Head, C. and Kriz, M.J. (1993). Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin. Orthop. Rel. Res. 293, 317–326. Griffith, D.L., Keck, P.C., Sampath, T.K. et al. (1996). Threedimensional structure of recombinant human osteogenic protein-1: structural paradigm for the transforming growth factor-B superfamily. Proc. Natl Acad. Sci. USA 93, 878–883. Hammonds, R.G., Schwall, R., Dudley, A. et al. (1991). Boneinducing activity of mature BMP-2B produced from a hybrid BMP-2A/2B precursor. Mol.Endocrinol. 5, 149–155. Heikinheimo, K. (1994). Stage-specific expression of decapentaplegic-Vg-related genes 2, 4, and 6 (bone morphogenetic proteins 2, 4, and 6) during human tooth morphogenesis. J. Dental Res. 73, 590–597. Heldin, C.H., Miyazono, K. and ten Dijke, P. (1997). TGFB signaling from cell membrane to nucleus through Smad proteins. Nature 390, 465–471. Hollinger, J., Mayer, M., Buck, D. et al. (1996). Poly (a-hydroxy acid) carrier for delivering recombinant human bone morphogenetic protein-2 for bone regeneration. J. Control. Rel. 39, 287–304. Hulth, A. (1989). Current concepts in fracture healing. Clin. Orthop. 249, 265–284.

Ilizarov, G.A. (1989). The tension–stress effect on the genesis and growth of tissues. Part 1. The influence of stability of fixation and soft tissue preservation. Clin. Orthop. Rel. Res. 238, 249–261. Jones, C.M., Lyons, K.M. and Hogan, B.L.M. (1991). Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111, 531–542. Khouri, R.K., Koudsi, B. and Reddi, A.H. (1991). Tissue transformation into bone in vivo. JAMA 266, 1953–1955. Lanyon, L.E., Goodship, A.E., Pye, C.J. and MacFie, J.H. (1982). Mechanically adaptive bone remodeling. J. Biomech. 15, 141–154. Luyten, F., Cunningham, N.S., Ma, S. et al. (1989). Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation. J. Biol. Chem. 265, 13377–13380. Luyten, F.P., Yu, Y.M., Yanagishita, M. et al. (1992). Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures. J. Biol. Chem. 267, 3691–3695. Lyons, K.M., Pelton, R.W. and Hogan, B.L.M. (1990). Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109, 833–844. Ma, S., Chen, G. and Reddi, A.H. (1990). Collaboration between collagenous matrix and osteogenin is required for bone induction. Ann. NY Acad. Sci. 580, 524–525. Mabie, P.C., Mehler, M.F. and Kessler, J.A. (1999). Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype. J. Neurosci. 19, 7077–7088. Marsh, D. (1998). Concepts of fracture union, delayed union, and nonunion. Clin. Orthop. Ref. Res. 355S, 822–830. McKibbin, B. (1978). The biology of fracture healing in long bones. J. Bone Joint Surg. 60B, 150–162. Nakase, T., Nomura, S., Yoshikawa, H. and Takaoka, K. (1994). Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J. Bone Min. Res. 9, 651–659. Nakashima, M., Nagasawa, H., Yamada, Y. and Reddi, A.H. (1994). Regulatory role of transforming growth factor-b, BMP-2 and BMP-4 on gene expression of extracellular matrix proteins and differentiation of dental pulp cells. Dev. Biol. 162, 18–28. Ozkaynak, E., Rueger, D.C., Drier, E.A. et al. (1990). OP-1 cDNA encodes an osteogenic protein in the TGF-b family. EMBO J. 9, 2085–2093. Ozkaynak,E.,Schnegelsberg,P.N.J.andOppermann,H.(1991). Murine osteogenic protein (OP-1): high levels of mRNA in kidney.Biochem.Biophys.Res.Commun.179,116–123. Ozkaynak, E., Schnegelsberg, P.N.J., Jin, D.F. and Opperman, H. (1992). Osteogenic protein-2. A new member of the TGF-b superfamily expressed in early embryogenesis. J. Biol Chem. 267, 25220–25227. Padgett, R.W., Wozney, J.M. and Gelbart, W.M. (1993). Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo. Proc. Natl Acad. Sci. USA 90, 2905–2909. Paralkar, V.M., Nandedkar, A.K.N., Pointers, R.H. et al. (1990). Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen. J. Biol. Chem. 265, 17281–17284.

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Paralkar, V.M., Vukicevic, S. and Reddi, A.H. (1991). Transforming growth factor b type 1 binds to collagen IV of basement membrane matrix: implications for development. Dev. Biol. 143, 303–308. Perren, S.M. (1979). Physical and biological aspects of fracture healing with special reference to internal fixation. Clin. Orthop. Rel. Res. 138, 175–195. Reddi, A.H. (1981). Cell biology and biochemistry of endochondral bone development. Collagen Rel. Res. 1, 209–226. Reddi, A.H. (1994). Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage. J. Cell. Biochem. 56, 192–195. Reddi, A.H. (1998a). Initiation of fracture repair by bone morphogenetic proteins. Clin. Orthop. Rel. Res. 355S, 566–572. Reddi, A.H. (1998b). Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat. Biotechnol. 16, 247–252. Reddi, A.H. and Huggins, C.B. (1972). Biochemical sequences in the transformation of normal fibroblasts in adolescent rat. Proc. Natl Acad. Sci. USA 69, 1601–1605. Reddi, A.H. and Anderson, W.A. (1976). Collagenous bone matrix-induced endochondral ossification and hemopoiesis. J. Cell. Biol. 69, 557–572. Riedel, G.E. and Valentin-Opran, A. (1999). Clinical evaluation of rhBMP 2/ACS in orthopaedic trauma: a progress report. Orthopaedics 22, 663–665. Ripamonti, U., Ma, S. and Reddi, A.H. (1992). The critical role of geometry of porus hydroxyapatite delivery system induction of bone by osteogenin, a bone morphogenetic protein. Matrix 12, 202–212. Sampath, T.K. and Reddi, A.H. (1981). Dissociative extraction and reconstitution of bone matrix components involved in local bone differentiation. Proc. Natl Acad. Sci. USA 78, 7599–7603. Sampath, T.K. and Reddi, A.H. (1983). Homology of bone inductive proteins from human, monkey, bovine, and rat extracellular matrix. Proc. Natl Acad. Sci. USA 80, 6591–6595. Shafvitz, A.B., Shore, E.M., Gannon, F.H. et al. (1996). Overexpression of an osteogenic morphogen in fibrodysphoia ossificans progressiva. New Engl. J. Med. 325, 555–559. Storm, E.E., Huynh, T.V., Copeland, N.G. et al. (1994). Limb alterations in brachypodism mice due to mutations in a new member of TGF-B superfamily. Nature 368, 639–642. Takaoka, K., Yoshikawa, H., Hashimoto, J. et al. (1993). Gene cloning and expression of a bone morphogenetic protein

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52 Autoimmunity and cytokines: pathogenesis and therapy Evangelos Th. Andreakos and Marc Feldmann Imperial College of Science, Technology and Medicine London, UK

Do not blame anybody for your mistakes and failures. Bernard M. Baruch

INTRODUCTION Although most self-reactive lymphocytes are eliminated by clonal deletion during their development in the thymus or in the periphery, some persist and can be detected in normal healthy individuals by various assays. Mechanisms of clonal anergy or clonal suppression operate to ensure that these cells do not usually attack self-tissues. However, in a number of common human diseases these mechanisms have been perturbed, resulting in the activation of selfreactive T and B cells and the generation of cellmediated and humoral immune responses against self-antigens. The response of the immune system against self-tissue is termed ‘autoimmunity’ and, when sustained, results in serious damage of organs that can sometimes be fatal. Autoimmune diseases affect 5–7% of the population, and the most common of them include rheumatoid arthritis (RA), systemic lupus erythematosus, Sjogren’s syndrome,

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

insulin-dependent diabetes mellitus, and Graves’ and Hashimoto’s thyroiditis. It is not known what combination of events triggers autoimmunity, but there is strong evidence for both genetic and environmental factors. Studies in autoimmune individuals and monozygotic twins have linked the susceptibility to autoimmune disease with the MHC genotype and particularly with the HLA class II antigens (Table 52.1). Thus, more than 80% of RA patients have the HLA-DR4 or HLA-DR1 genotype (Wordsworth and Bell, 1991) and more than 90% of insulin-dependent diabetics the HLA-DR3 or HLADR4 genotype (Bell et al., 1987). Other diseases such as ankylosing spondylitis are associated with HLA class I antigens (Nasution et al., 1997). The classical role of MHC molecules is in peptide binding and presentation to T cells (Townsend et al., 1989). Thus, MHC molecules may affect disease susceptibility by first ‘shaping’ the repertoire of naive T cells in the thymus, and then presenting antigenic

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TABLE 52.1 Susceptibility to autoimmunity is associated with the HLA genotype and the sex (adapted with permission from Janeway and Travers, 1997) Disease

Prevalent HLA alleles

Relative risk

Sex ratio (female:male)

Ankylosing spondylitis Acute anterior uveitis Goodpasture’s syndrome Graves’ disease Hashimoto’s thyroiditis Insulin-dependent diabetes mellitus Juvenile rheumatoid arthritis Multiple sclerosis Myasthenia gravis Pemphigus vulgaris Pernicious anemia Psoriatic arthritis Reiter’s syndrome Rheumatoid arthritis Sjogren’s syndrome Systemic lupus erythematosus Ulcerative colitis

B27 B27 DR2 DR3 DR5 DR4/DR3 and DR3/DQW8 B27/DR5 DR2 DR3 DR4 DR5 B7, B27 B27 Dw4/DR4 Dw3 DR3 B5

87.4 10.0 15.9 3.7 3.2 3.2 4 4.8 2.5 14.4 5.0 11.0 37.0 4.2 6 5.8 4.0

0.3 0.5 ~1 4–5 4–5 ~1 2 3 3 ~1 — 1 — 3 19 9 ~1

determinants of proteins to the same T cells in the periphery to initiate antigen-specific immune responses. Association of disease susceptibility with MHC loci, however, is not necessarily due to inappropriate antigen presentation. In addition to MHC molecules, MHC loci encode a wide variety of other proteins such as peptide transporters (Bahram et al., 1991), proteasome enzymes (Kelly A. et al., 1991), hsp70 and tumor necrosis factors (LT-a, LT-b. TNF-a). These proteins are also involved in the initiation of antigen-specific immune responses and can influence disease susceptibility in an MHC moleculeindependent fashion. It should be stressed, however that MHC genotypes alone do not determine whether individuals will develop autoimmunity, as identical twins that share all their genes are more likely to develop disease than MHC-identical siblings. Genetic factors other than MHC are also essential for susceptibility as, for example, systemic lupus erythematosus that is strongly associated with inherited homozygous deficiency of the early proteins (C1, C4 or C2) of the classical pathway of complement (Kolble and Reid, 1993). Moreover, the hormonal status of the individual does also affect disease susceptibility as many autoimmune diseases show a strong sex bias (Figure 52.1). More than 70% of the patients of Sjogren’s syndrome, SLE, autoimmune thyroid disease, rheumatoid arthritis, multiple sclerosis and myasthenia gravis are female,

implying that sex hormones are involved in disease pathogenesis (Whitacre, 2001). Sex hormones such as estrogen, progesterone and androgens are known to act directly to cells of the immune system, among their multiple functions, modulating antigen presentation, lymphocyte activation, cytokine production and/or cell homing. How exactly, however, these hormones influence the prevalence of autoimmune disease remains unclear. Females do develop stronger immune responses (Verthelyi, 2001) and so autoimmunity may be the price. Finally, the observation that autoimmune disease manifests in less than 50% of identical twins that share all their genes (e.g. the concordance rates of identical twins in rheumatoid arthritis is 15–35%) demonstrates that non-inherited factors are clearly of importance (Silman et al., 1993). Whether these are inherited agents or random events such as exposure to particular environmental conditions or pathogens is not known.

AUTOIMMUNE DISEASES CAUSE TISSUE DAMAGE INITIATED BY ANTIBODY OR T CELLS By definition, autoimmune diseases are due to sustained adaptive immune responses to self-antigens. As antigen is an intrinsic component of self-tissue,

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AUTOIMMUNE DISEASES CAUSE TISSUE DAMAGE INITIATED BY ANTIBODY OR T CELLS

Genetic predisposition Hormonal status Environmental factors

Initiation

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Antigen presentation T cell activation

CYTOKINES

Perpetuation

Antigen-presenting cells

Autoantigen reactive T cells

CYTOKINES

Disease EFFECTOR MECHANISMS

FIGURE 52.1 The development of autoimmunity consists of three stages, the initiation, the perpetuation and the disease stage.

autoimmunity results in extensive tissue damage. The nature of tissue damage, the pathology and the clinical expression of the disease depend on the antigen and tissue against which autoimmune responses are directed. Both antibody and cell-mediated immune responses can be involved. In autoimmune hemolytic anemia and autoimmune thrombocytopenic purpura, for example, the mechanisms of tissue damage are relatively understood and involve antibodies against autoantigen that induce cell destruction by direct lysis or most probably by accelerated clearance of autoantibodysensitized cells (Schreiber and Frank, 1972). Antibody interactions with complement and in particular with Fc receptors are well known to induce tissue damage (Ravetch and Clynes, 1998). In Hashimoto’s thyroiditis or systemic lupus erythematosus (SLE) antibodies initiate a powerful inflammatory response that results in antibody-dependent and direct T celldependent cytotoxicity (Podleski, 1972; Calder et al., 1973). Powerful inflammatory responses are also observed in Goodpasture’s syndrome where autoantibodies recognize extracellular antigens. Further, autoantibodies can cause disease when directed against cell-surface receptors by interfering with their function. Thus, in Graves’ disease autoantibodies to the thyroid-stimulating hormone receptor stimulate the production of excessive thyroid

hormone (Drexhage et al., 1981), and in myasthenia gravis autoantibodies to the a chain of the nicotinic acetylcholine receptor block neuromuscular transmission (Almon et al., 1974; Lennon and Lambert, 1980). Sustained autoantibody responses require the activation of autoantigen-specific CD4 T cells, cells that provide help to autoantigen-specific B cells. In addition, T cells can mediate tissue injury by themselves or by activating macrophages and inducing local inflammation. Such mechanisms seem to operate in rheumatoid arthritis and multiple sclerosis in which the disease tissues are heavily infiltrated by T lymphocytes and activated macrophages. Rheumatoid arthritis, in particular, seems to be initiated by CD4 T cells specific for antigens expressed in the joint. T cells are also involved in the maintenance of local inflammation. Accumulation of polymorphonuclear leukocytes and macrophages may ultimately destroy the joint. During that process autoantibodies are produced and include the rheumatoid factor, an IgM anti-IgG antibody that may up-regulate cytokine production and account for part of the tissue damage observed. CD8 T cells, on the other hand, are more important in other autoimmune diseases, such as insulindependent diabetes mellitus, where CD8 T cells attack and destroy the insulin-producing b cells of the pancreas.

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MODELS OF AUTOIMMUNITY A number of potential etiological mechanisms have been proposed to explain the induction of autoimmunity and it is possible that multiple mechanisms are involved, in different individuals. First, the ‘release of anatomically sequestered antigens’ model proposes that autoimmunity occurs as a result of the exposure of mature T cells to antigens that are sequestered from the circulation and are not thus seen by developing T cells in the thymus. Such antigens may be confined behind anatomical and immunosuppressive barriers such as the cornea, brain or testes, and include myelin basic protein (MBP) and thyroid peroxidase (TPO), two major autoantigens found in multiple sclerosis and thyroid autoimmunity respectively. Support for this theory comes from the clinical experience of the induction of autoimmune uveitis after eye injury (Gery and Streilein, 1994) or orchitis after vasectomy (Shahani and Hattikudur, 1981). The relevance of this concept, however, is unclear as there is increasing evidence that even ‘hidden’ antigens may at times be expressed in the thymus (Wekerle et al., 1996; Hanahan, 1998). The ‘cryptic self’ hypothesis is another model that has been proposed. It is based on the concept that T cell tolerance is generated against self-epitopes that are presented in the context of MHC in the thymus. However, self-reactive T cells may escape negative selection because they recognize cryptic epitopes of autoantigens (defined as epitopes not normally expressed when antigen is processed) rather than anatomically sequestered autoantigens. Cryptic epitopes may be due to ineffective antigen processing, dominance of a flanking epitope that competes for binding to the same MHC molecule, or aberrant expression of developmental or differentiation antigens. This model is comparable with the ‘Bottazzo–Feldmann hypothesis’ that autoimmunity occurs when MHC class II expression and antigenpresenting cell function is up-regulated and induced in cells not normally expressing class II molecules. Inducing factors may involve cytokines such as IFN released during local infection or trauma (Bottazzo et al., 1983) and may result in the presentation of previously sequestered antigens or cryptic epitopes. Support for this model comes from findings that local

IFN production in b islets is able to induce diabetes (Sarvetnick et al., 1990). Some thyroid-infiltrating TPO-specific T cell clones recognize cryptic epitopes which are presented by thyroid epithelial cells synthesizing TPO, but not by antigen-presenting cells taking up TPO as an exogenous antigen (Dayan et al., 1991; Quarantino et al., 1996). The relevance of this concept to other autoimmune diseases is not yet documented. A popular theory to explain autoimmunity is based on ‘molecular mimicry’. A number of viruses and bacteria possess antigenic determinants that are identical or homologous, closely related to normal host cell components. During an infection, this may lead to cross-reactivity between immune responses to selfand foreign antigens, and activation and clonal expansion of autoreactive cells in the periphery (Oldstone, 1989). Many autoimmune diseases have been associated with the infection of particular pathogens, including ankylosing spondylitis with mycobaterium Klebsiella (Fielder et al., 1995), Chagas disease with Trypanosoma cruzi (Van Voorhis and Eisen, 1989) and insulin-dependent diabetes mellitus with cocksackie virus (Ray et al., 1980; Baum et al., 1996). In addition, many proteins found in bacterial and parasitic pathogens have been shown to crossreact with mammalian proteins, such as the microbial heat-shock protein hsp65. Both antibody and T cell responses against hsp65 have been detected in rheumatoid arthritis (Thompson et al., 1991; De Graeff-Meeder et al., 1991) and insulin-dependent diabetes mellitus patients (Tun et al., 1994), and antihsp65 antibodies have been demonstrated to recognize glutamic acid decarboxylase, a pancreatic enzyme localized in the insulin-producing beta cells of the islets of Langerhans (Jones et al., 1993). However, definitive evidence of pathogenic molecular mimicry is lacking and bystander T cell activation that occurs during infection may actually be as important as molecular mimicry in inducing autoimmunity (Bottazzo et al., 1983; Horwitz et al., 1998; Benoist and Mathis, 2001). Numerous other theories and factors have also been proposed to help explain autoimmunity and include defective apoptosis (Vaux and Flavell, 2000; Eguchi, 2001; Beutler, 2001), TH1/TH2 cytokine imbalance (O’Garra et al., 1997; Singh et al., 1999) or neuroendocrine hormones (Cutolo and Wilder, 2000) as potential etiological factors. Despite that, no mecha-

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THE CYTOKINE SYSTEM IN RA

nism has been consistently shown to account for autoimmune disease. It may be that autoimmunity requires a combination of them rather than a single mechanism to occur.

STAGES OF AUTOIMMUNITY Autoimmunity is envisaged to contain three stages of the disease process (Figure 52.1). The first stage is ‘initiation’, during which autoimmunity is induced in genetically predisposed individuals and under the influence of specific presumably environmental factors. This stage is believed to involve a local inflammatory response with the release of cytokines and the activation of T cells, and is difficult to study as it is largely asymptomatic and usually goes unreported. Evidence for this is indirect and comes mainly from cancer patients where administration of IL-2 and IFN-a often induces autoimmunity, especially of the thyroid (Burman et al., 1986; Atkins et al., 1988). Although autoimmunity resolves after cessation of the cytokine therapy, it can be severe and occurs with the presence of autoantibodies and the association with the disease susceptibility MHC genotype. The second stage of autoimmunity is ‘perpetuation’ and involves the continual interaction between tissue antigen-presenting cells and autoantigen-reactive T cells. This results in the persistence of autoreactive lymphocytes and the maintenance of inflammation. In thyroid disease, for example, there is evidence that thyroid epithelial cells can present antigen and thyrocyte-specific T cells can be isolated from the thyroid infiltrate (Londei et al., 1984, 1985; Dayan et al., 1991). In addition to the initiation and perpetuation stages of autoimmunity, there is the late or tissue-damaging stage of autoimmunity. During this stage, cytokines and other inflammatory mediators released by both lymphocytes and antigen-presenting cells induce the production of autoantibodies, the formation of immune complexes, the activation of complement and the extravasation and activation of cytotoxic T cells, natural killer cells, macrophages and polymorphonuclear leukocytes. This leads to extensive damage of the tissues, ultimately resulting in disability or even death.

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THE CYTOKINE SYSTEM IN RA The late or tissue-damaging stage of autoimmunity is the most relevant therapeutically and the most accessible for study. It is usually at this stage that patients seek medical attention and that disease is detected or reported. As in all stages of autoimmunity, this stage involves the production of cytokines that specifically regulate the inflammatory response and the tissue damage and repair mechanisms. Realizing the importance of cytokines as mediators of inflammation and immunity (reviewed in Oppenheim and Feldmann, 2001), we have undertaken for the last 16 years the task of studying the role of cytokines in rheumatoid arthritis. RA offers some major advantages for this type of study over other autoimmune diseases, such as multiple sclerosis and insulin-dependent diabetes mellitus, because biopsy tissue from the disease site at the height of the inflammatory process can be obtained (reviewed by Feldmann et al., 1996b). A number of methods have been used to analyse synovial cytokine expression which include immunohistology of fresh ex vivo tissue (Chu et al., 1991a), in situ hybridization of synovial fluid products (Wood et al., 1992), and short-term culture of synovial membrane cells in the absence of extrinsic stimulation (Brennan et al., 1989a). Immunohistology is particularly useful when determining which cells are the major cytokine producers, whereas short-term culture of synovial membrane cells permits the quantitative analysis of proteins released, and their regulation, and is the one we used most extensively. Soon, our studies and those of other groups in the field made us realize that the synovium of RA patients is rich in the expression of cytokines; virtually every cytokine known can be detected as summarized in Table 52.2. The abundance of most known cytokines in the rheumatoid synovium was not surprising as this site is inflamed and contains numerous activated T cells, macrophages, endothelium, fibroblasts and plasma cells, all of which are able to produce cytokines. The plethora of cytokines expressed highlighted the complexity of determining which cytokines might be important in the pathogenesis of the disease. The quantity of the cytokines detected is not necessarily a reflection of their importance, as different cytokines exert distinct functions and have various properties.

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TABLE 52.2 Cytokines expressed in rheumatoid synovial tissue (adapted with permission from Feldmann et al. 1998) Cytokines

Expression mRNA protein

Reference

Pro-Inflammatory IL-1a,b





TNFa





LT IL-6

 

 

GM-CSF





M-CSF LIF

 

 

Oncostatin M IL-2 IL-3 IL-7 IL-9 IL-12 IL-15 IFNa/b IFN IL-17 IL-18 Immunoregulatory IL-4 IL-10

   ? ?      

   ? ?      

Brennan et al. (1989b); Buchan et al. (1988a); Chomarat et al. 1995; Deleuran et al. (1992); Firestein et al. (1990) Brennan et al. (1989b); Chu et al. (1991a); Di Giovine et al. (1988) Brennan et al. (1990b); Saxne et al. (1988) Field et al. (1991); Firestein et al. (1990); Helle et al. (1991); Hirano et al. (1988); Houssiau et al. (1988) Alvaro-Gracia et al. (1989); Haworth et al. (1991); Xu et al. (1989) Firestein et al. (1990) Lotz et al. (1992); Okamoto et al. (1997); Waring et al. (1993) Okamoto et al. (1997) Buchan et al. (1988b); Firestein et al. (1990) Firestein et al. (1990) — — Morita et al. (1998) McInnes et al. (1996) Hopkins and Meager (1988) Buchan et al. (1988b); Firestein et al. (1990) Chabaud et al. (1999); Kirkham et al. (1997) Yamamura et al. (1997)

 

 

IL-11





IL-13 TGFb

 

 

Chemokines IL-8





Groa MIP-1 MCP-1

  

  

ENA-78 RANTES Growth Factors FGF

 

 





PDGF





VEGF





Miossec et al. (1990) Cohen et al. (1995); Cush et al. (1995); Katsikis et al. (1994); Llorente et al. (1994) Hermann et al. (1998); Okamoto et al. (1997); Taki et al. (1998) Isomaki et al. (1996) Brennan et al. (1990a); Bucala et al. (1991); Chu et al. (1991b); Fava et al. (1991); Goddard et al. (1992); Lafyatis et al. (1989); Lotz et al. (1990); Miossec et al. (1990) Brennan et al. (1990b); Hosaka et al. (1994); Koch et al. (1991) Hosaka et al. (1994) Hosaka et al. (1994); Koch et al. (1994b) Akahoshi et al. (1993); Hachicha et al. (1993); Hosaka et al. (1994); Koch et al. (1992); Villiger et al. (1992) Koch et al. (1994a) Rathanaswami et al. (1993) Bucala et al. (1991); Goddard et al. (1990); Goddard et al. (1992); Sano et al. (1990); Sano et al. (1993) Remmers et al. (1990); Remmers et al. (1991); Sano et al. (1993) Fava (1994); Koch et al. (1994c)

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THE CYTOKINE SYSTEM IN RA

The fact, however, that most cytokines are expressed transiently and can be induced or inhibited by other cytokines suggested that a ‘cytokine network’ may exist in which cytokines regulate each other (Vilcek, 1997; Feldmann et al., 1996a). In the rheumatoid synovium, this network can be envisaged as a balance of pro-inflammatory (TNFa, IL-1, IL-6) and antiinflammatory (IL-10, IL-11) cytokines shifted towards the pro-inflammatory side (Figure 52.2). To understand the regulation of cytokine production in the rheumatoid joint, we decided to use shortterm cultures of rheumatoid synovial membranes and study the effects of neutralizing antibodies against specific cytokines. Short term (5–6-day) cultures of all the cells extracted from rheumatoid synovial membranes provided a good model of certain aspects of the disease, as they contain 30% T cells, 30–40% macrophages, and fewer endothelial cells, fibroblasts, dendritic cells, plasma cells and B lymphocytes. These cells reaggregate in vitro, and spontaneously release the inflammatory mediators which are also produced in vivo (reviewed in Feldmann et al., 1996b). This type of culture is much more representative of the synovium than the passaged ‘synoviocyte’ which selects for the fibroblast-like cells that probably represent 10% of the total synovium. Our work with synovial cultures yielded some very interesting observations. First, addition of a neutralizing anti-TNFa antibody, but not the closely related anti-LTa antibody reproducibly inhibited the production of IL-1 in these cultures (Brennan et al., 1989b). IL-1 was used as a readout because it had been previously shown to be important in cartilage and bone destruction (Gowen et al., 1983; Saklatvala et al., 1985). This result was unexpected, because many other signals present in

the synovium, such as IL-1 itself, GM-CSF, IFN or immune complexes were known to induce IL-1 in other systems. Second, addition of the anti-TNFa antibody also inhibited the production of other proinflammatory mediators too, such as GM-CSF, IL-6 and IL-8 (Haworth et al., 1991; Butler et al., 1995). These observations were consistent with those of others (Alvaro-Gracia et al., 1991) and indicated that many of the major pro-inflammatory cytokines produced in the rheumatoid synovium are linked in a network or cascade with TNFa at its apex (Figure 52.3), and hence suggested that inhibition of TNFa could be a good therapeutic approach. Similar studies by us and others in murine models of arthritis such as collagen-induced arthritis have also confirmed the importance of TNFa in the induction and maintenance of disease (Thorbecke et al., 1992; Williams et al., 1992; Piguet et al., 1992; Wooley et al., 1993). Interestingly, a ‘spontaneous’ model of arthritis was developed using a disregulated TNFa transgene carrying a deletion in the 3 untranslated region of the TNFa gene that is essential for its normal regulation (Keffer et al., 1991). The rheumatoid synovium was also found to have up-regulated a number of anti-inflammatory mediators that include IL-10 and IL-11. This prompted us to investigate their role in the regulation of proinflammatory cytokine production in rheumatoid synovial membrane cells. We found that neutralization of IL-10 by anti-IL-10 antibodies increased the spontaneous release of both TNFa and IL-1, as well as IFN, a cytokine barely detectable otherwise (Katsikis et al., 1994). In agreement with that, we found that addition of recombinant IL-10 to monocyte cultures down-regulated TNFa and IL-1 activity by inhibiting Anti-inflammatory

IL-11 IL-1 TNFa

GM-CSF

IL-10, IL-1ra, sTRF-R

IL-10 sTNF-R

IL-1ra

IL-6

Immune system

TNFa

IL-1

IL-6, IL-8, GM-CSF Pro-inflammatory

Pro-inflammatory

Anti-inflammatory

FIGURE 52.2 Cytokine disequilibrium in rheumatoid arthritis (modified with permission from Feldmann et al., 1996a).

FIGURE 52.3 Cytokine cascade in rheumatoid arthritis (taken with permission from Feldmann et al., 1996a).

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their expression and inducing the production of soluble TNF receptors and IL-1 receptor antagonist (de Waal Malefyt et al., 1991; Joyce et al., 1994). Similarly, neutralization of IL-11 by anti-IL-11 antibodies increased the production of TNFa in rheumatoid synovial membrane cultures, whereas addition of recombinant IL-11 inhibited the production of TNFa and matrix mettaloproteinases 1 and 3 (Hermann et al., 1998). In summary, IL-10 and IL-11 seem to negatively regulate pro-inflammatory cytokine production in the rheumatoid synovium. Other potentially inhibitory cytokines, such as IL-4 and IL-13, are poorly expressed. Another group of anti-inflammatory mediators are soluble TNF and IL-1 receptors, and the IL-1 receptor antagonist that specifically inhibit the function of TNFa and IL-1 respectively. The soluble p55 and p75 TNF receptors are elevated in RA plasma and synovial fluid, and are also spontaneously released in rheumatoid synovial membrane cultures (Cope et al., 1992). Neutralization of the soluble TNF receptors with monoclonal antibodies increases the bioactivity of TNFa. At the same time, the soluble IL-1 receptor and the IL-1 receptor antagonist are also increased in RA synovial fluid and synovial membrane cells (RouxLombard et al., 1992; Firestein et al., 1992; Deleuran et al., 1992). However, despite the production of natural TNF and IL-1 inhibitors, TNFa and IL-1 bioactivity and signaling (such as NF-B activation) is still detectable in rheumatoid synovial membrane cultures and in synovium by immunohistology (Marok et al., 1996; Sioud et al., 1998), suggesting that these mechanisms are insufficient in regulating the disease process. This supports the concept that rheumatoid arthritis is an imbalance between the production of pro-inflammatory and anti-inflammatory mediators (Figure 52.2).

THE CYTOKINE SYSTEM IN OTHER AUTOIMMUNE DISEASES The role of cytokines has also been studied in a number of other autoimmune diseases and has been recently reviewed in a book by Brennan and Feldmann (1996). In Crohn’s disease, cytokine production has been investigated by using immunohisto-

chemistry, whole biopsy cultures, and isolated lamina propria mononuclear cells (LPMC), and a large number of pro-inflammatory cytokines (TNFa, IL-1, IL-6) and chemokines (IL-8, MCP-1 and RANTES) were found to be elevated (Fiocchi, 1998). As in rheumatoid arthritis, soluble TNF receptors and the IL-1 receptor antagonist, the natural inhibitors of TNFa and IL-1, were also found to be increased (Noguchi et al., 1998) as was the immunoregulatory cytokine IL-10 (Autschbach et al., 1998). Addition of exogenous IL-10 has been shown to suppress the elevated proinflammatory cytokine production from Crohn’s disease LPMC when delivered in the in vitro cultures or when administered rectally to patients with active disease (Schreiber et al., 1995). T cell-derived cytokines can also be detected in Crohn’s disease tissue and are predominantly of a TH1 profile with elevated levels of IL-2 and IFNc (Mullin et al., 1992). Increased expression of IL-12 has also been demonstrated in LPMC from Crohn’s disease mucosa (Monteleone et al., 1997). Although, however, Crohn’s disease has many analogies to rheumatoid arthritis, the pro-inflammatory cascade with TNFa at its apex has not been described, although it is likely to operate in vivo as antiTNFa clinical trials have shown marked benefit (van Dullemen et al., 1995; Rutgeerts et al., 1999). The cytokine networks in other autoimmune diseases are less clearly defined. In Graves’ disease and Hashimoto’s thyroiditis, a large number of cytokines have been detected and include pro-inflammatory (TNFa, LT, IL-1, IL-6) and immunoregulatory cytokines (IL-10), chemokines (IL-8) and T cell-derived cytokines (IL-2, IL-4, IFN) (reviewed by GrubeckLoebenstein, 1996). Although thyroid epithelial cells can produce cytokines, most of the cytokines detected are due to the intrathyroidal lymphocytic infiltrate that is seen in thyroid diseases (GrubeckLoebenstein et al., 1989; Weetman and McGregor, 1994). In Sjögren’s syndrome salivary gland biopsies TNFa, LT, IL-1, IL-2, IL-4, IL-6, IL-10, TGFb and IFN can be detected by polymerase chain reaction and in situ hybridization (reviewed by Skopouli and Moutsopoulos, 1996). Similarly, elevated levels of TNFa, IL-1, IL-2, IL-6, IL-10, IFN and soluble TNF receptors levels are increased in sera from patients with systemic lupus erythematosus (reviewed by Smolen et al., 1996) as are TNFa, LT, IL-1, IL-2 and

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

ANTI - TNF a THERAPY IN RHEUMATOID ARTHRITIS

IFN in multiple sclerosis lesions from patients with active disease (reviewed by Baker et al., 1996). In summary, it is becoming increasingly clear that cytokines are essential for the development, maintenance and pathology of autoimmunity. More detailed reviews of cytokines in autoimmunity can be found in two recent books, Cytokines in Autoimmunity edited by Brennan and Feldmann (1996) and Cytokine Reference (also on the web) edited by Oppenheim and Feldmann (2001).

ANTI-TNFa THERAPY IN RHEUMATOID ARTHRITIS Based on various findings including the observation that TNFa is at the apex of the pro-inflammatory cascade in rheumatoid arthritis (Feldmann and Maini, 2001), clinical trials aimed at blocking TNFa activity with infliximab, a neutralizing anti-TNFa antibody, were initiated in 1992. Infliximab (formerly known as cA2) is a chimeric mouse Fv, human IgG1 monoclonal antibody of high affinity and neutralizing capacity (Knight et al., 1993). The first phase I/II study was an open (non-placebo controlled) trial of infliximab in long-standing active RA patients who failed all prior therapy averaging four disease-modifying drugs. The results were very encouraging with patients reporting alleviation of symptoms such as pain, morning stiffness, tiredness and reduction of swollen and tender

joints within a week or two (Figure 52.4). Inflammation markers, such as C-reactive protein (CRP), were also reduced. This response, however, was temporary and lasted 8–22 weeks. Readministration of infliximab induced further benefit (Elliott et al., 1993, 1994a). A double-blind, randomized, placebo-controlled clinical trial then followed and finally established the efficacy of infliximab in controlling the signs and symptoms of RA. A 60–70% reduction in the measures of disease activity such as swollen or tender joint counts and CRP was observed (Elliott et al., 1994b). Although infliximab was effective as a monotherapy in 60–70% of the patients, it was important to determine its effect with established therapies and to evaluate if combined anti-TNFa and anti-T cell therapy may provide additional therapy as it does in mouse models (Williams et al., 1999). Methotrexate (MTX) is one of the most effective and durable therapies for RA with effects on T cells too, so a phase II clinical trial was done to examine the effect of infliximab in patients with active disease despite therapy with MTX. Results from this study demonstrated a synergy between infliximab at low doses and MTX. The onset of the response was rapid, with a significant improvement in symptoms and signs by 2 weeks, and the majority of patients responding by 6 weeks (Figure 52.5). Over 60–70% change in individual parameters of disease activity was achieved, as well as joint protection, when hands and feet were examined by

0.02

0.001

0.001

100

20

0.002

120

0.001

24

p < 0.001

0.001

CRP (mg/l) 0.001

0.001

0.001

p < 0.01

Swollen joint count (0-28) 28

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1

2

3

4

6

8

80

16 60

12 8

40

4

20

0

0 Screen 0

1

2

3

Weeks

4

6

8

Screen 0

Weeks

FIGURE 52.4 Open-label treatment of rheumatoid arthritis with infliximab (with permission from Elliot et al., 1993). Patients received a total of 20 mg/kg of infliximab (2–4 infusions) within the first 2 weeks of the trial and were subsequently monitored for swollen joints and CRP levels for 8 weeks (Elliott et al., 1993). CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

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% Patients responding

AUTOIMMUNITY AND CYTOKINES : PATHOGENESIS AND THERAPY

1 mg/kg Infliximab

3 mg/kg Infliximab

10 mg/kg Infliximab

100

100

80

80

60

60

40

40

20

20

0

0 0 4 8 12 16

26 0

4 8 12 16

Placebo-MTX+

26 0

4 8 12 16

Infliximab-MTX-

((b) b)

26 Week

Infliximab-MTX+

ACR 20

Patients responding (%)

3 mg/kg

10 mg/kg

60

60

40

40

20

20

0

0

Improvement in Swollen Joints 80 3 mg/kg

Change from baseline (%)

Change from baseline (%)

80

60

40

20

10 mg/kg 60

40

20

0

0 0 2

6

10

14

18

Time (weeks) Infliximab every 4 weeks

22

26

30

0 2

6

10

14

18

22

26

30

Time (weeks) Infliximab every 8 weeks

Placebo

FIGURE 52.5 Efficacy of combination of infliximab and methotrexate (MTX) versus methotrexate and placebo. In (a), patients were treated with MTX (7.5 mg week1) and either placebo, 3 or 10 mg kg1 infliximab administered intravenously at time points indicated by the arrows, in a DMARD unresponsive patient group with active disease despite methotrexate therapy. Patients were examined for disease improvement at the indicated time points (with permission from Maini et al., 1998). In (b), patients were treated with MTX (15 mg week1) and either placebo, 3 or 10 mg kg1 infliximab every 4 or 8 weeks. After 30 weeks, the response of the patients was evaluated according to the ACR 20 criteria and the improvement in swollen joints (with permission from Maini et al., 1999). In (b), patients were treated with MTX (15 mg/week) and either placebo, 3 or 10 mg/kg infliximab every 4 or 8 weeks. After 30 weeks, the response of the patients was evaluated according to the ACR 20 criteria and the improvement in swollen joints (with permission from Maini et al. 1999). CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

ANTI - TNF a THERAPY OF OTHER DISEASES

autoradiographs after 54 weeks in a similarly designed phase III trial involving two doses of infliximab (3 mg and 10 mg) and high doses of MTX (Maini et al., 1999; van der Heijde, 1996). These observations formally confirmed the essential role of TNFa in RA and demonstrated that the combination of infliximab with MTX can prevent progressive joint damage over a 2-year period (Lipsky et al., 2000).

MECHANISM OF ACTION OF ANTI-TNFa THERAPY The efficacy of anti-TNFa therapy in RA, prompted us to investigate the mechanisms of action of infliximab in the anticipation that this will lead to a better understanding of the disease process. Some important observations were made by studying clinical trial serum and synovial samples. First, anti-TNFa down-regulated the expression of cytokines such as IL-1, IL-6, IL-8, MCP-1 and VEGF in the serum (Lorenz et al., 1996; Elliott et al., 1993; Paleolog et al., 1998; Taylor et al., 2000). The rapid kinetics of this reduction (e.g. IL-6 was normalized in less than 24 h) suggested that this is a direct consequence of TNFa blockade in the cytokine network rather than a reduction in the number of cytokine-producing cells (Charles et al., 1999) that might occur if anti-TNFa leads to cell death. These results confirmed our early observations in RA synovial membrane cultures (Brennan et al., 1989b). Second, anti-TNFa reduced the trafficking of leukocytes into the joints. This is probably due to decreased chemokine production and ICAM-1, E-selectin and VCAM-1 adhesion molecule expression (Taylor et al., 2000; Paleolog et al., 1996; Tak, et al., 1996). Third, anti-TNFa diminished angiogenesis in inflamed joints as assessed by computerized image analysis of the endothelium (Maini et al., 1999). Inhibition of VEGF production may partly account for that (Taylor et al., 2000). Finally, anti-TNFa restored hematological abnormalities observed in RA patients such as anemia and elevated platelet counts, although the mechanism by which this occurs is not clear (Davis et al., 1997). One possibility is that this is a consequence of IL-6 reduction. The mechanism of action of anti-TNFa therapy is documented in more detail in Feldmann and Maini, 2001.

1199

ANTI-TNFa THERAPY OF OTHER DISEASES The success of anti-TNFa therapy in RA suggested that TNFa may also be a good therapeutic target in a number of other autoimmune diseases in which similar mechanisms may operate. Crohn’s disease, a chronic inflammatory disease characterized by inflammation and granulomatous lesions of the intestinal mucosa, is one such example in which trials were initiated after disclosure of results with infliximab (Derkx et al., 1993). TNFa is elevated in mucosal biopsies as are a number of other cytokines, although it has never been shown that TNFa is at the apex of a pro-inflammatory cascade. Nevertheless, clinical trials were initiated using infliximab as the TNFa blocking agent. In an initial phase I/II open-label study, a single infusion of infliximab was sufficient to induce clinical and endoscopic improvement in eight out of ten patients with active steroid-resistant disease (van Dullemen et al., 1995). Persistent clinical response could be seen in 41% of the infliximab-treated patients compared with 12% of placebo control patients (Targan et al., 1997). Infliximab downregulated the endoscopic disease activity, as well as the inflammatory response in the mucosal layer (D’Haens et al., 1999; Baert et al., 1999). In the light of the studies in RA, the effect of multiple infusions of infliximab were also examined in patients that initially responded to the single infusion of infliximab (Rutgeerts et al., 1997). It was found that 51% of the infliximab patients remained in remission for the duration of the trial, compared with 21% placebo controls (Rutgeerts et al., 1999). Recently, infliximab obtained approval from the FDA and the European Authority for its use in humans for the treatment of Crohn’s disease. Currently, a number of clinical trials with antiTNFa blocking agents are being initiated in other autoimmune conditions too. In two open-label pilot studies in spondyloarthropathy, infliximab was found to be effective in both axial and peripheral arthritis (Van Den Bosch et al., 2000; Brandt et al., 2000). In a phase I/II clinical trial in juvenile arthritis, etanercept diminished disease activity in 74% of the patients compared with placebo (Lovell et al., 2000). Administration of etanercept also benefited patients with psoriasis, as etanercept-treated

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patients showed a significantly higher response as judged by the proriasis activity and severity index than placebo-treated patients (Mease et al., 2000). Psoriatic arthritis disease activity was also reduced in these patients.

OTHER ANTI-CYTOKINE THERAPY Anti-cytokine therapy has not been limited to TNFa but has been extended to other cytokines. In RA, clinical trials with IL-1 receptor antagonist have been performed and results have been positive, but less dramatic. IL-1ra administration revealed an improvement in clinical disease as judged by the ARC criteria (Bresnihan, 1998) and in joint destruction as determined by radiology (Jiang et al., 2000; Watt and Cobby, 2001). An alternative approach aiming to block IL-1 directly in the joints was recently used. It involved the transduction of synovial cells ex vivo with a retroviral vector expressing IL-1ra, and transduced cells were re-implanted in the same patient 1 week before joint replacement surgery. Appropriate untransduced synovial cells were also implanted to the same patient in a separate joint to serve as a placebo control. However, when IL-1ra expression was examined at the time of surgery, IL-1ra could be detected in both joints suggesting either that injected cells can migrate to neighbouring joints, or that that there is some communication between adjacent RA joints (Evans, 1999). Because of that, no further conclusions could be drawn. Anti-IL-6 therapy clinical trials have also been reported in RA. In one study, a murine neutralizing anti-human IL-6 antibody was used in a small number of patients and demonstrated a short-term improvement. In another open-label study, administration of a humanized monoclonal anti-IL-6 receptor antibody improved clinical symptoms of RA and normalized acute-phase proteins within 2 weeks (Yoshizaki et al., 1998). Although onset of the benefit was slower than that of anti-TNFa, randomized trials are under way to provide more information about its efficacy.

ANTI-INFLAMMATORY CYTOKINE THERAPY IN AUTOIMMUNITY In addition to the anti-cytokine therapy, there is an alternative approach to inhibit pro-inflammatory cytokine production that involves the use of immunoregulatory cytokines, such as IL-10, IL-11 and IFN-b. The efficacy of this approach has been tested in a number of autoimmune diseases with variable efficacy. In inflammatory bowel diseases, administration of IL-10 has demonstrated some modest clinical benefit in mild/moderate Crohn’s disease patients, but no effect in ulcerative colitis patients (van Deventer et al., 1997; Fedorak et al., 1998). Following administration of IL-11 in patients with Crohn’s disease, 42% of the treated patients demonstrated a clinical benefit compared with 7% of the placebo group (Sands et al., 1999). The dose of IL-11 used was well tolerated and was not associated with an increase in platelet counts (Sands et al., 1999). IL-10 and IL-11 have also been tested for their efficacy in RA. In two independent phase I clinical trials, RA patients treated with IL-10 or IL-11 showed no significant clinical improvement when compared with placebo (Maini et al., 1997; Moreland et al., 1999). More encouraging though, was the use of IL-10 in psoriasis. In an open-label study, nine out of ten patients demonstrated significant clinical benefit. In a double-blind, placebo-controlled study that followed, clinical improvement was mainly restricted in the skin but not the arthritic disease activity (psoriatic arthritis) (Asadullah et al., 1999; McInnes et al., 2001). IL-10-treated patients demonstrated suppressed monocyte function and TH1 but not TH2 cytokine production, as well as reduced T cell and macrophage infiltration and angiogenesis in the synovium (McInnes et al., 2001). The most successful story of immunoregulatory cytokine therapy is probably the use of IFN-b in multiple sclerosis. Administration of IFN-b decreases the relapse rates by 18–34% in patients with relapsing/remitting multiple sclerosis (Anonymous 1993, 1998a). This is of major importance in the management of multiple sclerosis, as the failure of patients to recover from relapses is a major cause of disability. IFN-b administration can also delay disease progression for 9–12 months in patients with secondary

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FUTURE THERAPY

progressive multiple sclerosis (Anonymous, 1998b). IFN-b therapy is now considered to be a major advance in the treatment of multiple sclerosis, although its cost and moderate efficacy has led to few patients benefiting from its use.

FUTURE THERAPY Although anti-TNFa therapy works well as a monotherapy, optimal efficiency appears to be achieved when anti-TNFa blocking agents are administered in combination with methotrexate. This has been demonstrated for both infliximab (Maini et al., 1998; Maini et al., 1999; Lipsky et al., 2000) and etanercept (Weinblatt et al., 1999) and suggests that combination therapy is an approach that can improve on antiTNFa therapy as expectations from patients augment. Studies that we have undertaken on animal models of arthritis demonstrated that the benefit of anti-TNFa can be increased by its combination with anti-T cell therapy. Co-administration of neutralizing anti-TNFa antibodies with T cell depleting anti-CD4 antibodies (Williams et al., 1994), for example, synergize to ameliorate disease, as does the co-administration of antiTNFa with anti-CD3 antibodies or CTLA-4Ig fusion proteins (Williams, unpublished data). Thus, it is possible that in human rheumatoid arthritis too, additional anti-T cell therapy involving anti-CD4 or anti-CD3 antibodies or T cell-inhibitory agents, such as cyclosporin may lead to increased therapeutic benefit in the patients. Anti-cytokine or immunoregulatory cytokine-based therapies have proved to be very successful in the treatment of certain autoimmune diseases, such as RA, Crohn’s disease and multiple sclerosis, but they have not been able to induce long-term disease remission in any case in the absence of continuous treatment. Their efficacy has been dependent on prolonged administration of the therapeutic agent. In RA, prolonged administration of anti-TNFa biologicals suffers from some major disadvantages. First, it requires injection of the anti-TNFa agent. Second and more importantly, chronic blockade of TNFa may seriously compromise normal immune functions, as TNFa is a multifunctional cytokine. TNFa-deficient mice, for example, demonstrate an increased risk of bacterial infections, such as Listeria monocytogenes

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(Marino et al., 1997), although no increased risk of infection was observed in RA patients treated with either infliximab or etanercept (Feldmann and Maini, 2001). In long term-treated patients the infection risk may be augmented. There is recent evidence that the incidence of tuberculosis is increased after TNFablockade in both RA and Crohn’s disease (Keane et al., 2001). However, by far the biggest practical drawback is the cost of the treatment. To design better therapeutic agents that may specifically block the ‘pathological’ TNFa expressed in the rheumatoid synovium without compromising the ‘physiological’ TNFa expressed as a result of normal immune function, we decided to investigate the intracellular signalling pathways that regulate TNFa production. We envisaged that the mechanisms involved in TNFa production in the rheumatoid disease would be different from those involved in normal immune function. This is made possible by the fact that the expression of the TNFa gene is under complex control: the 5 promoter region contains binding sites for multiple transcription factors that include NF-B, AP-1, NF-IL-6 and NF-AT (Shakhov et al., 1990; Goldfeld et al., 1991; McCaffrey et al., 1994) and hence may be involved in transcriptional control, whereas the 3 untranslated region contains AU-rich elements (ARE) that may be involved in posttranscriptional or translational control through the action of p38 mitogen-activated protein kinase (MAPK) and mRNAbinding proteins (Lee et al., 1994). Evidence, however, about the relative contribution of these factors in TNFa has been conflicting. In the murine RAW 264 macrophage cell line and bone marrow-derived macrophages, for example, NF-B was required for TNFa production (Shakhov et al., 1990; Han and Beutler, 1990). In contrast, in human lymphoid cell lines NF-AT rather than NF-B was essential for TNFa production (Goldfeld et al., 1991; Tsai et al., 1996). The main TNFa-producing cells in the rheumatoid synovium are macrophages with a small contribution from T cells and endothelial cells (Feldmann et al., 1996b). We first decided to investigate the role of NF-B in TNFa production in this cell-type. By developing an efficient adenoviral gene transfer technique to express wild-type or dominant negative forms of intracellular signalling molecules, we were able to examine the regulation of TNFa production in primary human macrophages (Foxwell

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et al., 1998). The results were very interesting: Overexpression of IBa, the natural inhibitor of NF-B abrogated TNFa production in response to LPS, PMA or UV light stimulation, but not zymosan or antiCD45 stimulation (Bondeson et al., 1999b). These findings suggested that the involvement of NF-B in macrophage TNFa production is stimulus-specific, and further added to the complexity of the regulation of TNFa. In RA, the stimulus that induces TNFa production is not known and signalling observations made in primary macrophages cannot be extrapolated into RA disease tissue. To overcome this problem we examined the role of NF-B directly in rheumatoid synovial membrane cultures. Adenoviral gene transfer again constituted a powerful approach to genetically modify RA cells, with 90% of the cells expressing the transgene of interest after infection at a multiplicity of infection of 40 (Foxwell et al., 1998; Bondeson et al., 1999a). Using flow cytometry, we found that CD14 CD3 synovial macrophages, CD14 CD3 synovial T cells and CD14 CD3 synovial fibroblasts were readily infected (Plate 52.6a) (see Plate section) (Bondeson et al., 1999a). This allowed us to block NF-B activation in RA cells by overexpressing IBa, and then examine the effect that had on TNFa production. We found that the levels of TNFa released in synovial membrane cell cultures was reduced by 70% (Plate 52.6b), suggesting that NF-B is rate-limiting for the expression of TNFa in RA. We also examined the effect of NF-B inhibition in the expression of other pro-inflammatory mediators and found that IL-6 production was inhibited by 90%, IL-1 by 40%, and IL-8 by 30–40% although this was more variable (Bondeson et al., 1999a). Interestingly, however, overexpression of IBa had only minimal effects on the production of the anti-inflammatory mediators IL-10 and IL-11, and only moderately inhibited the release of soluble TNF receptors. Even then, this was found to be an indirect effect arising from the inhibition of TNFa rather than a direct effect of NF-B in the regulation of their transcription. These results demonstrated that NF-B selectively plays a major role in the regulation of proinflammatory cytokine gene expression in RA synovial tissue without affecting anti-inflammatory cytokine gene expression. The effect, thus, of blocking NF-B in RA may be very beneficial, as it may restore

the cytokine equilibrium in the joints by diminishing pro-inflammatory mediators without reducing the anti-inflammatory ones. However, the means for doing so safely are not yet available, but as many companies are working on NF-B inhibitors, this may change in the near future. We investigated the role of NF-B in destructive processes that take place in the joint too. Matrix metalloproteinases (MMPs) are thought to be involved in the breakdown of human articular cartilage. Articular cartilage is primarily composed of collagen type II and proteoglycans, and MMP1, MMP13 and membranebound MMPs are capable of cleaving collagen type II (Knauper et al., 1997; Cowell et al., 1998). MMP3 lacks this capacity but is able to cleave other components of the extracellular matrix that include proteoglycans, fibronectin and laminin (Matrisian, 1990). The expression of MMPs has been previously proposed to be AP-1-dependent as studies in some systems have shown (Fini et al., 1998), although there was also evidence for AP-1-independent mechanisms also operating (Buttice et al., 1991; Quinones et al., 1994; Borghaei et al., 1998). When, however, we explored the effect of NF-B inhibition in rheumatoid synovial membrane cells, we found that the production of MMP-1, MMP-3 and MMP-13 was markedly down-regulated. This was in contrast to the production of TIMP-1, the inducible tissue inhibitor of MMPs enzymatic action, whose expression was up-regulated (Bondeson et al., 1999a). Whether this effect was due to a direct inhibitory effect of NF-B in their gene expression or due to an indirect effect through cytokine inhibition is not clear, although NF-jB binding sites have been detected in the promoter element of MMP-1 (Angel et al., 1987; Tewari et al., 1996). Nevertheless, these findings demonstrate that NF-B is essential not only for the inflammatory, but also for the destructive processes that operate in the rheumatoid joint. Another major pathway that controls inflammatory gene expression involves the action of mitogenactivated protein kinases (MAPK). With respect to TNFa expression, all three subtypes of MAPK have been reported to be involved, albeit to different extents. In monocytes/macrophages, p54 MAPK has been proposed to control TNFa production at the translational level (Swantek et al., 1997), In contrast, p42/44 MAPK regulates TNFa production at the tran-

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CONCLUSIONS

scriptional levels (Foey et al., 1998; Rutault et al., 2001), probably by phosphorylating Elk-1, resulting in increased transcription of c-Fos and hence increased formation of AP-1 (Karin, 1995). Finally, p38 MAPK is the best studied of the MAPK and affects TNFa expression at multiple levels that involve transcriptional, post-transcriptional and translational mechanisms (Ridley et al., 1998; Dean et al., 1999; Rutault et al., 2001). Transcriptional regulation by p38 MAPK is mediated through the activation of the ATF2, Alk-1, Sap-1, MEF2A, MEF2C and CHOP/GADD153 transcription factors (Wang and Ron, 1996; Han et al., 1997; Wasylyk et al., 1998; Zhao et al., 1999), whereas translational control requires the phosphorylation of the MAPK-interacting kinases Mnk1 and Mnk2 that in turn phosphorylate the eIF-4E component of the translation initiation complex (Waskiewicz et al., 1997), and the eIF-4E binding protein PHAS-1 (Lin et al., 1994). Post-transcriptional regulation of gene expression, on the other hand, involves the activation of MAPKAP kinase 2 that controls mRNA stability possibly by affecting the binding of ARE-binding proteins, such as TTP (Lai, 1999; Mahtani et al., 2001), AUF1 (Zhang et al., 1993), TIAR (Gueydan et al., 1999), Hel-N1 (Levine et al., 1993) and HuR (Myer et al., 1997; Dean et al., 2001). Transgenic mice, for example, that express TNFa transcripts lacking the ARE have an increased production of TNFa protein and develop chronic inflammatory polyarthritis and inflammatory bowel disease (Keffer et al., 1991; Kontoyiannis et al., 1999). With all these effects that p38 MAPK has on inflammatory gene expression, it is not surprising that inhibition of its activity markedly abrogates the production of TNFa, whereas inhibition of the other MAPK only partially does so. In addition, inhibition of p38 MAPK also reduces the production of IL-6 and IL-8 in primary human monocytes or macrophages (Ridley et al., 1998; Dean et al., 1999), establishing p38 MAPK as a primary target for the action of antiinflammatory drugs. To determine, however, whether the p38 MAPK pathway is also essential in the regulation of cytokine gene expression in rheumatoid arthritis synovium or other chronic inflammatory disease tissue the role of p38 MAPK needs to be addressed at the relevant disease tissues or models. Indeed, a study published only recently demonstrated that RPR200765A, a novel p38 MAPK inhibitor,

reduces the incidence and progression of arthritis in the rat streptococcal cell wall arthritis model when administered in either prophylactic or therapeutic dosing regiments (McLay et al., 2001). In contrast, a separate study using SP600125, a novel p54 MAPK inhibitor, in the rat adjuvant-induced arthritis model found that the inhibition of p54 MAPK activity only modestly decreases paw swelling, but almost completely prevents joint destruction as measured by radiography (Han, 2001). It may be that combinations of inhibitors of different MAPK is required for optimal therapeutic efficiency. Clinical trials of p38 MAPK inhibitors in RA are under way and so this hypothesis will be evaluated soon. In summary, the production of TNFa and other inflammatory or destructive mediators in the rheumatoid joint seem to be NF-B- and MAPKdependent. Both NF-B and p38 MAPK, in particular, are potential therapeutic targets for the treatment of rheumatoid arthritis. Further clinical trials involving inhibitors of IB kinases, the main kinases that activate NF-B, are expected to follow. As the understanding of the signalling pathways that regulate the inflammatory process in the rheumatoid joint increases, and as kinase inhibitors become more specific, less toxic, cheaper and easier to administer, this approach is likely to provide the future generation of anti-inflammatory drugs for the treatment not only of rheumatoid arthritis but of chronic inflammatory and autoimmune diseases in general. Although for many years kinase inhibitors were not selective enough to be used as safe and effective medicines, the recent success of the bcr-Abl tyrosine kinase inhibitor Gleevec (STI571) from Novartis for the treatment of chronic myeloid leukemia demonstrates that this is feasible and paves the way for other inhibitors too.

CONCLUSIONS The identification and cloning of numerous cytokines over the last 20 years has provided new challenges for the scientist of today of how to translate this large amount of knowledge into therapeutic benefit. Cytokines play a major role in a number of physiological and pathological processes including immunity, inflammation, repair, fibrosis and cancer. We and others realized that cytokines may also be important in

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autoimmunity, during disease development, but also disease perpetuation and tissue damage. Thus, we and others undertook the major task of elucidating which cytokines may be key or rate-limiting to the pathogenesis of rheumatoid arthritis, which lead to the definition of TNFa as a major therapeutic target. This was confirmed in clinical trials that we initiated over a decade ago, and also demonstrated a remarkable degree of benefit, of blocking a single molecule in a complex disease. The success of anti-TNFa therapy in rheumatoid arthritis prompted anti-TNFa clinical trials in a number of related diseases, including Crohn’s disease, psoriatic arthritis and ankylosing spondylitis, which also proved to be highly beneficial. This seems to be the beginning of a new wave of target therapies for a number of related inflammatory diseases that is going to be based on the increased understanding of disease pathogenesis and the role cytokines play in that. Initially, this may take the form of anti-cytokine blocking agents, such as neutralizing antibodies and soluble receptors, but subsequently more specific therapeutic agents targeting the intracellular mechanisms involved in cytokine gene expression may predominate. Durability of the benefit, safety and pharmacoeconomic issues will determine whether this early promise will materialize and whether it will prove to be a major breakthrough to the treatment of incurable and painful diseases.

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Tsai, E.Y., Yie, J., Thanos, D. and Goldfeld, A.E. (1996). Celltype-specific regulation of the human tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF2/JUN. Mol. Cell. Biol. 16, 5232–5244. Tun, R.Y., Smith, M.D., Lo, S.S. et al. (1994). Antibodies to heat shock protein 65 kD in type 1 diabetes mellitus. Diabet. Med. 11, 66–70. Van den Bosch, F., Kruithof, E., Baeten, D. et al. (2000). Effects of a loading dose regimen of three infusions of chimeric monoclonal antibody to tumour necrosis factor alpha (infliximab) in spondyloarthropathy: an open pilot study. Ann. Rheum. Dis. 59, 428–433. van der Heijde, D.M. (1996). Plain X-rays in rheumatoid arthritis: overview of scoring methods, their reliability and applicability. Baillières Clin. Rheumatol. 10, 435–453. van Deventer, S.J., Elson, C.O. and Fedorak, R.N. (1997). Multiple doses of intravenous interleukin 10 in steroidrefractory Crohn’s disease. Crohn’s Disease Study Group. Gastroenterology 113, 383–389. van Dullemen, H.M., van Deventer, S.J., Hommes, D.W. et al. (1995). Treatment of Crohn’s disease with anti-tumor necrosis factor chimeric monoclonal antibody (cA2). Gastroenterology 109, 129–135. Van Voorhis, W.C. and Eisen, H. (1989). Fl-160. A surface antigen of Trypanosoma cruzi that mimics mammalian nervous tissue. J. Exp. Med. 169, 641–652. Vaux, D.L. and Flavell, R.A. (2000). Apoptosis genes and autoimmunity. Curr. Opin. Immunol. 12, 719–724. Verthelyi, D. (2001). Sex hormones as immunomodulators in health and disease. Int. Immunopharmacol. 1, 983–993. Vilcek, J. (1997). Forty years of interferon, forty years of cytokines. Cytokine Growth Factor Rev. 8, 239. Villiger, P.M., Terkeltaub, R. and Lotz, M. (1992). Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes. J. Immunol. 149, 722–727. Wang, X.Z. and Ron, D. (1996). Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 272, 1347–1349. Waring, P.M., Carroll, G.J., Kandiah, D.A. et al. (1993). Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 36, 911–915. Waskiewicz, A.J., Flynn, A., Proud, C.G. and Cooper, J.A. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16, 1909–1920. Wasylyk, B., Hagman, J. and Gutierrez-Hartmann, A. (1998). Ets transcription factors: nuclear effectors of the RasMAP-kinase signaling pathway. Trends Biochem. Sci. 23, 213–216. Watt, I. and Cobby, M. (2001). Treatment of rheumatoid arthritis patients with interleukin-1 receptor antagonist: radiologic assessment. Semin. Arthritis Rheum. 30, 21–25. Weetman, A.P. and McGregor, A.M. (1994). Autoimmune thyroid disease: further developments in our understanding. Endocr. Rev. 15, 788–830. Weinblatt, M.E., Kremer, J.M., Bankhurst, A.D. et al. (1999). A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. N. Engl. J. Med. 340, 253–259.

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53 Cytokines and cancer Megan A. Cooper and Michael A. Caligiuri Ohio State University, Columbus, OH, USA

No natural phenomenon can be adequately studied in itself alone, but to be understood must be considered as it stands connected with all nature. Sir Francis Bacon (1561–1626)

INTRODUCTION General properties of all cancers include uncontrolled cell growth and, in most instances, the ability to metastasize, or spread to other areas of the body (Hanahan and Weinberg, 2000). Cytokines have been extensively investigated in the search for both a cause and cure for cancer. Depending upon the type of tumor, roles have been discovered for numerous cytokines in both the genesis and the treatment of cancer. Thus, a careful consideration of the factors responsible for the development and progression of cancers and the cytokine milieu of the tumor environment is critical when considering cytokine or anticytokine therapy for this disease.

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

PRINCIPLES OF CANCER Carcinogenesis The transformation of a normal cell into a malignant cell, or carcinogenesis, can take weeks to years (Figure 53.1). This malignant transformation can be the result of a hereditary defect, but most cases arise from mutations of somatic cells (Liotta and Liu, 2001b). There are numerous events that can lead to cancercausing mutations including the activation of protooncogenes, inactivation of tumor suppressor genes, and mutations of DNA repair and cell cycle genes (Figure 53.1). These mutations can occur during normal cellular division; following increased cellular division due to proliferative signals such as those during inflammation; and/or as the result of external carcinogens (Hanahan and Weinberg, 2000; Liotta and Liu, 2001b). Tumor progression results from the overgrowth of cancerous cells that, in effect, become

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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FIGURE 53.1 A schema of the process of carcinogenesis. The malignant transformation of a normal somatic cell can take weeks to years. In most instances, a normal cell acquires a transforming mutation via multiple mechanisms, as highlighted. This transformed cell, or neoplastic cell, can then in certain cases exhibit uncontrolled growth and expansion, resulting in cancer. unstoppable, in that they do not rely on normal cellular mechanisms for regulation of proliferation, but instead demonstrate uncontrolled growth. These cancer cells oftentimes bear little resemblance to the original cell type and do not carry out normal cellular functions.

Classification of cancers Cancer can be loosely classified based on its cell of origin. Those cancers arising from epithelial cells are termed carcinomas and cancers of mesenchymal origin, lymphomas, leukemias, or sarcomas. For the purposes of this discussion, cancers will be classified as solid tumors, such as tumors arising in organs such as the lung or breast, and hematologic malignancies, such as tumors arising in the blood, bone marrow, or lymph.

Current treatment strategies for cancer Traditional therapy for solid and hematologic malignancies have included various combinations of chemotherapeutics, radiation, surgery and bone marrow transplantation (DeVita et al., 2001). Because of the side-effects of many of these therapies and the

inability of cancer treatments to routinely cure most cancers, biologic therapies, including cytokine therapy, are currently being pursued in a variety of malignancies. The ultimate goal of most cytokine therapy is to activate and enhance an effective anti-tumor response. In addition to anti-cancer therapy, cytokine therapy is currently used for supportive therapy of cancer- or treatment-related side-effects including anemia, neutropenia and thrombocytopenia.

THE RELATIONSHIP BETWEEN CYTOKINES AND CANCER It is now evident that cytokine signals have the potential to be both deleterious and beneficial in the context of cancer (Old, 1996; Balkwill and Mantovani, 2001). The body’s response to inflammation is not unlike that of its response to tumors (Dvorak, 1986). For example, many similarities have been noted between the processes of wound healing and tumorigenesis and numerous associations between inflammation and tumors have been established. Some of these include links between infection with papillomavirus, schistosomiasis, Helicobator pylori and

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hepatitis with cervical, bladder, stomach and liver cancer, respectively (reviewed in Kuper et al. (2000) and Balkwill and Mantovani (2001)). Cytokines and inflammation are intimately linked and in each of these infections, the pro-inflammatory environment created by cytokines and chemokines is thought to play a major role in the promotion of tumor development and progression. Perhaps the most compelling evidence of a link between inflammation and cancer is the observation that persons who regularly take non-steroidal anti-inflammatory drugs (NSAIDS) have a 40–50% lower risk of fatal colon cancer (Thun et al., 1991). How can cytokines produced during the inflammatory process lead to the development of cancer? There are multiple mechanisms by which inflammation and cytokines might result in tumor development, including the promotion of cell survival and proliferation, tumor cell differentiation and cell migration (Balkwill and Mantovani, 2001). Two examples of inflammatory cytokines in inflammation and cancer include TNF-a and macrophage migration inhibitor factor (MIF).

TNF-a and NF-jB in cancer TNF-a is induced in response to numerous pathogens and is one of the first cytokines released in the proinflammatory cascade (Beutler and Cerami, 1989; Ruddle, 1992). TNF-a has been detected in multiple solid and hematologic malignancies (reviewed in Balkwill and Mantovani, (2001)). A number of different intracellular signals are induced by TNF-a and includes signals for both cell survival through NF-jB and cell death through caspase activation (Plate 53.2) (see Plate section). NF-jB is a key regulator of cell survival and promoter of carcinogenesis in multiple tumor types (Wang et al., 1996; Baldwin, 2001). In addition to cell survival, NF-jB induces the transcription of pro-inflammatory cytokines, such as IL-1 and IL-6, thus potentiating a pro-inflammatory cytokine loop. Constitutive expression of NF-jB has been discovered in primary leukemias and lymphomas (Bargou et al., 1997; Kordes et al., 2000; Guzman et al., 2001). A recent study of NF-jB expression in primary AML samples found that inhibition of NF-jB activity by treatment with a proteasome inhibitor resulted in the apoptosis of primary AML, but not normal cells (Guzman et al., 2001). Thus in the setting of chronic

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inflammation, a shift in the balance between antiapoptotic and pro-apoptotic signaling by cytokines such as TNF-a has the potential to favor cell survival and proliferation (Plate 53.2). The escape from apoptosis by a single proliferating cell will in some instances increase the likelihood of incurring an oncogenic mutation, ultimately leading to the formation of a tumor.

MIF and p53 inactivation The p53 tumor suppressor gene is lost or inactivated in a wide variety of cancers (Levine et al., 1991). Regulation of normal cellular growth and death depends upon an intact p53 and its mutation, inactivation, or loss results in dysregulation of these functions allowing for the continued survival of cells and consequent increased risk of cancer-causing mutations (CordonCardo and Prives, 1999). Hudson and colleagues demonstrated that a cytokine, MIF, can down-regulate expression of this gene by interfering with its transcriptional activity (Hudson et al., 1999). In the presence of MIF, p53 is still detected, but is unable to activate downstream signaling molecules, including the pro-apoptotic Bax protein. MIF is produced locally by T cells and macrophages during inflammation and promotes the activation of these immune effectors, including T lymphocyte proliferation (Calandra and Bucala, 1997; Mitchell and Bucala, 2000). The authors propose that MIF production during inflammation and the subsequent downregulation of normal p53 function could initially result in the beneficial survival of inflammatory cells to promote clearance of infection and minimize damage to normal host cells (Hudson et al., 1999; CordonCardo and Prives, 1999). However, in the case of chronic inflammation, the long-term down-regulation of p53 would likely result in the survival and proliferation of cells that would normally undergo apoptosis, creating a favorable scenario for malignant transformation. In addition, local production of nitric oxide associated with inflammation can induce chromosomal damage, to which cells with impaired p53 function would not be able appropriately to respond. MIF protein has been found to be increased in primary human tumors including breast, prostate, and brain cancers (Meyer-Siegler and Hudson, 1996; Bini et al., 1997; Meyer-Siegler et al., 1998; Markert et al., 2001),

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suggesting a possible role for autocrine production of this cytokine in tumor progression. In addition, a role for MIF in angiogenesis and tumor neovascularization has been shown in an in vivo model (Chesney et al., 1999). Therefore there are multiple mechanisms by which this pro-inflammatory cytokine might promote tumorigenesis.

CYTOKINES IN CARCINOGENESIS AND TUMOR PROGRESSION A number of cytokines and cytokine receptors have been directly linked to the development of human cancers. Some of these, such as the inappropriate levels of IL-2 and the IL-2R associated with HTLV-1 infected cells, have been implicated in promoting cell proliferation and survival that promotes carcinogenesis (Waldmann, 2000). Other cytokines, including protooncogenes encoding receptor tyrosine kinases or their ligands, have been more directly linked to the development of cancer (Blume-Jensen and Hunter, 2001). In addition, cytokines can promote the devel-

opment of a rich tumor stroma and growth of new blood vessels, thus supporting tumor cell growth and metastasis. The development of some cancer-associated side-effects, such as cachexia, has also been attributed to excessive amounts of inappropriate cytokines. Shown in Table 53.1 is a partial list of cytokines that are overproduced in cancer and thought to play a role in the development and/or progression of human cancers including IL-1, IL-6, IL-9, IL-10, TGF and TNF. The complexity of cytokine production during cancer and the uncertainty of the significance of every cytokine found in cancer make it impossible to review all of the associations between cytokines and cancer. For example, Hodgkin’s lymphoma cells have been shown to produce numerous cytokines including IL-1, IL-4, IL-5, IL-6, TNF-a, GM-CSF, TGF-b (Gruss et al., 1997), however little is known about the role of each particular cytokine in this disease.

IL-2 and adult T cell leukemia Adult T cell leukemia (ATL), is a hematologic malignancy most commonly associated with infection by the human T cell lymphotrophic virus type 1 (HTLV-1)

TABLE 53.1 Examples of cytokines and cytokine receptors associated with human cancers Cytokine or receptor

Associated cancers

Reference(s)

Cytokines/Receptors overexpressed IL-2 and IL-2R Adult T cell leukemia (ATL) IL-6 Multiple myeloma B cell malignancies Castleman’s disease IL-10 B cell malignancies IL-15 Cutaneous T cell lymphoma (CTCL) Large granular lymphocytic leukemia (LGL) Multiple myeloma TGF-bR mutations Colon, breast, lymphoma, and other cancers TNF-a Lymphoma Ovarian cancer MCP-1 Ovarian cancer

Waldmann et al. (1984) Bataille et al. (1989); Barille et al. (2000) Tosato et al. (1993) Yoshizaki et al. (1989) Khatri and Caligiuri (1998) Leroy et al. (2001); Dobbeling et al. (1998) Zambello et al. (1997) Tinhofer et al. (2000) Hata, (2001); Wong and Lai (2001) Warzocha et al. (2000) Naylor et al. (1993) Negus et al. (1995)

Genetic polymorphisms IL-1b Gastric carcinoma Pancreatic cancer TNF Prostate cancer Multiple myeloma Adult T cell leukemia (ATL) Chronic lymphocytic leukemia (CLL) Non-Hodgkin’s lymphoma

El-Omar et al. (2000) Barber et al. (2000) Oh et al. (2000) Davies et al. (2000) Tsukasaki et al. (2001) Demeter et al. (1997) Warzocha et al. (1998)

TGF, transforming growth factor; TNF, tumor necrosis factor; PDGF, platelet-derived growth factor; EGFR, epidermal growth factor receptor; CSF-1R, colony-stimulating factor-1 receptor; VEGFR, vascular endothelial cell growth factor receptor. CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

CYTOKINES IN CARCINOGENESIS AND TUMOR PROGRESSION

(Waldmann, 2000). The HTLV-1 retrovirus encodes a trasncription factor, tax, which deregulates the IL-2 and IL-2Ra genes following infection of T cells (Sodroski et al., 1984). The autocrine feedback loop created by excess IL-2 and IL-2Ra on infected T lymphocytes increases the proliferation of these cells, thus increasing the risk of a mutagenic event. Subsequently, the malignant T cells in ATL constitutively express the high affinity (IL-2Rabc) and intermediate affinity (IL-2Rbc) IL-2 receptors, in distinct contrast to the majority of resting T and NK cells which only express the intermediate IL-2R (Waldmann et al., 1984). Initially, the proliferation of HTLV-1-infected T cells is IL-2-dependent, but eventually these T cells and leukemic cells can often proliferate in an IL-2independent manner. This is likely due to an additional virus-induced mutation, such as constitutive activation of downstream IL-2 Jak/Stat signaling pathways, which allows uncontrolled growth (Arya et al., 1984; Migone et al., 1995). Therefore autocrine production of IL-2 contributes to the early stages of malignant transformation. The expression of the IL-2Ra by HTLV-1-infected cells and ATL has been used to develop experimental treatments for this disease and antibodies blocking this receptor have been tested in vivo. This relationship between IL-2 and ATL illustrates a mechanism of leukemogenesis whereby autocrine production of a cytokine as the result of infection can contribute to the initial stages of malignant transformation.

IL-6 and multiple myeloma Associations have been established between IL-6 and hematologic malignancies, including multiple myeloma (Kawano et al., 1988; Zhang et al., 1989; Klein et al., 1995) and B cell lymphomas (Tosato et al., 1993), as well as Castleman’s disease (Yoshizaki et al., 1989), a benign atypical lymphoproliferative disorder with poor prognosis asociated wth HHV8 infection. Multiple myeloma is a malignant plasma cell disorder in which IL-6 is believed to be one of the key mediators of tumor cell survival and proliferation (Bataille and Harousseau, 1997; Hawley and Berger, 1998; Treon and Anderson, 1998). In vitro and in vivo studies have shown that this cytokine plays a major role in the pathogenesis of multiple myeloma (reviewed in Hawley and Berger (1998)) and disease severity can be

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correlated with IL-6 levels (Bataille et al., 1989; Zhang et al., 1989). IL-6 is produced in large amounts by the tumor microenvironment and to a lesser extent by the multiple myeloma cells themselves. As a result, serum levels of IL-6 are elevated in patients with active disease (van Zaanen et al., 1996; Bataille and Harousseau, 1997). It is generally thought that paracrine, rather than autocrine, production of IL-6 in the bone marrow microenvironment is the primary mechanism of IL-6 release in vivo (Klein et al., 1989; Bataille and Harousseau, 1997). Through cell contactdependent mechanisms and release of soluble factors, multiple myeloma cells can stimulate IL-6 production by stromal cells (Bataille and Harousseau, 1997), potentiating a positive feedback loop accounting for the high levels of IL-6 observed in this disease (Figure 53.3). The association between multiple myeloma and IL-6 illustrates a mechanism whereby paracrine production of a cytokine is necessary for the survival and progression of a malignancy.

IL-10 and B cell malignancies IL-10 production in various B cell malignancies has been reported including acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt’s lymphoma, non-Hodgkin’s lymphoma, and EBV- and HIV-associated lymphomas (reviewed in Khatri and Caligiuri (1998)). The significance of IL-10 in the development of B cell malignancies is not clear, but there is good experimental evidence to suggest that this cytokine plays a key role in these cancers. When mice with severe, combined immune deficiency (SCID) are injected with human peripheral blood lymphocytes they develop a spontaneous human EBV-associated B cell lymphoproliferative disorder (EBV-LPD). In this model, mice that went on to develop EBV-LPD had significantly higher serum levels of human IL-10 compared with animals that did not develop the malignancy (Baiocchi et al., 1995). Furthermore, serum-free medium supplemented with IL-10 was able to prevent programmed cell death and promote cell growth of fresh human tumors from these animals in vitro. Similarly, primary cells from patients with non-Hodgkin’s lymphoma proliferate in vitro in response to IL-10, an effect that was increased with the addition of other cytokines, including IL-6 and IL-2 (Voorzanger et al., 1996). Malignant B cells

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FIGURE 53.3 Production of IL-6 by bone marrow stromal cells potentiates the growth and progression of multiple myeloma. The cytokine IL-6 is thought to be involved in the survival, proliferation and progression of multiple myeloma as dicussed in the text. These cancer cells express the IL-6R and serum levels of IL-6 are elevated in patients with multiple myeloma. Bone marrow stromal cells are likely the source of IL-6 in this disease, and through cell contact-dependent mechanisms and release of soluble factors, multiple myeloma cells themselves stimulate IL-6 production by stromal cells. from chronic lymphocytic leukemia themselves express a functional IL-10 receptor (Jurlander et al., 1997) and have been shown to produce IL-10 that can act in an autocrine fashion to promote proliferation and survival (Beatty et al., 1997). Aside from IL-10’s effects on cell survival and proliferation, there are several other potential mechanisms whereby this cytokine can promote the progression of B cell malignancies. Infection with the Epstein–Barr virus (EBV) is associated with a high percentage of B cell malignancies, HIV-associated malignancies, and post-transplant lymphoproliferative disorder. The effective control of a viral infection, including EBV, requires a type 1 adaptive T cell immune response and associated cytokines, such as the IFNs (Biron et al., 1998). However, IL-10 promotes a type 2 adaptive immune response, which is generally characterized as immunosuppressive and not conducive for an effective anti-viral response (O’Garra, 1998; Glimcher and Murphy, 2000). In addition to IL-10 associated with

B cell malignancies, EBV carries in its genome the BCRF-1 gene that encodes a protein similar in structure to IL-10, called viral IL-10 (vIL-10). This homolog of IL-10 can elicit many of the same functions as IL-10, although it has a significantly lower affinity for the IL-10R (Liu et al., 1997). As this virus evolved, it is quite possible that it captured this gene from the human genome as a mechanism to promote survival and proliferation of EBV-infected cells (Khatri and Caligiuri, 1998). Both IL-10 and vIL-10 have been shown to down-regulate the transporter protein 1 (TAP1) in B cells (Zeidler et al., 1997). This resulted in a decreased expression of MHC class I due to impairment of peptide transportation and loading on to class I (Zeidler et al., 1997). There is thus the potential for decreased recognition and lysis of transformed B cells and/or EBV-infected B cells in the presence of IL-10 in vivo because CD8 cytotoxic T lymphocytes require MCH class I molecules for recognition of virally infected cells. Therefore IL-10’s mechanism of action in B cell malignancies is likely to be a combi-

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nation between the promotion of tumor cell survival and proliferation, as well as tumor immune evasion.

Cytokine polymorphisms and cancer Polymorphisms of cytokines and cytokine receptors can alter the expression patterns of these genes resulting in abnormally high or low cytokine production. Genetic polymorphisms of the proinflammatory IL-1b and TNF genes have been shown to be associated with several cancers (shown in Table 53.1). Infection and inflammation associated with Helicobacter pylori is a risk factor for gastric cancer (Blaser, 2000). El Omar and colleagues (2000) identified a group of patients with an increased inflammatory response to H. pylori and increased risk for gastric cancer. The authors found that these patients had a significantly higher rate of an IL-1b polymorphism thought to result in increased IL-1b production during infection (El-Omar et al., 2000). A polymorphism of IL-1b has also been associated with a poor prognosis in pancreatic cancer and correlates with increased production of IL-1b by patient PBMC in vitro (Barber et al., 2000). Polymorphisms of the TNF family members TNF-a and/or lymphotoxin-a (LT-a, also known as TNF-b) predicted to result in high production of these cytokines have been associated with multiple cancers including prostate cancer (Oh et al., 2000), multiple myeloma (Davies et al., 2000), ATL (Tsukasaki et al., 2001), and chronic lymphocytic leukemia (CLL)

(Demeter et al., 1997). Similarly, genetic polymorphisms associated with increased production TNF-a and LT-a were found to be poor prognostic indicators in non-Hodgkin’s lymphoma (Warzocha et al., 1998). While it is likely that these polymorphisms are not the sole factors leading to the development of disease, these findings do strongly suggest a role for the proinflammatory cytokine network in cancer. In addition, identification of cytokine polymorphisms in cancer patients could aid in designing effective anticytokine treatment strategies.

Protooncogenes associated with cytokine signals Protooncogenes are those genes with a normal cellular function that, when genetically altered, have the potential to become oncogenic. Oncogenic mutations generally target genes that regulate cellular growth, signaling and differentiation (Liotta and Liu, 2001a). One potent class of oncogenes related to cytokines and cytokine signaling are the receptor tyrosine kinases (RTKs) and their ligands (reviewed in BlumeJensen and Hunter (2001)). The RTKs and their ligands are a large family of factors critical for cellular differentiation and growth and their expression is normally very tightly regulated (Paulson and Bernstein, 1995). Mutations of these genes can result in constitutive activation with subsequent oncogenic growth and differentiation signals. Table 53.2 lists some of the major

TABLE 53.2 Select receptor tyrosine kinase protooncogenes related to dysregulation of cytokines or cytokine signaling Cytokine or receptor Ligands PDGF-b Receptors C-kit CSF-1R EGFR family

FLT-3 PDGFR-a PDGFR-b VEGFR

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Gene

Associated cancers

SIS

Astrocytoma, osteosarcoma

C-KIT FMS ErbB1 ErbB2/HER2 ERBB3 ERBB4 FLT3 ITD PDGFR-a Tel-PDGFRb FLT-1

Stromal cell tumors, leukemia, lung cancer Leukemia Mammary, brain, lung and ovarian cancer Breast, ovarian, mammary, gastric, lung and colon cancer Mammary cancer Mammary cancer Leukemia Glioma, brain and ovarian cancer Chronic myelogenous leukemia (CML) Tumor angiogenesis

References: Blume-Jensen and Hunter (2001); Bloomfield and Caligiuri (2001). CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

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cytokines and cytokine receptors of the RTK family that can function as oncogenes. In addition, numerous signaling molecules associated with RTKs, such as the janus kinase Jak1 or the ABL kinase, can be oncogenic (Blume-Jensen and Hunter, 2001). There are various mechanisms by which these genes can become oncogenic. For example, a loss of the coding region for the extracellular domain of the EGFR leads to an oncogenic mutation (Arteaga, 2001). The mutation associated with the FLT3 gene involves an inframe duplication of the gene and a protein product with additional amino acids in the juxtamembrane region of the receptor resulting in constitutive signaling (Nakao et al., 1996). This FLT3 internal tandem duplication (ITD) is often associated with acute myeloid leukemia (AML) and its expression in the absence of the wild-type allele can predict an adverse clinical outcome in this disease (Whitman et al., 2001). Expression of the ErbB2/neu receptor (also known as HER2/neu), a member of the EGFR family, by breast cancers has been the basis for biologic therapies targeting this receptor with the monoclonal antibody trastuzumab (Shak, 1999). The finding that multiple RTK family members have the capacity to become oncogenic, stresses the importance of the ability to regulate expression of growth factors and their receptor activation in normal cellular differentiation and growth.

CYTOKINES IN THE TUMOR MICROENVIRONMENT The composition of the tumor microenvironment influences cancer cell proliferation and metastasis. The growth of solid tumors often requires a fertile tumor stroma consisting of endothelial cells, mast cells, host immune cells and fibroblasts (Wernert, 1997). Tumor and host cell-derived cytokines and chemokines can either stimulate or inhibit tumor cell growth and metastasis, thus the overall balance of activating inhibitory signals influences the progression of disease. The importance of cytokines in the tumor stroma has been comprehensively reviewed elsewhere, and a brief overview of these interactions is presented here (Dvorak, 1986; Wernert, 1997; O’Byrne et al., 2000; Balkwill and Mantovani, 2001). One example of a cytokine produced by the tumor

microenvironment is IL-6, which is secreted by bone marrow stromal cells and contributes to the progression of multiple myeloma, as discussed earlier (Hawley and Berger, 1998). Host immune cells associated with tumor stroma include tumor-associated macrophages (TAM), tumor-associated dendritic cells (TADC), and/or tumor infiltrating lymphocytes (TIL), which are often drawn to the tumor site by chemokines produced by cancer cells. While TAM, TADC and TIL have the potential to mount an antitumor response, each of these cell types is often deficient in many of the functions of their non-tumor associated counterparts and also produce cytokines, such as IL-10 and TGF-b that stimulate tumor progression and are themselves immunosuppressive in part (Ardestani et al., 1999; Mocellin et al., 2001). Most solid tumors such as breast cancer, colon cancer, or glioblastomas are also dependent on angiogenesis for continued growth and expansion or metastasis and the cytokine vascular endothelial growth factor (VEGF) produced in the tumor microenvironment is critical in this regard (Folkman, 1995; Cross and Claesson-Welsh, 2001). Indeed, pre-clinical models of angiogenesis inhibitors such as angiostatin have shown promising results in animal models and clinical testing of these agents is currently under way (O’Reilly et al., 1994; Bergers et al., 1999; Folkman et al., 2001). Thus, cytokines produced by tumor stroma can stimulate tumor progression and inhibition of those cytokines is one potential strategy for the treatment of cancer.

CHEMOKINES AND TUMOR METASTASIS There is now good evidence to suggest that chemokines play a role in tumor cell metastasis (Murphy, 2001; Strieter, 2001; Liotta, 2001). The chemokine receptor, CXCR4, and its ligand, stromal cell-derived factor 1 (SDF-1), govern the pattern of metastasis in a murine model of breast cancer (Muller et al., 2001). In this model, expression of CXCR4 was up-regulated following establishment of a local tumor and migrating cancer cells selectively invade those tissues expressing high amounts of SDF-1 (Muller et al., 2001). Thus the elucidation of the chemokine receptor profile of cancer cells could lead to a predic-

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ANIMAL MODELS OF CYTOKINES AND CANCER

tion of tumor cell metastasis and serve as a target for immunotherapy aimed at blocking receptor activation.

CYTOKINE MEDIATED SIDE EFFECTS OF CANCER: CACHEXIA The wasting syndrome, cachexia, associated with a variety of cancers and other chronic diseases such as HIV contributes significantly to the morbidity and mortality of cancer. This disorder is characterized by loss of both muscle and adipose tissue and in contrast to anorexia is generally unresponsive to nutritional therapy (Barton, 2001). Multiple cytokines have been proposed to mediate cachexia including TNF-a, IL-1, IL-6, IL-11, IFN-c, and leukemia inhibitory factor (LIF) (Oliff et al., 1987; Tisdale, 1997; Argiles and Lopez-Soriano, 1999; Barton, 2001). The exact mechanism of cytokine-induced cachexia is unknown. A recent report demonstrated that TNF-a plus IFN-c induction of the transcription factor NF-jB resulted in skeletal muscle degeneration through suppression of MyoD mRNA (Guttridge et al., 2000). These data suggest that treatment with inhibitors of NF-jB (discussed below) might be beneficial for the treatment of cancer-related cachexia.

ANIMAL MODELS OF CYTOKINES AND CANCER Numerous cytokine transgenic (tg) murine models have been engineered over the years including animals

that overexpress cytokines and animals with targeted disruptions of cytokine genes. These transgenic models have taught us a great deal about the normal functions of cytokines in vivo and in certain cases have resulted in animal models of cancer, some of which are outlined in Table 53.3. Overexpression of cytokines (tg models) has resulted in the development of cancer, while the elimination of other cytokine genes can result in a decreased or increased incidence of cancer. The IL-15tg mouse develops a hematologic malignancy, and the TNF-a-deficient mouse has a decreased incidence of inducible skin cancer.

Overexpression of IL-15 leads to leukemia: an example of chronic inflammation resulting in cancer The pleiotropic cytokine IL-15 is required for the differentiation of NK cells and is involved in the survival and/or expansion of NK cells and some subsets of T cells. The IL-15 gene is normally tightly regulated at the post-transcriptional level and the protein product is often difficult to detect in vivo (Fehniger and Caligiuri, 2001). Two groups independently developed murine models in which overexpression of IL-15 resulted in a T-NK hematologic malignancy, emphasizing the importance of effective host control mechanisms for immunoregulatory cytokines (Brewer, 2001). First, Fehniger et al. (2001a) described the construction of a mouse that lacks the complex post-transcriptional regulatory elements of the wild-type gene and consequently overexpresses murine IL-15. All of these animals exhibited early benign proliferation of NK cells and CD8 T cells. Approximately half of the animals went on to develop a fatal clonal lymphocytic

TABLE 53.3 Examples of transgenic murine models of cytokines in the genesis of cancer Genetic alteration

Cancer

Reference(s)

huIL-6 and husIL-6R tg IL-7 tg IL-9 tg muIL-15 tg HMG1-C/T tg (↑ IL-15) TGF-a tg

Hepatocellular carcinoma B and T cell lymphomas Thymic lymphoma T/NK leukemia T/NK lymphoma Liver tumors Mammary cancer Resistant to induced skin cancer Resistant to B cell tumors

Maione et al. (1998) Rich et al. (1993) Renauld et al. (1994) Fehniger et al. (2001a) Baldassarre et al. (2001) Jhappan et al. (1990) Sandgren et al. (1990) Moore et al. (1999) Hilbert et al. (1995)

TNF-a/ IL-6/

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leukemia between 16 and 20 weeks of age (Fehniger et al., 2001a). It is hypothesized that the early proliferation of lymphocytes observed in these animals makes these cells more susceptible to mutagenic events and can lead to an oncogenic mutation ultimately resulting in fulminant malignant transformation (Plate 53.4) (see Plate section) (Fehniger et al., 2001b). In support of this hypothesis, heterogeneity of tumor phenotype between animals with T, NK/T and NK tumors was observed (Fehniger et al., 2001a). In a subsequent study, Baldassarre et al. (2001) reported that transgenic mice over-expressing a truncated form of the high mobility group protein I-C (HMGIC/T) gene develop a T-NK lymphoma at approximately 12 months of age. The authors went on to demonstrate that the development of the lymphoma was associated with an increase in IL-2/IL-15 signaling pathways and that HMGI-C/T can bind directly to the IL-15 promoter and enhance the transcription of this cytokine (Baldassarre et al., 2001). The HMGI-C/T tg mice developed cancer at a significantly later time point that IL-15tg mice (12 months versus 16–20 weeks), likely due to the lower expression of IL-15 in these animals. Together, these studies suggest that chronic stimulation with the pro-inflammatory cytokine IL-15 can result in the development of a lymphocytic leukemia/lymphoma and suggest an association between excessive proliferation of IL-15 responsive cells and cancer. These results illustrate a mechanism whereby chronic inflammation and prolonged exposure to a cytokine can lead to the development of cancer.

A murine model of TNF-a and skin cancer Work by the Balkwill laboratory has shown that mice deficient in TNF-a (TNF-a/) are resistant to chemically induced skin carcinogenesis. Under multiple tumor-inducing regimens and in two genetic backgrounds, mice deficient in TNF-a had a significantly lower incidence of skin carcinogenesis and progression. Furthermore, animals deficient in the TNF-ainducible chemokine monocyte chemoattractant protein 1 (MCP-1) also had a lower incidence of carcinogenesis, although not to the same extent as TNFa/ mice (Moore et al., 1999). TNF-a was found to be important for the early stages of chemically induced

skin carcinogenesis. Shortly after carcinogen treatment of wild-type mice, an increase in TNF-a protein was detected in the epidermis and inflammation and proliferation of keratinocytes was evident. In TNFa/ mice, there was a marked reduction in this early inflammatory process, suggesting that the proliferation of the keratinocytes may be regulated by TNF-a (Moore et al., 1999). TNF-a was not involved in the later stages of the tumor and had no effect on tumor progression. Therefore TNF-a is a potent mediator of skin carcinogenesis and a potential target for anti-cytokine therapy for the prevention of this cancer or treatment of the early stages of malignant transformation.

BIOLOGIC THERAPIES AIMED AT BLOCKING CYTOKINE SIGNALING Given the wealth of associations between inappropriate cytokine production and signaling during cancer, there have been several attempts to neutralize cytokines and to block cytokine signaling for the treatment of cancer.

Specific inhibitors of cytokines In a limited number of cancers with well-established links to cytokines, anti-cytokine therapy has been tested in clinical trials. While no anti-cytokine therapies are currently approved for the treatment of cancer, various anti-cytokine, soluble cytokine receptors, or blocking or anti-cytokine receptor monoclonal antibodies have been examined in clinical trials, including anti-IL-6 and IL-6R, anti-IL-2Ra and antiEGFR (Bataille et al., 1995; Nishimoto et al., 2000; Waldmann, 2000; Ciardiello and Tortora, 2001). For example, IL-6 is involved in the pathogenesis of multiple myeloma and other B cell malignancies, as well as Castleman’s disease. Various clinical trials testing anti-IL-6 and anti-IL-6R monoclonal antibodies in these diseases have been conducted (Bataille et al., 1995; van Zaanen et al., 1996; Moreau et al., 2000; Nishimoto et al., 2000; Haddad et al., 2001). Thus far these studies have been inconclusive; however two small studies have offered encouraging results. Nishimoto et al. (2000) found that treatment with a

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BIOLOGIC THERAPIES AIMED AT BLOCKING CYTOKINE SIGNALING

humanized anti-IL-6R antibody resulted in clinical improvement of Castleman’s disease in a study of seven patients. In a phase II trial of relapsed or refractory multiple myeloma patients, Moreau et al. (2000) reported improved response when an anti-IL-6 murine monoclonal antibody in combination with dexamethasone was administered prior to autologous stem cell transplantation.

Generalized approaches to anti-cytokine therapy It is likely that multiple cytokines contribute to the growth and progression of tumors. Thus, a more generalized approach to anti-cytokine therapy of cancer is also being pursued and may eventually be a more promising approach to biologic therapy of cancer than single agents. Examples of this are agents designed to block NF-jB such as the proteasome inhibitors (Karin, 1999). As discussed earlier in this chapter, NF-jB is constitutively activated in a number of cancers and is up-regulated by several proinflammatory cytokines including TNF-a, IL-1 and IL-6 and was recently associated with muscle wasting in cachexia (Guttridge et al., 2000). Inhibition of this pro-inflammatory signaling molecule in patients with cancer has the potential to inhibit cytokinedependent survival and proliferation of tumor cells, as has been shown in vitro and in pre-clinical in vivo models with proteasome inhibitors (Hideshima et al., 2001; Guzman et al., 2001; Sunwoo et al., 2001). For example, the proteasome inhibitor PS-341 prevented the proliferation of human multiple myeloma cells both in vitro and in an in vivo murine model (Hideshima et al., 2001; LeBlanc et al., 2001). This inhibitor, which appears to work in part by inhibition of NF-jB-induced IL-6, was recently examined in patients with multiple myeloma in a phase II clinical trial with encouraging preliminary results (Richardson et al., 2001). The antiangiogenic drug thalidomide is currently being tested in a variety of cancers with the most encouraging results for this agent in the treatment of relapsed or chemotherapy-resistant multiple myeloma (Singhal et al., 1999; Kaufmann et al., 2001). Aside from its anti-angiogenic properties, thalidomide potently inhibits pro-inflammatory cytokine produc-

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tion, including TNF-a, IL-6 and VEGF. NF-jB is also be inhibited by thalidomide through inhibition of IjB kinase (Keifer et al., 2001). Thus, down-regulation of cytokines and pro-inflammatory mediators associated with multiple myeloma and other cancers is thought to be a mechanism responsible for this drug’s beneficial effects (Thomas and Kantarjian, 2000). Because if its anti-cytokine effects, thalidomide is also being tested clinically for the treatment of cytokinemediated disorders associated with cancer, such as cachexia and graft-versus-host.

Tyrosine kinase inhibitors The RTK family of cytokines and cytokine receptors can function as oncogenes and are constitutively activated in numerous cancers (see Table 53.2). Small molecular inhibitors of tyrosine kinase activity have been developed and are currently undergoing evaluation as clinical therapeutic agents. For example, a FLT3 tyrosine kinase inhibitor has been identified that inhibits growth of primary AML cells harboring the FLT3-ITD gain-of-function mutation (Levis et al., 2001). This chemical inhibitor, AG1295, specifically induced apoptosis of FLT3-ITD AML blasts in vitro and had no effect on cells without the mutation. Another inhibitor of FLT3, herbamycin A, inhibited leukemogenesis of a FLT-3-ITD transformed cell line in vivo (Zhao et al., 2000). These promising results suggest that clinical use of a FLT3 inhibitor may be beneficial for the treatment of patients with AML. One example of a tyrosine kinase inhibitor currently being used clinically is the drug STI571, which inhibits the BCR-ABL tyrosine kinase found in a high percentage of patients with CML (Topaly et al., 2001; Druker et al., 2001). As many as 98% of patients with CML treated with this tyrosine kinase inhibitor had a complete hematologic response (Druker et al., 2001). C-kit and the PDGFR are also inhibited by STI571 making this drug a good candidate for treatment of cancers associated with these RTK oncogenes (see Table 53.2). Numerous clinical and pre-clinical studies are ongoing to identify additional novel inhibitors of tyrosine kinases for the treatment of cancer (Fry et al., 1994; Buchdunger et al., 1995; Wood et al., 2000; Ciardiello et al., 2001; Solorzano et al., 2001; Hidalgo et al., 2001).

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CYTOKINES IN THE THERAPY OF CANCER Some of the earliest evidence for the potential of immune activation for cancer therapy resulted from experiments performed by Dr William Coley in the late 1800s. Coley administered a bacterial extract to untreatable cancer patients and reported a cure rate ranging from 10 to 40%, unheard of with other therapies used at that time (Coley, 1991; Starnes, 1992). We know now that bacterial DNA is a potent activator of the pro-inflammatory cytokine cascade and the innate immune response via activation of Toll like receptors (Krieg and Wagner, 2000). Administration of bacille Calmette–Guérin (BCG) bacteria is a treatment option for bladder cancer (Lamm, 2000; Herr et al., 2001).

IMMUNE CELLS INVOLVED IN TUMOR REJECTION An effective host anti-tumor immune response requires coordinate engagement of both innate and adaptive immune effectors (Pardoll and Topalian, 1998; Mumberg et al., 1999; Dallal and Lotze, 2000; Soloski, 2001). Evidence for this comes primarily from observations in murine models, where these immune cells have been shown to be important for an antitumor response in vivo. For example, animals deficient in IFN-c have a higher rate of chemically induced and spontaneous tumors when compared with control animals (Kaplan et al., 1998), suggesting that this cytokine and the induction of a TH1 response are critical for tumor surveillance. Numerous animals models have shown that NK cells or CD8 cells, both of which are cytotoxic effectors and secrete immunoregulatory cytokines, are required for resistance to or treatment of certain tumors (Hanson et al., 2000; Shankaran et al., 2001; Kawarada et al., 2001). Each of the immune cells listed above has a unique cytokine and cytokine receptor repertoire and they perform distinct functions in vivo. NK cells have the ability to lyse target tumor and virally infected cells without prior sensitization (Cooper et al., 2001). Unlike T lymphocytes, NK cells do not rearrange their receptors, but have developed the ability to recognize self-MHC class I molecules through a unique class of

NK receptors (NKRs) that can inhibit or activate NK cell killing (Lanier, 2001). Transformed cells that have down-regulated normal host MHC class I or tumor cells expressing MHC-like antigens recognized by activating NKRs become susceptible to NK cell lysis. As opposed to NK cells, CD8 T cells mediate cytotoxicity following recognition of an antigen associated with MHC class I molecules on the tumor cell surface. CD4 cells recognize antigens processed by MHC class II molecules and type 1 cytokines (e.g. IFN-c, IL-2) they are capable of producing and are critical for priming an efficient CD8 cytotoxic response. T cells exhibit immunologic memory and can recognize tumor-specific antigens. Expansion of CD8 or CD4 T cells has been the focus of many efforts to create cancer vaccines (Greten and Jaffee, 1999; Rosenberg, 2001). DC also play a critical role in the activation of tumor-specific T cells as they have the ability to act as potent antigen-presenting cells and stimulate T cell expansion (Dallal and Lotze, 2000). Thus, cytokine therapy is aimed at mobilizing and activating this diverse group of immune cells.

CYTOKINE THERAPY Cytokine therapy has not been found to be widely effective in human cancers (Brunda et al., 1996; Burke and Balkwill, 1996; Oppenheim et al., 1997). A variety of cytokines have been tested in clinical trials, however only a few cytokines are currently approved in the United States for use in cancer: IL-2 and IFN-a for cancer therapy and G-CSF, GM-CSF, IL-11 and erythropoietin (EPO) for supportive care of cancer- and treatment-related side-effects (shown in Table 53.4). Accepted use of other cytokines with promising results in clinical trials, such as TNF-a for isolation perfusion treatment of sarcoma, melanoma, or liver cancer (Lejeune et al., 1998; van Der Veen et al., 2000), is often limited by complexity of delivery or adverse side-effects.

IL-2 immunotherapy IL-2 was an early candidate for the immunotherapy of cancer and approved for use in renal cell cancer by the FDA in 1992 (reviewed Figlin (2000)). Rosenberg and colleagues first utilized high doses of IL-2 to treat

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TABLE 53.4 Cytokines approved for use in cancer Cytokine

Clinical indications

IL-2 IFN-a GM-CSF G-CSF IL-11 EPO

Melanoma, renal cell carcinoma Malignant melanoma, renal cell carcinoma, Kaposi’s sarcoma, follicular lymphoma, CML Supportive therapy: reconstitution of myeloid cells Supportive therapy: reconstitution of neutrophils Supportive therapy: chemotherapy-associated thrombocytopenia Supportive therapy: anemia-associated with cancer

patients with metastatic renal cell carcinoma (reviewed in Rosenberg (2000)). Such high-dose IL-2 therapy resulted in a significant clinical response (~20%) in this rapidly fatal disease (Rosenberg et al., 1998b; Fisher et al., 2000), thus demonstrating the concept that immunotherapy with cytokines has the potential to cure cancer. However, this regimen also resulted in very significant toxicity. Based upon this clinical efficacy, the FDA approved IL-2 for use in metastatic renal cell carcinoma, and later in 1998 for metastatic melanoma. Recent reports have confirmed the durability of the responses obtained with highdose bolus IL-2 in metastatic renal cell carcinoma and melanoma (Fisher et al., 2000; Atkins et al., 2000). The precise mechanism of high-dose IL-2 activity is not known, but is presumed to be mediated by augmentation of inadequate T cell responses against tumors (Rosenberg, 2000). However, other possibilities may be activation of other immune cells expressing the IL-2 receptor complex, such as macrophages and NK cells. The side-effects associated with high-dose IL-2 therapy has limited its use in many cancers. Alternative regimens of intermediate- and low-dose IL-2 or combinations of IL-2 and other biologic therapies are currently being evaluated in clinical trials (Caligiuri, 1993; Smith, 1997).

IFN-a immunotherapy IFN-a is currently used alone and/or in combination with various chemotherapeutic or biologic agents (e.g. IL-2) as adjuvant therapy or for the treatment of several cancers, including malignant melanoma, renal cell carcinoma, follicular lymphoma, chronic myelogenous leukemia (CML), and Kaposi’s sarcoma associated with HIV infection (Salgaller and Lodge, 1998; Krown, 1998; Russell-Jones, 2000; Fossa, 2000). There are varying reports of the efficacy of IFN-a in

many of these cancers. For example, in one large study adjuvant therapy with high-dose IFN-a following surgical resection of primary melanoma resulted in a significant improvement in relapse-free and overall survival in patients with high-risk disease, however severe toxicities were observed (Kirkwood et al., 1996). Other reports of low-dose IFN-a adjuvant therapy have shown that for intermediate-risk melanoma patients there is a disease-free survival benefit, but no overall survival benefit (Grob et al., 1998; Pehamberger et al., 1998). These differences are likely due to the doses of IFN-a used in these studies. Several randomized trials have shown IFN-a alone to be an effective therapy for the early phases of CML, and in combination with chemotherapeutics for the later stages of this disease (Hehlmann et al., 1994; Ohnishi et al., 1995; Guilhot et al., 1997; Kantarjian et al., 1999). The mechanism of IFN-a’s anti-tumor effect is currently unknown, with possibilities including antiproliferative effects and immunomodulation.

A role for cytokines in supportive therapy of cancer Perhaps the greatest utility of cytokines in cancer care has been for the management of cancer- and cancer treatment-associated side-effects, such as decreased white blood cell count, anemia and thrombocytopenia (Griffin, 2001). Administration of GM-CSF and G-CSF following chemotherapy or bone marrow transplantation can speed up recovery of myeloid cells, which can significantly reduce the risk of infection (Vose and Armitage, 1995). Anemia associated with cancer or occurring as the result of cancer therapy can often be alleviated by administration of EPO (Kasper, 2001). IL-11 can induce generation of platelets and is approved clinically for the treatment and prevention of thrombocytopenia following

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chemotherapy in high-risk patients (Schwertschlag et al., 1999). Additional cytokine growth factors are being pursued in clinical trials for cancer treatmentrelated side-effects, including IL-7 for lymphoid recovery following bone marrow transplantion (Alpdogan et al., 2001); stem cell factor following autologous transplant for the recovery of immune cells (Weaver et al., 1996); and IL-3 and thrombopoietin for thrombocytopenia (Yamamoto et al., 1999; Goldberg, 2000).

NOVEL STRATEGIES FOR CYTOKINE THERAPY While cytokines, as single agents, have not been highly successful in the treatment of most cancers, the utility of cytokines in combination with additional biologic therapies is now being considered in pre-clinical and clinical trials. Examples include administration of multiple cytokines, cytokines in combination with monoclonal antibodies, and cytokines as vaccine adjuvants (Sondel and Hank, 1997; Pawelec et al., 1999; Greten and Jaffee, 1999). There are also several novel mechanisms for cytokine administration such as cytokine gene therapy and cytokine–antibody fusion proteins, and cytokine– toxin fusion proteins are also being investigated, several of which are discussed in subsequent chapters (Penichet and Morrison, 2001; Kreitman, 1999). Various cytokine therapies employing these strategies are currently being tested in pre-clinical and clinical trials. For example, the combination of the cytokines IL-2 and GM-CSF was recently shown to be an effective therapy for a human EBV-lymphoproliferative disorder in a pre-clinical murine model (Baiocchi et al., 2001). Phase I and II clinical trials evaluating cytokine combinations such as IL-2 plus GM-CSF and IFN-a or IL-2 plus IL-12 have been reported or are ongoing with varying success (de Gast et al., 2000; Westermann et al., 2001; Correale et al., 2001). Addition of cytokines such as IL-2 to monoclonal antibody therapy has the potential to expand and activate immune effectors such as NK cells, which can then recognize and lyse antibody-coated cells. Various clinical trials evaluating such strategies are ongoing, including administration of IL-2 in combination with an anti-CD20 antibody (rituximab) for the treatment

of B cell malignancies (Porcu et al., 2001) or in combination with an anti-Her2/neu antibody (Herceptin) for the treatment of Her2/neu positive malignancies (Fleming et al., 1999). Tumor cells engineered to secrete a variety of cytokines such as IL-2, IL-4, GM-CSF, and IL-12 have been engineered as one strategy for cancer vaccines in an attempt to enhance the immune response to poorly antigenic peptides expressed by cancer cells (reviewed in Greten and Jaffee, 1999). An alternative vaccine strategy is to simply administer cytokines in combination with vaccines in order to enhance the immune response. For example, administration of IL-2 in combination with peptide vaccines to patients with metastatic melanoma appears to result in a higher level of cancer regression than IL-2 alone (Rosenberg et al., 1998a, 1999).

CONCLUSION The inflammatory process and in particular, proinflammatory cytokines have been implicated in the process of carcinogenesis and tumor progression. Cytokines produced by tumor cells or tumor stroma can stimulate the survival, proliferation and metastasis of cancer cells. While numerous cytokines have been evaluated in clinical trials, the greatest potential for cytokine therapy appears to be in combination with other biologic therapies, including monoclonal antibodies, cellular therapies and cancer vaccines.

ACKNOWLEDGMENTS We wish to thank Drs Todd Fehniger, Robert Baiocchi, Jaquelyn Ward and Susan Whitman for helpful discussions and scientific contributions. Portions of this work were supported by grants CA68458, P30CA16058, CA68458 and CA077658 from the National Cancer Institute.

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54 Interferon a: biology, pharmacology and therapy for chronic viral hepatitis Geoffrey M. Dusheiko Royal Free and University College School of Medicine, London, UK

When you have a great and difficult task, something perhaps almost impossible, if you only work a little at a time, every day a little, suddenly the work will finish itself. Isak Dinesen

INTRODUCTION

INTERFERON PROTEINS

Interferons (IFNs) are a family of secretory proteins synthesised by most higher organisms in response to viral challenge and non-viral inducing substances. The seminal observation by Isaacs and Lindeman in 1957 of a virus-resistant state induced in virusinfected cells, the further elucidation of the IFN (IFN) system, together with advances in recombinant DNA biotechnology, have led to the subsequent development of alpha interferons as therapeutic agents for viral hepatitis (Isaacs and Lindenmann, 1987). The IFNs were the first cytokines identified to be produced for antiviral therapy by recombinant DNA technology. Considerable success has been achieved in the treatment of chronic viral hepatitis.

The interferons are a system of related proteins, derived from a multigene family. There are at least 24 IFNa encoding genes, but only single beta and gamma genes. Naturally occurring IFNas comprise at least 23 predominantly non-glycosylated proteins of molecular weights between 17 000 and 22 000. There are four main groups: alpha, beta, omega and gamma. The IFNs have been classified into two types, based on receptor specificity. Type 1 IFNs (viral IFNs) include IFNa (leucocyte) IFNb (fibroblast) and IFN. Type II IFN is also known as immune IFN, i.e. IFN. The type 1 IFN genes have been mapped to the short arm of chromosome 9 in the human. There may be a critical antiviral role for IFNb (Deonarain et al., 2000). The single IFN genes maps to the long arm of chromosome 12 in the human. IFNa 1 (alpha and gamma) genes code for 165–166 amino acids. The gamma IFN gene encodes a protein of 174 amino acids. IFNa and b are induced in most nucleated cells in culture in

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

INTERFERON a: BIOLOGY , PHARMACOLOGY AND THERAPY FOR CHRONIC VIRAL HEPATITIS

IFN-c

IFNGR1 Jak-1

Tyk-2

Jak-1

91

91

JAK STAT pathway proteins The signal transducer and activator of transcription (STAT) family of proteins are cytoplasmic transcription factors that are phosphorylated and activated by the Janus family of tyrosine kinase (JAK) enzymes in response to cytokine stimulation (Figure 54.1). Receptor associated JAKs are activated following binding of IFNs to the cognate receptor. The activated JAK-1 and Tyk-2 kinases lead to the phosphorylation and dimerisation of STAT proteins, and subsequent translocation of the phosphorylated STAT forms to the nucleus. The upstream controlling element of the IFN stimulatable genes (ISGs) contains an element known as the IFN-stimulatable response element (ISRE). The primary activator of IFN-induced gene expression is the constituted complex known as IFNstimulated gene factor 3 (ISGF-3) which activates the transcription of IFN a/b inducible genes through the IFN-stimulated response element (ISRE) (Cohen et al., 1988; Rutherford et al., 1988). The ISGF-3 trimeric complex binds to a cis-acting element ISRE, found in IFN a/b inducible genes.

STAT 1 p

STAT 2

The cellular activities of IFNa are mediated by the products of the IFN-inducible genes. Samuel has reviewed the transcriptional activation of cellular gene expression and IFN-mediated signaling (Samuel, 2001). There are species-specific co-factors for activation of IFN function (Rashidbaigi et al., 1986; Jung et al., 1987). The expression of IFNa and b is controlled by regulatory elements within their 5 flanking promoter and enhancer sequences. A number of factors binding to these regulatory domains have been identified, including IFN response factors (IRF1 1 and IRF2) (Harada et al., 1989; Goodbourn, 1990).

STAT 1

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91

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91 p

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NUCLEUS

IFN SIGNALING

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response to virus infection or to double-stranded RNA (dsRNA). IFN act via species-specific surface target cell receptors. The alpha, beta and omega IFNs share a common receptor, which is encoded on chromosome 21. IFN is bound to distinct receptor subunits encoded on chromosome 6 and 21.

IFNAR2

1234

FIGURE 54.1 Schematic diagram of IFN signaling by the JAK-STAT pathway, IFNa and IFNb are depicted by the diamond, and IFN by the sphere. From Samuel (2001) with permission of the publisher.

For IFN, the phosphorylated STAT factor binds to a different cis-acting element designated gamma activated sequence (GAS) that is commonly found in IFN inducible genes. The cascade of this pathway is supported by data following experimental disruption of STAT and JAK genes. The IFN regulatory factor (IRF) family of transcriptional regulators are important regulators of IFNa and b gene promoter and ISRE. IRFs and STATS can function in conjunction to establish the signal transduction and gene regulatory sequences that lead to an antiviral state. Additional pathways of signal transduction involving RNA dependent protein kinase (PKR), mitogen activated protein kinase and other kinases may be active. Thus an elaborate cross-signaling and cellular negative regulatory system of IFN signaling exists (Samuel, 2001).

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INTERFERONS AND HOST DEFENCE

INTERFERONS AND HOST DEFENSE

Antiviral mechanisms of interferons The IFN response is an early host defense that precedes the onset of the immune response. Interferons interfere with several stages of the viral life cycle, although the actual mechanism varies with different viruses. Virus infection activates the transcription of a large number of cellular genes (Chang and Laimins, 2000). Protein phosphorylation, RNA degradation, RNA editing are thus modulated. IFN also induces a form of nitric oxide synthase (iNOS2) and the MHC class I and II molecules. IFNs thus induce several enzymes and proteins, the best characterised of which are the 25 oligoadenylate synthetases (25A synthetases), double-stranded (ds) RNA dependent protein kinases (PKR), RNase L and the Mx protein GTPases (Figure 54.2). IRF-3 is a key transcriptional activator induced by viral infection. The protein is a subunit of the double-stranded RNA activated transcription factor complex. IRF-3 is involved in the transcriptional activation of the IFNa

IFNa genes can be divided into two groups: immediate–early response, and IFNas, that are synthesised more slowly and require proteins synthesis (Marie et al., 1998). The natural IFNa producing cell is the precursor dendritic cell (Siegal et al., 1999; Tough and Kamath, 2001). IFN is produced by cells of the immune system including natural killer cells, CD4 TH1 cells and CD8 T cells following antigen-specific stimulation, and acts via a separate cell receptor. IFN is critical for innate and adaptive immunity. IFN has important immunomodulatory effects that intensify post-immune responses. These include activation of macrophages, natural killer cells and cytotoxic T-lymphocytes. The anti-inflammatory properties of IFNa are mediated via inhibition of the production of tumour necrosis factor interleukin-1 and IL-8 and stimulation of the production of IL-10.

Antiviral Actions of Interferon Multiple-Cell Responses

Single-Cell Responses IFN Protein Kinase PKR

Adenosine Deaminase ADAR1

Oligoadenylate Synthetase OAS

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Nitric Oxide Synthase NOS2 arginine

citrulline  NO

MHC Ag and Viral Ag Interaction (Cell Surface)

Virus-Specific Cytotoxic T-Cell Response Lysis of Infected Cells

RNA Degradation

FIGURE 54.2 Functions of selected IFN inducible proteins. From Samuel (2001) with permission of the publisher. CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

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INTERFERON

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BIOLOGY , PHARMACOLOGY AND THERAPY FOR CHRONIC VIRAL HEPATITIS

and IFN b promoters and IFNa and b responsive genes.

25 oligoadenylated synthetase and RNAse L The 25 A system consists of three enzymes: dsRNA dependent 25 A synthetase, RNase L (25 A dependent endoribonuclease), and 25 A phosphodiesterase (Kerr, 1987). 25 A synthetase is induced by type I IFN and activated by binding dsRNA. There are at least four forms of 25 A synthetase, located in different subcellular sites, which add to the complexity of this system. The enzyme synthesises 25 oligoadenylates of a general structure, abbreviated as 2–5A. RNase L, a latent endogenous ribonuclease, is activated by binding 2–5A oligonucleotides. Activated RNase L leads to breakdown of messenger and ribosomal RNA blocking viral translation (Williams and Kerr, 1978). The human RNase L protein is 741 amino acids, and is constitutively present in most cell types (Zhou et al., 1993). A phosphodiesterase catalyses the degradation of oligoadenylates. Three size forms of OAS termed OAS1, OAS2 and OAS3 have been identified in human cells (Samuel, 2001). They are encoded from a single gene. OAS proteins are activated during viral infection, but the activator RNA has not been precisely defined. The three different OAS forms are all induced by IFNa, b and .

Protein kinase PKR PKR is an IFN-inducible RNA dependent protein kinase (previously known as eIF-2a kinase). In the presence of ds RNA, dsRNA-dependent protein kinase catalyses the phosphorylation of several protein substrates, including the translation factor eukaryotic initiation factor 2 alpha (EIF-2a) (Samuel, 1979). Changes in protein phosphorylation mediated by the IFN-inducible PKR play an important role in the antiviral effects of IFNs and the control of protein translation. The serine phosphorylation of eIF2a leads to an inhibition of translation, which leads to the inhibition of protein synthesis: EIF2a causes irreversible binding of the guanine nucleotide recycling factor which is required for continued translation (Samuel, 1979).

PKR is a 551 amino acid protein with a molecular weight of 62 kDA. A number of RNA activators of PKR function have been elucidated, but the selectivity of these effectors has not been fully ascertained. IFNa is an efficient inducer of two PKR mRNAs in human cells (Meurs et al., 1990; Thomis et al., 1992). The functional significance of the three forms of PKR mRNA transcripts is unknown.

RNA dependent adenosine deaminase (ADAR1) Postranscriptional RNA modifications, such as deamination of adenosine to produce inosine, constitute an important mechanism of altering viral and cellular RNAs. ADAR1 is an IFN-inducible RNA adenosine deaminase that has been shown to edit viral RNA transcripts. RNA A to I transitions destabilize the ds RNA helix by disrupting base pairing, producing RNA transcripts that differ from their template, thus altering the protein coding capacity of the edited transcript and the sequence of replicated RNAs. Extensive editing may result. For example, RNA editing plays an essential role in the production of two hepatitis D proteins from one open reading frame, which have different functions in the life cycle of the circular ssRNA HDV (Lai, 1995). Human ADAR1 is a 1226 amino acid protein, encoded by a single gene on human chromosome 1q21.1-21.2. (Wang et al., 1995). Two immunologically related forms of ADAR are found in human cells. ADAR1 transcripts are increased by IFN treatment. Both IFNa and  lead to increased transcription of the protein.

Protein Mx GTPase The third important component system that has been characterized is the Mx system. The Mx1 gene encodes a protein which is strongly induced by type 1 IFN, but not by gamma IFN. Mx proteins are GTPases that belong to the superfamily of dynamin-like GTPases. The Mx proteins possess GTP binding motifs and have intrinsic antiviral processes. These may influence cellular transport processes, or may inhibit transcription of viral RNA. The net effect is impairment of nucleocapsid transport or transcription. The human genome contains two related Mx

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VIRAL ANTAGONISTS

proteins, MxA and MxB that are induced by IFN a/b (Aebi et al., 1989; Haller et al., 1998; Frese et al., 2001). The human mXA protein is 662 aa, whereas the mxB protein is 715 aa. Both proteins map to chromosome 21. The mouse genome, also encodes two Mx proteins.

Major histocompatibility complex proteins Virus-specific, MHC-restricted recognition and killing of infected cells by activated T cells is a key component of the host response to viral infection. Other antiviral mechanisms include the activation of expression of the class I major histocompatibility antigen (MHC) genes by all IFNs. However, only gamma IFN is an efficient inducer of class II MHC molecules. The effects of increasing and enhancing the expression of MHC at the cell surface, and thereby amplifying viral antigen display recognition are key components in recovery from viral infection. An essential role of IFN signaling and the Stat-1 transcription factors suggest an essential role of IFN signaling and the Stat-1 factor for basal expression of the MHC classs I antigens (Lee et al., 1999). The MHC classs II transactivator factor CIITA is the prime regulator of MHC class II expression. Expression of CIITA is inducible by IFN (Chang et al., 1994; Steimle et al., 1994).

NOS (nitric oxide synthase) and other proteins NOS catalyses NADPH dependent oxidation of L-arginine to produce nitric oxide (NO) and citrulline. Inducible NOS (iNOS) is inducible by gamma IFN. NO produced by iNOS plays a key role in immunological defences as an antimicrobial and antiviral agent, by affecting the antimicrobial function of activated macrophages. NO and derived reactive intermediates can affect killing of infected cells (Michel and Feron 1997; Nathan, 1997). DNA microarrays, representing several thousand distinct genes indicate that of the probe set, genes are identifiable as candidate genes that are differentially regulated by IFN treatment. It will be important in the future to identify other genes that are transcriptional targets of IFNs, and that are differentially expressed

in hepatitis B and C infection (Bigger et al., 2001). Interestingly, profile analysis of gene expression using oligonucleotide-based microarray gene chips suggest that the most abundant IFNa induced mRNA in HT1080 cells is the IFN-inducible P56 protein (Der et al., 1998). However, the role of P56 in the antiviral mechanism of IFN is unclear.

VIRAL ANTAGONISTS Viral antagonists of IFN signaling have been reported (reviewed in Samuel, 2001). IFNs modify the cellular and humoral immune response: expression and activation of natural killer cells and T lymphocytes are affected. Early and late events in the viral life cycle, such as uncoating of viruses, or integration of proviral DNA, are modified by IFNs. However, viruses may decrease IFN production from infected cells, and certain viruses may have evolved mechanisms of inhibition of IFN production (McNair and Kerr, 1992). Several DNA and RNA viruses encode proteins that impair the activity of the the JAK-STAT signaling pathway. Numerous mechanisms apply that affect or block IFN signaling, or circumvent the IFN-induced antiviral state. Viruses that have evolved mechanisms that interfere with class II expression, could conceivably contribute to persistent infection through escape from immune surveillance. Transgenic mice studies and studies in chimpanzees suggest that antiviral mechanisms of clearance of hepatitis B may involve both target lysis and inhibition of hepatitis B replication by non-cytolytic mechanisms (Guilhot et al., 1993; Cavanaugh et al., 1998; Guidotti et al., 1994, 1999). Inactivation of hepatitis B by CTL elaborated cytokine expression conceivably occur via by elimination of HBV nucleocapsids and destabilisation of HBV RNAs. It has been indeed been suggested that a rationale for IFN treatment of chronic hepatitis B is a relative defect in adult carriers in the production or action of IFN, or an inhibition by hepatitis B of the activation of the IFN system (Onji et al., 1989; Twu et al., 1988). In particular, IFN is not detectable in the serum of patients chronically infected with HBV. Proteins of HBV may affect the inducibility of IFNb by dsRNA in cotransfection experiments. HBV core and HBeAg proteins are able to inhibit IFN production. (Twu

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INTERFERON a: BIOLOGY , PHARMACOLOGY AND THERAPY FOR CHRONIC VIRAL HEPATITIS

et al., 1988). The mechanism of action of core protein is not yet known, but the carboxy terminal region may function by binding directly to DNA elements within the promoter region. The enhancer 1 region of hepatitis B has an ISRE element that is a target of IRF (Nakao et al., 1999). IFN treatment suppresses the activity of the HBV enhancer. Mutation of the HBV vISRE reduces the suppressive effect of IFNa. Other mechanisms may also operate: HBV DNA has been detected in lymphoid cells from bone marrow and T cells, and may be responsible for direct inhibition. HBeAg may have an indirect effect on IFN production by circulating lymphocytes. Against the fact that HBV inhibits IFN, however, is the evidence that endogenous IFN is indeed activated, and that elevated 25 synthetase concentrations can be detected during spontaneous HBeAg seroconversion or with IFN treatment (Quiroga et al., 1988; Heathcote et al., 1989). Hepatitis C antagonism of PKR and eIF2a phosphorylation is linked to inhibition of antiviral response (Figure 54.3). Type 1b HCV resistance to IFN therapy is associated with changes in the NS5a gene region. The HCV envelope hypervariable region (HVR1) located at the 5 end of the E2 gene, shows changes in the HVR1 quasi-species major variants during antiviral therapy. The underlying mechanism is unknown. Patterson and Samuel suggests that IFN-mediated selection for resistant virus resulting from editing of HCV viral RNA catalysed by the IFN inducible RNA editing enzyme ADAR1 could play a role (Patterson and Samuel, 1995).

Hepatitis C viral genome 5

core

E1

E2

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PKR-binding eIF-2a

IFNa

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Phosphorylation

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IFNa sensitivity Inhibition of HCV replication

Interferon Resistance and PKR

FIGURE 54.3 Putative IFN resistance of hepatitis C virus, and PKR.

The HCV envelope protein E2 and non-structural protein NS5A both inhibit PKR under experimental conditions, although the data are controversial. Experimental expression of the HCV polyprotein in UHCV-11 cells impairs signal transduction through the JAK-STAT pathway which could have the effect of influencing IFN-mediated regulation of many relevant genes.

THERAPEUTIC USE OF ALPHA INTERFERONS IN CHRONIC VIRAL HEPATITIS IFNas are naturally occurring substances that are now used therapeutically for their properties of inducing an antiviral state in cells, and inhibiting cellular proliferation and immunomodulation. Alpha, beta and gamma IFNs were recombinantly cloned and became available for therapeutic use after 1981. Several preparations of IFNa are currently available, two of which are recombinant (Roferon A, alfa-2a, Hoffmann La Roche; Intron A, a-2b, Schering), (and are more widely used) and one of which is prepared from a lymphoblastoid cell line (Wellferon, alfa 1n, Burroughs Wellcome). The latter is not in wide use. Recombinant methionyl human IFN consensus is a novel, nonnaturally occurring type 1 IFN that was synthesised at Amgen Inc. In vitro, consensus IFN (Infergen) displays a greater specific antiviral activity than other type 1 IFNs. Consensus IFN has also been licensed for the treatment of hepatitis C. Twelve kd and 40 kd pegylated IFNas have recently been licensed for the treatment of hepatitis C: the FDA has licensed pegylated a-2b (PEG-Intron, Schering) and pegylated IFNa 2a (Pegasys, Roche) for the treatment of chronic hepatitis C. Therapeutic preparations consist either of a mixture of IFN species derived from virus-stimulated Namalwa cells (Wellferon), highly purified and presented as a frozen, clear, colourless liquid, or recombinant DNA-produced single-component IFNa species (Intron A, Roferon-A, Con-IFN), presented as a white, freeze-dried powder with water for reconstitution. All are administered parenterally, either intramuscularly or subcutaneously. If IFN is specifically indicated in patients with bleeding problems cautious intravenous administration may be considered.

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THERAPEUTIC USE OF IFN a

Clinical pharmacology IFNs are perhaps best known for their antiviral activity, but they have a multitude of other effects. IFNs bind to specific cellular IFN receptors and inhibit the replication of a wide variety of RNA and DNA viruses. IFN also has antiproliferative effects on both normal and malignant cells and the effects on malignant cells may be due to a re-imposition of the normal control mechanisms for cell growth. IFN also has immunodulatory effects on the immune system. IFNs bind to their natural cellular receptors, downregulating the level of high affinity receptors and rendering the cell temporarily refractory to further IFN activation. The dose varies, depending on the clinical indication, from a generalised therapeutic range of 1.5–10 mega units given daily or three times per week for most indications, up to much higher doses in AIDS-related Kaposi’s sarcoma. Dosage is determined by a balance between therapeutic efficacy and patient tolerance and both can be highly variable from individual to individual. In toxicity studies, doses up to 200 mega units m2 body surface area have been used. In general, doses above 10 mega units m2 are associated with a significant incidence of unacceptable side-effects. The pharmacological actions induced by binding of the IFN ligand to its cellular receptor are the induction of a series of cellular proteins and enzymes with potential antiviral properties in that cell, and concurrent inhibition of cell proliferation. Certain cell types, such as natural killer (NK) cells, are activated by IFNa and this may contribute to IFN anti-neoplastic properties. IFNs are intimately involved in normal cell cycle events, as well as in natural defense against viruses and tumours, and have widespread effects on all tissues studied, including alterations of cellular growth rate, membrane fluidity, display of cell surface proteins, etc. The specific activities are dose-dependent and the stimulatory effect observed on some cellular activities may at higher doses become overwhelmed by the antiproliferative effects. In vitro, at very high dosages, IFN is toxic to homologous cells.

Pharmacokinetics The preferred assay method is a bioassay for the total activity of the drug. Bioassays detect the antiviral

activity of the drug and are compared with international standards which are sensitive down to one unit of IFN. Absorption from the gastrointestinal tract and presystemic metabolism are unknown. The true elimination half life is 3–4 h, but peak plasma levels are seen 4–8 h after injection. It is not known whether IFN appears in breast milk. Active drug is receptor bound and probably internalised and plasma levels are not a relevant measure of therapeutic effect. Concentrations 1–5% of plasma level have been detected in CSF. IFN crosses the placenta and enters the fetal circulation. IFN is excreted in the urine, reabsorbed by the proximal tubules and undergoes lysosomal degradation in these cells. IFN bound to its target cells is thought to be internalised and degraded in the target cell.

Pharmaceutics Wellferon (Wellcome, UK) is supplied as a frozen 1-ml volume of liquid containing either three million units or ten million units IFNa-N1. The drug is not in wide therapeutic use. Intron A (Schering, USA) is provided as a powder for reconstitution in units of either three million, five million or ten million units. Intron A vials include 1 mg ml1 human albumin, 20 mg ml1 glycine and sodium phosphate. Roferon-A (Roche) is provided as a powder for reconstitution in either three million or nine million units (18 million in USA) IFN a-2a Roferon-A vials include 5 mg ml1 human albumin and 3 mg ml1 phenol. Consensus IFN (Infergen) is a 166 aa protein with similar pharmacokinetics to alpha 2a and alpha 2b IFN.

THERAPEUTIC USE OF IFNa The knowledge that IFNs exert an important endogenous antiviral effect led to their utilisation in patients with chronic viral hepatitis. Recombinant IFNas have since been approved for clinical use worldwide. Chronic viral hepatitis caused by hepatitis B, C or D may lead to cirrhosis, hepatocellular failure and hepatocellular carcinoma. The morbidity of these diseases has necessitated a prolonged search for effective therapy. Although a substantial number of antiviral compounds has been evaluated for the treatment of

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chronic viral hepatitis, few have achieved clinical applicability. IFNa has been widely studied, and remains a mainstay of treatment. Several new nucleoside and nucleotide analogues, including lamivudine, and adefovir dipivoxil, have been licensed for the treatment of hepatitis B, and other nucleoside analogues are in phase III trial. Nucleoside analogues, alone or in combination with IFNa, may prove to be useful adjuncts to the treatment of chronic hepatitis B and C. A number of other cytokines, including thymosin, and IL-12 have been evaluated.

HEPATITIS B Hepatitis B is caused by the hepatitis B virus (HBV), a large (42 nm) enveloped DNA virus. HBV exists as a 42 nm particle in serum, containing an outer envelope component of HBsAg and an inner nucleocapsid component of hepatitis B core antigen (HBcAg). The genome of HBV has been cloned and four open reading frames delineating the four gene products of HBV have been identified (Galibert et al., 1979), all of which appear to represent functional genes. The three envelope regions of the genome encode HBsAg, preS1 and pre-S2 polypeptides. The C region encodes HBcAg and HBeAg, the P gene encodes the viral polymerase, and the X gene encodes a small protein with transactivating activity (HBxAg) (Siddiqui et al., 1986). Pres-S may play a role in attachment of HBV to hepatocyte receptors. Nucleocapsid antigens (HBcAg and HBeAg) expressed on the cell membrane contain important targets of the immune response and cytolytic T cells in acute hepatitis B. These antigens are products of a single gene region on the virion genome (Stahl et al., 1982). The C gene has two initiation codons and therefore two gene regions (pre-core and core) and two potential molecular forms (HBcAg and HBeAg). Initiation of translation at the first site (nucleotide 1814) produces a 312 amino acid polypeptide (p25) which has a signal peptide directing it to the endoplasmic reticulum, where the signal piece is removed by signal peptidase to cleave the N terminal 19 aa residues, as well as the C terminal 34 residues. The resultant polypeptide of 159 amino acids, is secreted as HBeAg (p15-18), a soluble protein that is the product of 10 residues coded by the pre-core

region and 149 residues coded by the C gene (Ou et al., 1986). Translation from the second initiation codon (nucleotide 1901) results in unprocessed polypeptides (p23, 183 amino acids), which are assembled into core particles within the liver (P21). HBcAg is a 27 nm particulate nucleocapsid protein. HBcAg is not detected in serum, but is an intracellular virion associated nucleocapsid antigen that is the product of the C gene alone. HBeAg therefore has substantial amino acid homology to HBcAg, and the proteins are crossreactive at the T cell level. Studies using recombinant HBcAg and HBeAg have shown that HBcAg can be detected in liver, usually in the nuclei of hepatocytes. HBeAg is detectable as a soluble 17 kDa protein in serum. The polymerase encoded by the viral P gene has both DNA-dependent and RNA-dependent polymerase activity. Quantitative detection of HBV DNA correlates with infectivity, and is an important means of determining the presence of viral genomes, and determining response to antiviral therapy. Amounts of HBV DNA are expressed in pg ml1, or alternatively, genome equivalents ml1. At least 103 virions have to be present in a specimen to be detected by polymerase chain reaction (PCR). PCR permits the detection, cloning and sequencing of HBV genomes from such patients. It is at least 104 times more sensitive than dot blot hybridisation assays for HBV. Although serious hepatitis may occur as a result of acute hepatitis B, the optimal outcome of acute hepatitis B is eradication of infected hepatocytes, curtailment of viral replication, and rapid hepatic regeneration. While acute and chronic infection are undoubtedly due to viral infection, the nature and pattern of infection suggest that immunological factors are important. Peak HBV replication occurs well before peak cellular injury in acute hepatitis, suggesting that the disease represents immune lysis of infected hepatocytes. Intense hepatocyte lysis occurs in acute hepatitis B, apparently as a result of a polyclonal, multispecific cytotoxic lymphocyte immune response. The specific epitopes recognised by B, as well as T cells are currently being mapped. In addition, experimental evidence suggests that elimination of viral DNA from infected hepatocytes is dependent upon cytokine-mediated reduction of replicative intermediates. Hepatitis B virus has a propensity to cause chronic

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HEPATITIS B

infection, particularly in those hosts affected early in life. An inverse relationship exists between the age of infection and the probability of chronic disease. In neonates, specific suppression of the cell-mediated immune response may favour infection, perhaps because of transplacental exposure to maternal HBeAg, inducing tolerance (within the thymus) to epitopes that are usually the target of the cytotoxic T cell response, at a time when the immune system is ontogenically ‘immature’. Immunological tolerance may be overcome later in life. This altered T cell tolerance may coincide with the appearance of raised ALT levels (Hsu et al., 1992). During chronic HBV infection, there is some evidence of failure of IFN production and activation of the infected liver cells so that viral protein synthesis is not decreased and there is poor enhancement of HLA protein display. Such a mechanism might result in failure of presentation of HLA antigens on the cell surface, and hence poor presentation of viral peptides to the immune system. Varying levels of HBV replication may be found during chronic infection. There is a spectrum of disease in patients ranging from minimal hepatitis to rapidly progressive liver injury and cirrhosis. There is little correlation between the severity of illness, and the amount or level of viral replication or viral antigen production. Patients with the highest level of virus in liver and serum typically have the mildest disease. Large amounts of HBsAg and HBcAg can be found in liver biopsies with minimal evidence of cellular necrosis (Bianchi et al., 1979). These findings suggest that HBV is not cytopathic and that failure of clearance of virus is due to a failure of an adequate immune response. It is an oversimplification to say that host factors alone determine the outcome of HBV infection, or that the virus is totally non-cytopathic: for example in transplanted, immunosuppressed patients, high levels of HBsAg may be associated with a the syndrome of fibrosing cholestatic hepatitis (Lau et al., 1992). Some patients with chronic hepatitis B have a remission in disease, marked by disappearance of HBV DNA and HBeAg from serum with improvement in serum aminotransferases, despite persistence of HBsAg. HBeAg seroconversion can occur with the loss of HBeAg and acquisition of HB envelope antibody, at which time a marked decrease in HBV DNA concentrations occurs from levels of 7–10 log10 copies ml1 to

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5 log10 copies ml1. This change is usually accompanied by normalisation of serum alanine transaminase (ALT). Measurement of serum HBV DNA in patients with chronic hepatitis B by polymerase chain reaction (PCR) demonstrates that the loss of HBeAg is associated with a decrease rather than complete clearance of HBV DNA in the serum (Kaneko et al., 1989). After some years, however, a proportion of patients may remain HBeAg negative or anti-HBe positive and experience periodic exacerbation of disease activity accompanied by higher serum levels of HBV DNA (106 copies ml1). These patients are infected with variants of HBV not expressing HBeAg, so-called HBeAg minus or pre-core mutant disease. PCR amplification and subsequent sequencing of genomic DNA in patients with this form of chronic hepatitis B has indicated that one or more nucleotide substitutions in the pre-C region of the genome account for the absent expression of HBeAg. A point mutation from guanine (G) to adenine (A) at nucleotide 83 (G1896A) creating an in-frame TAG stop codon converting coding 28 position converting codon 28 for tryptophan (TGG) to a stop codon (TAG) has been the most common finding, with or without additional point mutations in succeeding codons. This substitution prevents the production and secretion of HBeAg. In most patients there is a requirement for active viral replication in the pathogenesis of liver injury, and improvement in histology follows IFN-induced or spontaneous seroconversion to anti-HBe. Conversely, cells which do not express nucleocapsid antigens may be protected from the host immune response. Why some patients develop severe disease and not others has yet to be explained. Core mutations may also be relevant.

Treatment of acute hepatitis B Most cases of acute symptomatic hepatitis B resolve with clearance of HBsAg and complete healing of the hepatic injury. Ninety-five percent of adults recover following acute infection; the other 5% remain chronically infected. Although non-specific symptoms may be improved, there is no evidence that anti-viral therapy with IFNa accelerates this healing or clearance of virus or that early treatment with IFN prevents the development of chronic disease. At present,

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therefore, neither IFNa nor any other antiviral agent should be routinely used to treat patients with typical uncomplicated acute hepatitis B.

effective in less than half of cases of chronic hepatitis B, and is relatively expensive. IFNa requires administration by injection, and is not without side-effects. Nonetheless, recombinant IFN has been licensed for treatment of chronic hepatitis B in many countries.

Fulminant hepatitis B Low levels of viral replication are found in patients with fulminant hepatitis, suggesting that the pathogenesis of the disease is due to an exaggerated immune response to HBV. Recent evidence suggests that HBV pre-core mutants are more likely to induce fulminant disease. This is not invariable however, and the pathogenesis is uncertain (Kosaka et al., 1991; Liang et al., 1991). IFNa has not been found to be beneficial in fulminant hepatitis (Levin et al., 1989). Currently the optimal, albeit drastic therapy of fulminant hepatitis B is orthotopic liver transplantation, once prognostic factors indicate that survival is unlikely. Lamivudine or other nucleoside analogues may be indicated for patients with subacute hepatic necrosis, whose acute disease is progressing with ongoing viral replication.

Antiviral therapy for chronic hepatitis B Interventional treatment of chronic hepatitis B is targeted at patients with active disease and viral replication, preferably at a stage before signs and symptoms of cirrhosis or significant injury have occurred. Eradication of the infection is possible in only a minority of patients. Permanent loss of HBV DNA and HBeAg is considered a response to antiviral treatment, as this result is associated with an improvement in necro-inflammatory damage, and reduced infectivity. It is possible, but unproven, that the accompanying reduction in histologic chronic active hepatitis lessens the risk of cirrhosis and hepatocellular carcinoma. HBV DNA may still be detectable in serum and liver after loss of HBeAg and HBsAg induced by antiviral therapy, but this is often not associated with progressive disease (Marcellin et al., 1990b). Therapy for hepatitis B became possible with the demonstration that IFNas inhibit HBV replication and that prolonged therapy can lead to a remission in disease, although the mechanism of action of IFNs in chronic hepatitis B infection is poorly understood. Unfortunately, IFN therapy of chronic hepatitis is

Treatment of HBeAg-positive chronic hepatitis B Early pilot studies pointed out the inhibitory effect of recombinant alpha2a IFN upon HBV replication and the therapeutic potential of this drug (Dusheiko et al., 1985). Later, in controlled, dose-finding studies, predictors of response could be tentatively defined, dose ranges narrowed and therapeutic strategies outlined (Dusheiko et al., 1986). A response to IFNa is defined by a loss of HBV DNA and HBeAg from serum, fall of serum aminotransferase activities into the normal range and subsequent improvement in liver histology. HBeAg may be not become undetectable for some months after HBV DNA is negative. Although residual HBV DNA can be detected by PCR in serum, the disease appears to be ameliorated. Approximately 20% of patients who respond to treatment with clearance of HBeAg will also clear HBsAg within a year of therapy, and up to 65% may later clear HBsAg after 6 years of follow-up (Korenman et al., 1991). Response is generally associated with histological reduction in inflammation. (Brook et al., 1989). In large series of studies, more chronic carriers treated in international multicentre, randomised trials became negative for HBeAg, and were negative for HBV DNA, compared with untreated patients. A significantly higher percentage of patients treated with 10 million units m2 also became HBsAg-negative (Ryff, 1993). Response rates in most of these studies were higher in carriers with higher baseline serum aminotransferase levels those in those without elevated aminotransferases. The subclinical exacerbation of the hepatitis frequently seen in responders suggests that IFN acts by augmenting the immune response to HBV, perhaps triggered by the inhibition of viral replication, as well as the effects of IFN on cytotoxic T cells. Based on the data obtained from these studies, indications for treatment and definitions of response can be formulated. (Hoofnagle 1990a, 1990b; Lok et al., 2001). The immediate goal of therapy is to eradicate viral replication and ameliorate the underlying

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liver disease. Recommendations for therapy should be cautious, because therapy is effective in only 30 to 50% of patients, the optimal dose and duration of therapy remain unclear, and side-effects can be problematical. Patients with high serum ALT concentrations and relatively low HBV DNA concentrations are the ideal candidates for this treatment. However, many patients are not candidates for therapy with IFNa because of poor prognostic features or doselimiting side-effects. Also, IFN-a is contraindicated in patients with advanced liver disease, as hepatic decompensation may occur. Anti-HIV-positive patients tend to have higher levels of HBV replication (Bodsworth et al., 1989), and are less responsive. Although these factors provide some predictive information, none of these criteria are absolute, and individual carriers, for example ethnic Chinese, with active disease, or those patients with anti-HIV but normal CD4 lymphocyte counts may respond, making the prediction of treatment outcome somewhat difficult. The major advantage of IFN-a is that long-term HBeAg seroconversion occurs in a proportion of patients after a finite course of 16 weeks of treatment. Treatment is administered at 5 million units (MIU day or 9–10 MIU) three times weekly. Prospective studies have shown that Asians have a low rate of response to IFNa and they frequently relapse (Lok et al., 1989). For these reasons, IFNa therapy should be limited to those oriental patients with active disease and elevations in serum aminotransferases. Furthermore, these patients should be treated only after explanation of the low possibility of response. Inactive HBsAg positive carriers with HBsAg, but with normal serum aminotransferases and concentrations of HBV DNA in serum of less that 105 ml1 should not be treated, as IFNa has no effect on HBsAg alone, and serum aminotransferases may actually worsen during treatment. The role of IFN antibodies in reducing response is controversial, but neutralising antibodies may be one variable which affects response to IFN. Alpha N1 is a natural mixture of IFNa produced by human lymphoblastoid cells after viral stimulation. a-2a and 2b are recombinant preparations produced in E. coli. a2a has a lysine molecule at position 23 instead of an arginine present in a-2b. A higher proportion of patients with chronic hepatitis (20%) treated with a-2a develop neutralising antibody (20% versus 6.9% versus

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1.2% among patients with alpha N1). Thus a-2a IFN may be more immunogenic (Antonelli et al., 1991; Milella et al., 1993). In some patients loss of efficacy or ‘breakthrough’ may correlate with antibodies, and a neutralising effect can be attributed in some patients who have a breakthrough after an initial response, and in whom response occurs after treatment with an alternative IFN. However, it is still difficult to draw firm conclusions regarding the clinical significance of IFN antibodies. The patients treated are often heterogeneous, other factors affect the response and patients may respond despite developing antibodies. There are also different assays for measuring antibodies including immunoassay, immunoradiometric assays and antiviral neutralisation bioassays. The prescription of IFNa is also limited by its relatively high cost, but an economic evaluation of the cost of IFNa for chronic hepatitis B suggests that some benefit can be obtained (Garcia de Ancos et al., 1990). IFNa should not be used with decompensated hepatitis B because of the risk of inducing severe hepatic necrosis. Asian children, or other children who are infected perinatally, have mild disease activity, and respond poorly to IFN treatment. They appear to be profoundly tolerant to HBV, perhaps as a result of HBeAg tolerising the neonatal thymus, and thereby impairing the clonal T cell response to HBV (Milich et al., 1990). In contrast, children with active disease and high serum aminotransferases respond to IFN therapy similarly to adults and usually accept treatment well (Ruiz-Moreno et al., 1990). The appropriate dose of IFN in children is 5 million units m2 s.c. three times weekly for 4 months. Patients who do not respond to therapy with IFNa represent a difficult management problem. Lamivudine or other nucleoside and nucleotide analogues, used singly, but more likely in combination, are currently being studied. Phase III trials combining lamivudine and adefovir, for example, are in progress.

Patients with anti-HBe positive chronic hepatitis Patients who are anti-HBe-positive, but who have hepatitis B core antigen in hepatocytes and histologic chronic active hepatitis due to infection with HBV pre-core mutants affecting the expression of HBeAg,

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may have severe disease. In parts of Europe, and elsewhere, disease caused by this variant of hepatitis B is more common than disease caused by ‘wild type’ of HBeAg-positive chronic hepatitis. In preliminary studies, approximately 25% of patients have longterm responses (Brunetto et al., 1989). In a trial of IFNa2a therapy with 9 million units given i.m. three times weekly for 16 weeks, 33% of anti-HBe-positive carriers lost hepatitis B core antigen, compared with none of the controls. In a second study of lymphoblastoid IFN (5 million units m2 for 6 months) in 30 patients, at 18 months, 53% of the treated patients had responded, versus 17% of the controls (Fattovich et al., 1992). Histologic improvement was seen in 53% and hepatitis B core antigen disappeared in 66%, compared with 33% of untreated patients. Longer term evaluation has indicated that patients have tended to relapse with time. Thus although response rates are high, many patients relapse when treatment is stopped. Approximately 40% to 60% of anti-HBe-positive patients with increased serum HBV DNA concentrations respond initially, but durable responses are only observed in 15% to 25% of patients. The disease can be somewhat heterogeneous, thereby complicating the analysis of the results of IFNa trials. There have been 18 trials of IFN for anti-HBe-positive chronic hepatitis B (summarised by Alberti et al., in Lok et al., 2001). These include four randomised controlled trials. The 12 month sustained response rate has varied from 10 to 47% (averaging 24%) in treated versus 0% in untreated patients. The endpoint used for most studies was persistently negative HBV DNA (by hybridisation) and persistently normal ALT. Longer duration of therapy (12 months versus 6 months) was associated with improved response. The response is not necessarily durable and up to 50% of patients become DNA positive after 5 years of follow-up. However, up to 15–32% of treated patients may become HBsAg-negative.

Patients with advanced cirrhosis Patients with cirrhosis and ascites, encephalopathy or persistent jaundice from chronic hepatitis B have a dire prognosis without treatment, but are prone to developing multiple, serious side-effects from higher (2–10 lg) of IFNa, particularly bone marrow suppression, neutropenia and an increased incidence of seri-

ous bacterial infections (Kuo et al., 1991). Low doses of IFN (1–3 million units three times a week) could be considered in these patients (Rakela et al., 1990). This treatment should only be administered cautiously be physicians with experience of IFN, who can monitor the patient weekly, and where liver transplantation is available. (Nucleoside analogue therapy with lamivudine has proven important in patients with decompensated cirrhosis, prior to liver transplantation.)

Immunosuppressed patients Chronic hepatitis B is common among patients on renal dialysis, patients with malignancies receiving chemotherapy and patients who have kidney, heart or liver transplants. IFN has little effect in patients with major immune deficiencies (Davis, 1989). Retrospective analyses have suggested that patients with anti-HIV do not respond to IFN as well as non-HIVinfected patients. However, good responses may occur in some patients with anti-HIV but normal peripheral CD4 T lymphocyte counts, so that perhaps anti-HIV alone should not exclude patients from therapy. Likewise, lamivudine therapy or therapy with adefovir dipivoxil improves responsiveness in immunosuppressed patients. However, antiviral resistance is common in patients receiving lamivudine for longer than 1 year.

Extrahepatic disease Glomerulonephritis caused by chronic hepatitis B virus has been treated with recombinant a 2b IFN at a dose of 5 million units subcutaneously (LiskerMelman et al., 1989) daily. In patients in whom HBeAg disappeared and aminotransferases fell into the normal range, urine protein excretion also decreased and resolution of the glomerulonephritis occurred. The effects of IFN therapy have not been as well studied in this group of patients.

Treatment of hepatitis B with other IFNs and cytokines The pharmakinetics, tolerance and anti-viral effect of human recombinant IFN have been studied in patients with chronic active hepatitis B, but most

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ANTIVIRAL TREATMENT OF HEPATITIS C VIRUS

studies suggest that the antiviral effect of IFN is less than that of IFNa (Marcellin et al., 1990a). In eight patients treated with a combination of gamma and IFNa, IFN alone had minimal inhibitory effects upon serum levels of hepatitis B virus DNA (Lau et al., 1991). Troublesome side-effects were also encountered. When combined, the two IFNs showed little synergistic effect in inhibiting HBV DNA (Di-Bisceglie et al., 1990). A study of gamma and IFNb treatment of 20 patients with chronic hepatitis B was successful in reducing viraemia in five of 10 patients treated for 23 days. The IFNb was given intravenously, and the IFN subcutaneously (Caselmann et al., 1989). Thymosin is an immune stimulant which is known to enhance suppressor T cell activity and in vitro B cell synthesis of IgG. Peptide preparations of thymosin have been evaluated in controlled trials, and HBV DNA has been noted to become negative in some of these patients with chronic hepatitis B (Mutchnick et al., 1991; Chien et al., 1998). It is given parenterally.

ANTIVIRAL TREATMENT OF HEPATITIS C VIRUS A viral agent responsible for most cases of posttransfusion hepatitis was recognised after serological tests for hepatitis A and hepatitis B viruses became available in 1974 (Feinstone et al., 1975). HCV is an enveloped flavivirus with a plus strand RNA genome of 9.5 kb that encodes a precursor protein of 3000 aa. Cleavage of this polyprotein precursor generates both viral structural proteins, including the envelope glycoproteins E1 and E2 and several nonstructural proteins. The enigmatic name of non-A, non-B (NANB) hepatitis was given to classify this form of hepatitis. In 1989, Houghton and co-workers succeeded in isolating nucleic acid of the major NANB agent by recombinant DNA technology (Kuo et al., 1989; Choo et al., 1989). A viral protein (5-1-1) was expressed in bacteria from the open reading frame of the cDNA clone. Immunoblot analysis was performed on bacterial lysates using serum obtained from chimpanzees which had been experimentally infected with NANB hepatitis. A radioimmunoassay was then devel-

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oped for detection of antibodies in patients with chronic NANB hepatitis, and most sera from a panel of well-characterised cases of NANB hepatitis reacted specifically, while controls were negative. This virus has been given the nomenclature hepatitis C virus (HCV). Newer tests for diagnosis of hepatitis C include detection of antibodies to several recombinantly expressed HCV antigens, or peptides (c100-3, c22, c33, c200, NS5) and detection of HCV RNA in serum by polymerase chain reaction amplification. Several isolates of hepatitis C have been cloned. The sequence divergence of these isolates indicates that there are several major genotypes of hepatitis C, and component subtypes. Geographic localisation of these types has been reported. The natural history of the disease is uncertain. The virus has a disturbing propensity to cause chronic infection. The risk of further progression is probably cumulative, but is influenced by other cofactors including age of acquisition, alcohol abuse and coinfection with HIV infection. The infection causes systemic disease, and may be associated with a number of systemic complications including a form of autoimmune hepatitis, cryoglobulinaemia, porphyria cutanea tarda, lymphocytic sialedinitis, and membranous glomerulonephritis. It is thought that approximately 15% to 40% of individuals infected with HCV spontaneously recover. Most hepatitis C-positive infected individuals with mild non-specific symptoms have elevated serum ALT levels and either minimal to moderate fibrosis and mild or moderate necro-inflammatory lesions in the liver. Many of these may not develop advanced liver disease. However, about 10% to 20% of infected patients have progressive liver disease and go on to develop cirrhosis 10 to 20 years after acquiring the infection. They are at risk of complications of cirrhosis, including clinical sequelae of portal hypertension and hepatocellular carcinoma. There is a high prevalence of hepatitis C in haemophiliacs, thalassaemics, haemodialysed patients, transplant recipients and intravenous drug abusers. Intrafamilial transmission may occur, but appears to be relatively infrequent in developed countries. In some countries, patients with type II autoimmune CAH appear to have a high frequency of genuine exposure to HCV, and antibodies to liver and kidney microsomal antibodies are present in patients. These

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patients may also have circulating antibodies to a pentadecapeptide (Gor), an epitope of normal hepatocytes. This phenomenon may represent an autoimmune response peculiar to type C hepatitis (Mishiro et al., 1990). This association has some therapeutic implications, as in some of these patients the disease may be aggravated by IFNa and is responsive to corticosteroids.

Treatment of acute hepatitis C The management of acute sporadic or transfusionrelated hepatitis C includes conventional supportive treatment and specific antiviral therapy. The disease may be relatively silent in the acute phase, as most (75%) patients are not jaundiced, and have only nonspecific symptoms. Thus the disease is not often apparent. Current tests should enable a diagnosis to be made in many if the disease is suspected. The diagnosis may require testing for HCV RNA, as some patients may not have anti-HCV at the time that serum aminotransferases are elevated. Approximately 50–60% of patients will still have elevated serum aminotransferases 6 months after diagnosis (Alter et al., 1992). Therapeutic trials of IFNa have been undertaken. Most have included treatment with IFNa for 12 weeks of therapy, and shorter trials have not resulted in lasting benefit by reducing the rate of chronic disease. These studies might indicate an amelioration of the severity of the chronic hepatitis lesion. A trial of IFNb in Japan, given intravenously for 1 to 3 months, did significantly reduce the risk of chronic hepatitis, however (Omata et al., 1991). Recent studies have made clear the fact that treatment benefits those patients who have been treated early, but it may be reasonable to allow 1–3 months to determine which patients might convalesce spontaneously (Jaeckel et al., 2001). If a diagnosis of acute hepatitis C can be made, and the patient does not appear to be convalescing 2–4 months after the onset of the disease, IFNa can be considered at a dose of 3–6 million units three times a week for at least 6 months. It is not yet clear whether pegylated IFN would offer an advantage, or whether combination treatment is essential (Hoofnagle, 2001).

Treatment of chronic hepatitis C IFNa has been standard treatment for more than a decade. Therapeutic trials of IFNa indicate that a proportion of patients may respond to treatment with this agent. Larger, placebo controlled studies have indicated that approximately 50% of patients will have normal serum aminotransferases after treatment courses of IFNa of approximately 3 million units three times a week for 6 months (Davis et al., 1989). However, after stopping treatment, one half of the responsive patients promptly relapsed. Serum aminotransferases usually increase in patients who are HCV RNA positive at the end of therapy, although in some cases the relapse may be delayed for several months. The low response rate to IFN is not fully understood, but could include factors such as HCV genotype, functional antagonism of one or more cellular components involved in the signalling or action of IFN (Polyak et al., 1999; Tan et al., 1999; Taylor et al., 1999). Other patients do not respond to treatment, and no improvement in serum ALT can be discerned. Some patients may actually worsen on treatment with IFN, and develop increased serum aminotransferases. A positive anti-HCV antibody in patients with autoimmune disease remains a pitfall in diagnosis, which has implications for treatment. Immunosuppressed patients and patients with HIV may respond although long-term responsiveness is uncertain. This is of particular importance in liver transplant patients.

Combination therapy with IFN and ribavirin Ribavirin is a synthetic guanosine nucleotide analogue, which possesses a broad spectrum of activity against both DNA and RNA viruses in vitro and in vivo. Ribavirin shows only modest activity against hepatitis C, but it increases the activity of IFNa when the two agents are used in combination. This combination has been shown to produce sustained virologic responses in about 40% to 50% of patients. However, fewer than 30% of patients infected with HCV type 1 have a sustained virologic response. Ribavirin per se does not cause a marked decline in HCV RNA. The drug exerts its action after intracellular phosphorylation to mono-, di- and triphosphate nucleotides. The precise mode of action probably includes pertubation

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ANTIVIRAL TREATMENT OF HEPATITIS C VIRUS

of intracellular nucleoside triphosphate pools, interference with the formation of the 5 cap structure of viral mRNA by competitive inhibition of both guanyltransferase and methyltransferase capping enzymes, direct inhibition of the viral mRNA polymerase complex, and possibly enhancement of macrophage inhibition of viral replication. The major sideeffects of the drug that have been reported include anaemia, a metallic taste, dry mouth, flatulence, dyspepsia, nausea, headaches, irritability, emotional lability, fatigue, insomnia, skin rashes and myalgia. Mild reversible anaemia is common. Modest increases in uric acid have been reported. Rebetron has been approved for hepatitis C. Rebetron is the name of a combination of two different pharmaceutical drugs: ribavirin (Rebetol) and a-2b IFN (Intron A). The recommended dosage of Rebetron combination therapy is 3 million international units of Intron A injected subcutaneously three times weekly, and ribavirin is 1000–1200 mg administered orally in divided daily doses. Patients weighing less than 70 kg should receive 1000 mg daily; patients weighing more than 70 kg should receive 1.2 g daily. Roferon A is a brand name of IFNa manufactured by Hoffman La Roche. The response rates to Roferon A plus ribavirin are comparable with those observed with the combination of Intron A plus ribavirin. Consensus IFN (Infergen) is a cloned combination of various IFN molecules. Guidelines recommend antiviral therapy for those patients with chronic hepatitis C who are at highest risk of developing cirrhosis, i.e. patients with persistently increased serum ALT levels, detectable levels of HCV RNA and histological evidence of portal or bridging fibrosis or inflammation and necrosis. Recent clinical guidelines including the EASL guidelines published in 1999 recommend that patients with moderate inflammation or moderate fibrosis should be considered for combination therapy with IFNa plus ribavirin (EASL, 1999). The duration of therapy depends on the genotype and level of viraemia. Patients with genotype 2 or 3 are treated for 6 months, as there is no advantage in prolonging treatment beyond this point. For patients with genotype 1, current data suggest that 6 months is sufficient, if the level of viraemia is low (less than 2 million copies per ml); 12 months of treatment is recommended if the level of viraemia is high (more than 2 million copies

per ml). Treatment should be continued only in patients with type 1 infection in whom HCV RNA has disappeared after 6 months of therapy. In naive patients in whom ribavirin is contra-indicated IFN monotherapy (3 million units or 9 lg three times a week) should be administered for 12 months. It is important to manage the side-effects of therapy. Careful clinical monitoring is suggested as an alternative to antiviral therapy for patients with less severe histological changes in whom cirrhosis may not develop. Studies have also shown that combination therapy with IFNa plus ribavirin is beneficial in patients relapsing on IFNa monotherapy.

Pegylated interferons Limitations in the effectiveness of IFNa had been attributed to its rapid systemic clearance and short plasma elimination half-life of about 8 h. IFNa is rapidly absorbed with peak serum concentrations observed in 7–12 h, followed by a rapid decline. The terminal elimination half-life of IFNa ranges from 3–8 h, with serum concentrations decreasing to below the limit of detection with 24 h of administration (Khakoo et al., 1998) (Figure 54.4). Plasma concentrations of recombinant IFNa decrease to trough levels between each administration. Pegylated subcutaneous

Pharmacokinetics of alpha interferon and ribavirin

Week 1 Intron A

30

Riba + Intron Week 6 Intron A

20

Riba + Intron

10

0 0

6

12 Hours

FIGURE 54.4 Pharmacokinetics ribavirin.

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of

24

IFNa

and

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formulations of IFNa have been developed, which are produced by the covalent attachment of recombinant IFNa to branched 40-kDa or 12 kDa polyethylene glycol moieties. The PEG moieties are inert long chain amphiphilic molecules produced by linking repeating units of ethylene oxide. These molecules protect the IFN protein from enzymatic degradation reducing systemic clearance. A PEG mass of approximately 40–50 kDa is required to retard glomerular filtration. Pegylation thus changes the pharmacokinetics and pharmacodynamics of IFNas; the improved drug concentrations and sustained exposure is clinically advantageous. A stable linkage between the PEG moiety and the drug is necessary to maintain these pharmacological properties. The various pegylated IFNas extend t1/2 to a similar degree, but other pharmacokinetic parameters are affected differently. The 40 kDa branched pegylated a-2a IFN has a decreased volume of distribution (Vd), compared with standard IFN. The 40 kDa IFNa is associated with a greater reduction in renal clearance. The sustained absorption and reduced clearance of subcutaneously administered PEG 40 kDa and PEG 12 kDa allows for more convenient dosing, and results in higher sustained virological response rates (Jen et al., 2001). The efficacy of pegylated a-2a has been investigated in several controlled trials, and the outcome compared with standard IFN or IFN and ribavirin, based on virological, biochemical and histological responses.These were dose finding, as well as multi-national phase II/III and phase III trials. Initial dose finding studies were conducted to determine the appropriate dosage of pegylated a-2A IFN (Glue et al., 2000). In these trials sustained virologic responses were significantly higher in patients with chronic hepatitis C treated with pegylated IFNa than in those treated with IFNa-2a three million units three times weekly (Reddy et al., 2001). Pegylated IFNa has been proven to be significantly more effective in patients with hepatitis C than standard IFN. Zeuzem and others studied 531 patients who were randomised to receive either standard IFNa 2a or pegylated IFNa 2a. Patients in the pegylated IFN group received 180 lg of the drug once weekly for 48 weeks. Therapy with PEG IFN was associated with a higher rate of virological response than with standard IFN. After 72 weeks of treatment (end of follow-up period) 39% of the pegylated IFN grouper had a sus-

tained virological response compared with 19% of the standard IFN group (Zeuzem et al., 2000). A study by Heathcote and others was also conducted among patients with chronic hepatitis C and cirrhosis. Patients were randomised to receive either 3 million units of standard IFNa 2a or randomly assigned to receive treatment with 90 lg and 180 lg of pegylated a-2a IFN. At the end of 72 weeks the rates of sustained virological response were 8% for the standard IFN group, 15% for the 90 lg pegylated offer IFN group and 30% for the 180 lg pegylated IFN group (Heathcote et al., 2000). A recently published phase III study compared the efficacy of pegylated a-2b IFN plus ribavirin with standard a-2b IFN plus ribavirin for 48 weeks. The three arms of the study were (1) non-pegylated a-2b IFN (3 MIU three times weekly) plus ribavirin (1 to 1.2 g day1), (2) pegylated a-2b IFN (1.5 lg kg1 week1) plus ribavirin (800 mg day1), and (3) pegylated a-2b IFN (1.5 lg kg1 week1) plus ribavirin (1 to 1.2 g/day1) for 4 weeks then a reduction in dose of pegylated a-2b IFN to 0.5 lg kg1 week1 for 48 weeks. A high dose of pegylated IFN (1.5 lg kg1 plus ribavirin) was more effective than standard IFN plus ribavirin for the treatment of HCV, but the differences were relatively small. Sustained virological responses were observed in 54% of those treated in the high-dose pegylated a-2b IFN arm versus 47% of those treated with standard IFN, or the lower doses of pegylated IFN. Among patients with type 1 infection, the best response was observed in those treated with 1.5 lg kg1 pegylated IFN (42%) versus 34 and 33% for those treated with 0.5 lg kg1 and standard IFN. The sustained virological response was 79–80% for patients with type 2 and 3 infection, in all treatment groups (Manns et al., 2001) (Figure 54.5). A randomised phase III trial of pegylated a-2a IFN and ribavirin has been published in abstract form (Fried, 2001) (Figure 54.6). The authors compared the efficacy of (1) pegylated a-2a IFN 180 lg week1 plus placebo; (2) pegylated a-2a IFN 180 lg week1 plus ribavirin (1–2 g day1); or (3) conventional alpha 2b IFN plus ribavirin (1–2 g day1) for 48 weeks. Sustained virological responses for all genotypes were greater in recipients of pegylated a-2a IFN plus ribavirin (56%) compared with recipients of pegylated a2b IFN and ribavirin (45%) or standard a-2a IFN (30%). In this study, differences were observed in

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ANTIVIRAL TREATMENT OF HEPATITIS C VIRUS

Sustained Virological Response PEG a-2b interferon and ribavirin vs IFN and ribavirin 100

Percent response 82 80 79

75 54 50

47 47

42 34 33

25

0 All PEG 1.5  R

Genotype 1

2 and 3

PEG 0.5  R

IFN  R Manns et al. Lancet 2001

FIGURE 54.5 Chart showing sustained virological response rates in patients receiving either of two doses of pegylated IFNa-2b and ribavirin, versus standard IFNa-2b and ribavirin (Manns et al., 2001).

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patients infected with genotypes 1 and non 1: 76% of patients with type 2 and 3 responded to pegylated a2a IFN and ribavirin versus 61% and 45% of those who received a-2b IFN and ribavirin or standard a-2a IFN, respectively. Likewise, 46% of patients with type 1 HCV responded to pegylated a-2a IFN and ribavirin versus 37% and 21% of those who received a-2b IFN and ribavirin or standard a-2a IFN, respectively. In most of these trials, the primary end point was the proportion of patients with a sustained virological response (HCV RNA levels undetectable by PCR, i.e. less than 100 copies ml1 24 weeks after stopping treatment). Liver biopsies were also performed to assess the histological response as a secondary end point in several of these trials. Quality of life indices were also improved in patients as measured by a standardised 36 question short form (SF-36) in patients with chronic hepatitis C and cirrhosis. Thus these agents appeared to be useful new treatment options that are more convenient for patients.

Viral kinetics 40 Kda PEG interferon a-2a Sustained virological response by genotype 76 Percent response

70

61 46

80

60 50

45

37

40 30

21

20 10 0

Genotype 1

Genotype 2,3

PEG (40kd) IFN IFN a-2b and ribavirin PEG (40kd) and ribavirin

Fried MW et al. DDW abstract 2001

FIGURE 54.6 Chart showing sustained virological response rates in patients treated with PEG IFNa-2a (40 kDa) versus standard IFNa-2b and ribavirin versus PEG IFNa-2a and ribavirin (Fried, 2001).

IFNa treatment has previously been suggested and characterised as causing two phases of viral decline. An initial decline during the first 24 to 48 h that may reflect treatment-induced inhibition of hepatitis C replication and a degradation of free virus and a secondary decline that is a stable exponential decay and thought to be related to the rate of degradation of infected cells. The rate of degradation of cells infected with hepatitis C has been found to be genotypedependent, with the first phase of viral decline being significantly steeper and the second phase of viral decline significantly faster in patients infected with non-1 genotypes. The second phase of hepatitis C RNA decline is low in the majority of patients who do not have a virological response at the end of treatment and conversely is high in those patients who had a sustained virological response. Early virus elimination is less pronounced after a single subcutaneous 180 lg dose of pegylated IFNa than the viral decline described previously in patients receiving a high-dose daily regimen of IFNa. However, after a second dose of pegylated IFNa, patients sensitive to IFNa, had a substantial decrease

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(greater than 90%) in initial viral load (Neumann et al., 1998, 2000).

Antiviral treatment and liver transplantation for hepatitis C Cirrhosis due to hepatitis C is becoming an important indication for liver transplantation, and the number of patients being transplanted for decompensated hepatic disease has increased in the past 5 years. It has become apparent that recurrence of hepatitis C virus infection is common, and that 90% or more of patients transplanted will again be HCV RNApositive. The recurrence is rapid, and HCV RNA can be found in the engrafted liver within 2 weeks of the transplant. A number of studies have investigated the biochemical, virological and histological outcome of the transplantation (Read et al., 1991; Poterucha et al., 1992; Féray et al., 1992; Wright et al., 1992; Ferrell et al., 1992; Zignego et al., 1993). Episodes of biochemical hepatitis are more common in patients with recurrence of HCV than in control groups of patients transplanted for cirrhosis not due to chronic hepatitis. Histological hepatitis is also more common, and chronic hepatititis is seen in the approximately 60% of patients at 3 years. Cirrhosis occurs in about 10% of patients after 2–3 years. Thus although the recurrence is associated with relatively mild disease, follow-up of these patients has been short, and severe sequelae, including cirrhosis and even possibly hepatocellular carcinoma may become more common with the passage of time. This provides the rationale for consideration of treatment. There is as yet little information regarding these patients. Preliminary studies have indicated that in some treated patients, levels of ALT may improve, along with a decline in concentrations of HCV RNA. As with chronic hepatitis, many patients are unresponsive, and biochemical deterioration may occur. Moreover, IFN is a powerful modulating agent, and there is concern that rejection may be increased with this treatment. Relapse rates are likely to be high. Carefully considered controlled trials are therefore indicated. The timing and dose of treatment will require assessment.

SIDE-EFFECTS OF a INTERFERONS The major early side-effects of IFN include an influenza-like syndrome, chills, fever, malaise, muscle aches and rigors. Later side-effects include malaise, muscle aches, headaches, poor appetite, weight loss, increased need for sleep, psychologic effects, (irritability, anxiety, depression) hair loss, thrombocytopaenia, and leukopaenia (Dusheiko, 1997). Unusual, or severe side-effects include seizures, acute psychosis, bacterial infections, autoimmune reactions, thyroid disease, proteinuria, myocardiopathy, skin rashes and IFN antibodies. Patients should be monitored at 1- to 4-week intervals during treatment, and blood counts and serum aminotransferases should be measured at these intervals. HBV DNA and HBeAg should be measured monthly to monitor the progress of treatment. Thyroid functions should be measured before, during and at the end of therapy. IFNa produces undesirable adverse effect on bone marrow in patients with cirrhosis, thus limiting the use of the drug in some patients. Exacerbation of cirrhosis-related neutropenia and thrombocytopenia has been observed in patients with chronic hepatitis C receiving IFNa. The side-effects of pegylated a-2 IFN is broadly similar to that of IFNa-2a in patients with chronic hepatitis C. The adverse events reported in trials of pegylated IFNa trials were typical of those reported in previous trials of IFNa. Headache, fatigue, pyrexia, myalgias, rigors and alopecia were among the most common adverse events reported in patients receiving pegylated IFNa. Psychiatric events were the most frequent serious adverse events, including severe depression and personality disturbance, as well as psychosis in one patient in these trials. Marked decreases in both neutrophil and platelet counts occurred in recipients of pegylated a-2a IFN. These effects are usual managed by a reduction in dose. In non-cirrhotic patients the neutrophil count decrease to less than 500 mm3 in 12 patients receiving treatment with pegylated a-2 IFN and in four patients receiving a-2A. The dose of study medication was modified because of the development of neutropenia in 11% and 7% of pegylated a-2a and IFNa2A recipients, respectively. Anaemia was documented in three patients.

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All of the IFNs are contraindicated in patients with major depressive illness, autoimmune disorders, and marked thrombocytopenia or leucopaenia. IFN is not recommended for the treatment of pregnant women with chronic hepatitis C. New treatment strategies are being evaluated. These include triple therapy with IFNa, amantadine plus ribavirin, therapy with pegylated IFNa plus ribavirin, IFN plus thymosin alpha. DNA vaccine strategies are being evaluated. A number of new molecules to inhibit HCV protease, helicase and polymerase are in active development.

ANTIVIRAL TREATMENT OF CHRONIC HEPATITIS D The hepatitis delta virus (hepatitis D, HDV) is a viroidlike agent, which resembles the satellite RNA viruses of higher plants. The agent requires concomitant infection with hepatitis B virus to activate disease, although infection may be possible in the absence of hepatitis B, at least in liver transplant recipients. HBV provides surface antigen capsid to HDV, enabling transmission of HDV via the same routes as hepatitis B. The HDV genome has a similar organisation to that of plant viroids. It is a single covalently closed RNA virus of negative polarity that exists as a highly basepaired rod structure. A sequence of approximately 1700 nucleotides has been found. The viroid contains consensus oligonucleotide sequences found in other viroid-like molecules. The agent can undergo reversible self-cleavage and ligation. HDV infection is found to varying degrees in areas where HBV infection is endemic, and in high risk groups. Perhaps 5% of the world’s HBV carriers are also infected with HDV. Clinically there is a spectrum of disease, and in some coinfected or superinfected persons, HDV appears to be a pathogenic agent and to aggravate the underlying HBV infection. There is much interest in the study of pathogenicity caused by this agent. The diagnosis is made serologically by high titres of IgG and IgM anti-hepatitis D, by the finding of hepatitis D virus (HDV) RNA in serum, or the presence of HDAg in serum or liver tissue. A number of investigators have evaluated IFN treatment of chronic type D hepatitis. The hepatitis D agent is sensitive to IFN, and doses of 3–10 million units three times weekly

for 6 months results in repression of hepatitis D and amelioration of biochemical signs of hepatitis, with a decrease in serum aminotransferases (Rizzetto et al., 1986; Rosina et al., 1991). Coincident with this decrease, HDV RNA disappears from serum in approximately 50% of cases. Unfortunately many patients relapse when treatment is stopped.

CONCLUSIONS Considerable progress has been made in understanding the mechanism of actions of IFNas leading to improved therapies for viral hepatitis. We require further basic understanding of the strategies that viruses have evolved to antagonise the actions of IFNs, and the host cellular immune response. New tools and approaches for the study of these infections are being developed that should enable either cure, or suppression of disease activity in a high proportion of patients.

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55 Haemopoietic cytokines John L. Lewis and Myrtle Y. Gordon Imperial College Faculty of Medicine, Hamersmith Hospital, London, UK

Why grass is green, or why our blood is red Are mysteries which none have reach’d unto John Donne, Of the Progress of the Soul. The Second Anniversary

INTRODUCTION Haemopoiesis is the process of making all of the types of mature cells (red cells, white cells and platelets) that circulate in the bloodstream (Figure 55.1) and takes place to a large extent in the bone marrow of the pelvis and sternum in normal adult humans. The majority of the cells in the bone marrow are dividing and maturing cells, which can be recognised by their morphological appearance on stained smears. These are the progeny of a smaller number of more or less lineage-specific progenitor cells so that erythrocyte production is fed by an erythroid progenitor, granulocyte and macrophage production by a granulocyte– macrophage progenitor and platelet production by a megakaryocyte progenitor. The lineage-specific progenitors are themselves produced by a much smaller population of haemopoietic stem cells, which are capable of self-renewal and of producing progenitor cells to sustain all lineages of haemopoietic cell differentiation. The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

The mature blood cells have a finite lifespan in the circulation and need to be replaced continuously. Clearly, failure of replacement will lead to a fall in the numbers of cells in the blood, but the sequelae are more serious for some lineages than for others. Neutrophils have a half-life in the peripheral circulation that can be measured in hours, but the half-life of red cells is about 120 days. Consequently, a failure in neutrophil production will become evident more rapidly than a failure in red cell production. Also, there is a very close relationship between neutrophil count and susceptibility to life-threatening infections. Reasons for individuals being or becoming cytopenic include congenital or idiopathic bone marrow failure syndromes and exposure to environmental or therapeutic toxins. The haemopoietic growth factors and cytokines comprise the soluble regulators of blood cell production. Although the existence of a humoral regulator of haemopoiesis had been suggested as early as 1906, no other haematologically active cytokines were

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Pluripotent stem cell Lymphoid lineage

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FIGURE 55.1 Hierarchical model of haemopoiesis. CFU-GEMM, colony-forming unit granulocyte–erythroid– monocyte–macrophage; CFU-GM, colony-forming unit granulocyte–macrophage; CFU-Eo, colony-forming unit eosinophil; BFU-E, burst-forming unit erythroid; CFU-Mk, colony-forming unit megakaryocyte; CFU-G, colonyforming unit granulocyte; CFU-M, colony-forming unit monocyte/macrophage; CFU-E, colony-forming unit erythroid. discovered until the 1960s. Molecular cloning and the production of recombinant cytokines were developed in the 1980s. Immediately, their potential for the treatment of clinical cytopenias was realised and there was a remarkably rapid transition from ‘bench to bedside’. The clinical applications of haemopoietic growth factors and cytokines have diversified since the results of the first clinical trials were published in 1987–1988 (Groopman et al., 1987; Gabrilove et al., 1988a; Morstyn et al., 1989) and now include mobilisation of haemopoietic stem cells into the bloodstream for harvesting and transplantation, ex vivo expansion of stem cell numbers for transplantation purposes and stem cell maintenance during the manipulations required for gene therapy protocols.

The list of applications is likely to grow and include other cytokines in the future as a result of the recently discovered and unexpected plasticity of haemopoietic stem cells (Mertelsmann, 2000; Jackson et al., 2001; Goodell et al., 2001). There are now a large number of cytokines that have been identified and have effects on the haemopoietic system. Not all of them have had equal exposure in the clinical arena because there always has been some emphasis on factors that can improve neutrophil numbers and reduce susceptibility to life-threatening infections. In this chapter, we will first discuss aspects of haemopoietic cell and cytokine biology that underlie the effects of cytokines in clinical practice. This will include the regulation of haemopoietic cell function by cytokines and how the

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cytokines themselves are regulated. We will also discuss aspects of cytokine physiology and the coordination of the pleiotropic actions of multiple cytokines. In the second part of the chapter, we will discuss the application of haemopoietically active cytokines to the clinical treatment of patients with haematological problems.

A BRIEF HISTORY OF THE DEVELOPMENT OF CYTOKINE RESEARCH The existence of growth factors with specificity for the cells of the haemopoietic system was first suggested by Carnot and Deflandre in 1906 (Carnot and Deflandre, 1906). They postulated that a substance they called hemopoietine was responsible for the production of red blood cells in response to anaemia. They injected plasma from anaemic rabbits into normal rabbits and observed an increase in the peripheral blood red cell count. However, its specificity for erythropoiesis was not recognised until 1948 and it was renamed erythropoietin to reflect that fact. No equivalent cytokine for white cell production (granulocytes and monocyte–macrophages) was discovered until the 1960s. This coincided with the development of clonogenic assays for the progenitors of the mature blood cells. Research groups in Australia and Israel (Pluznik and Sachs, 1965; Bradley and Metcalf, 1966) found that mouse bone marrow cells that had been immobilised in semi-solid agar in vitro formed discrete colonies of granulocytes and monocyte–macrophages when they were supplied with certain feeder cells, tissue fragments or medium conditioned by various tissues. The factor contained within these additives was called colony-stimulating factor (CSF) for operational reasons. By 1970, an equivalent assay for human cells had been developed by Pike and Robinson (Pike and Robinson, 1970). As assays became available for clonogenic cells belonging to other haemopoietic cell lineages (erythroid, basophil, eosinophil, megakaryocyte) so did opportunities to extend research into the corresponding colony-stimulating factors and to define more precisely the target cells they acted upon. The next challenge was to biochemically characterise the CSFs and to obtain sufficient quantities of

purified protein to confirm that the in vitro activities in colony assays reflected an ability to stimulate blood cell production. The effort required for these tasks should not be underestimated. The starting material was large volumes of human urine or conditioned medium requiring many thousand-fold purification and yielding insufficient product to treat a mouse, let alone a human patient. Meanwhile, other evidence for in vivo actions of the CSFs was obtained by using the colony assays to measure CSF levels in mice with haematological perturbations when fluctuations in CSF levels might be expected to occur. Fortunately, the problem of insufficient material started to be solved in the early 1980s with the advent of recombinant DNA technology. This advance meant that it was possible to perform more detailed work on the cell and molecular biology of haemopoietic cytokines in vitro and in vivo, the structure of cytokines and their corresponding receptors, and the elucidation of intricate signal transduction pathways activated by cytokines binding to their receptors. The results of the first clinical trial of granulocyte– macrophage colony-stimulating factor (GM-CSF) were published in 1987 and showed that injected GMCSF could elicit dose-dependent increases in white blood cells in cytopenic AIDS patients (Groopman et al., 1987). This was followed by trials of granulocyte colony-stimulating factor (G-CSF) in patients with cancer or lymphoma (Gabrilove et al., 1988a; Morstyn et al., 1989).

HAEMOPOIESIS Haemopoiesis is the process whereby all mature blood cells are produced. These cells need to be produced in vast numbers because they have a limited lifespan which, for neutrophil granulocytes, is measured in hours. All of the different types of blood cells are derived from a small population of haemopoietic stem cells, which are generally considered to be capable of maintaining their numbers through selfreplication when they divide. They are also pluripotent in terms of the multiple lineages of blood cells they are capable of producing. In other words, stem cell progeny may themselves be stem cells or be committed to enter the pathways of differentiation leading to blood cell formation. The end stage of

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haemopoietic cell development consists of terminal differentiation to form neutrophil, eosinophil and basophil granulocytes, monocytes/macrophages, erythrocytes, megakaryocytes (platelets) and T lymphocytes (Figure 55.1). This concept of a haemopoietic hierarchy has been evolving since Maximow first proposed, in 1924 (Maximow, 1924), that blood cell formation results from the activity of several differentiation pathways, starting with a pluripotent stem cell. In 1938, Downey suggested a hierarchical model similar to the one shown in Figure 55.1 (Downey, 1938) and further details were supplied by the development of clonogenic in vitro assay systems (see above). Today, the hierarchical scheme provides the basic framework for much of the thinking (and controversy) surrounding the regulation of the haemopoietic system. Areas of debate include mechanisms of stem/progenitor cell self-renewal and lineage selection/commitment (stochastic versus deterministic) and which elements of regulation may be determined intrinsically or extrinsically. These need to be considered in the present context because they are relevant to the action of haemopoietic cytokines.

Stochastic versus deterministic regulation The term stochastic has been used in connection with haemopoiesis for more than 40 years (Blackett and Gordon, 1999), but its application remains controversial today (Metcalf, 1998; Enver et al., 1998). It refers to processes that appear random but are associated with a probability (P) distribution. In the context of haemopoiesis it means that the fate of individual cells cannot be predicted and only observation of a large number of events will reveal the overall behaviour (i.e. value of P) of the stem/progenitor cell population. Radioactive decay is the quintessential example of a stochastic process because the half-life is known very precisely, but the exact moment at which an individual atom will decay is completely unpredictable. For example, radioactive carbon has a half-life of 5770 years, so that an atom may disintegrate within a microsecond or not until tens of thousands of years later. Stochastic regulation is often considered synonymous with intrinsic, genetic control of cell fate, in

spite of the fact that it is not relevant to discussions about whether cells respond to extrinsic regulators (Blackett and Gordon, 1999). Deterministic regulation, on the other hand, refers to an instructive role of the environment (cytokines, adhesive interactions, etc.) in determining cell fate. Also, further confusion has been generated by the failure to consider selfrenewal and differentiation separately from commitment and lineage selection. These are different biological processes and may be regulated differently.

Stem/progenitor cell renewal The stochastic theory of stem cell self-renewal originates from early transplantation experiments in mice by Till and McCulloch (Till and McCulloch, 1961). They injected bone marrow cells into irradiated recipients and discovered that they contained a population of cells capable of forming macroscopic colonies in the recipients’ spleens. They proved that these spleen colonies (CFU-S) were clones of cells by using chromosomal aberrations induced by low doses of ionising radiation and found that individual colonies contained cells belonging to several lineages of haemopoietic cell differentiation. They also found that retransplantation of individual colonies into secondary recipients gave rise to spleen colonies in the secondary mice indicating that CFU-S were capable of self-renewal. Thus, the CFU-S assay became an operational assay for a self-renewing stem cell capable of producing up to one million cells belonging to several lineages. When they analysed the results of retransplanting spleen colonies into secondary mice, Till and colleagues found that the numbers of secondary colonies were highly variable and fitted a skewed (gamma) distribution (Siminovitch et al., 1963). This skewed distribution could be explained by the CFU-S having a certain probability of differentiating into one of the maturation lineages and losing their clonogenicity (P  0.4), or alternatively remaining as colony-forming cells (i.e. self-renewal) (P  0.6). Cell death as a possible contributing factor was not considered at the time. Till and colleagues indicated that the probability of stem cell renewal of 0.6 was intrinsically fixed. However, under steady-state conditions, P must be 0.5 so that stem cell numbers do not change. The increase in self-renewal from the steady

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state value of 0.5 to 0.6 in spleen colony formation is consistent with the rapid regeneration of haemopoiesis in irradiated mice. This fact alone should have been sufficient to refute the conclusion that stem/ progenitor cell self-renewal is controlled intrinsically and is not subject to change. More recently Abkowitz et al. (1996) have shown that stochastic effects may occur in haemopoiesis in vivo. They used Safari cats, a cross between Geoffrey cats (a South American wild cat) and domestic cats. The parental cats have distinct phenotypes of glucose-6-phosphate dehydrogenase (G6PD). Due to X chromosome inactivation, female cats have somatic cells that contain either the domestic cat G6PD or the Geoffrey cat G6PD. The female Safari cats are balanced heterozygotes with equal numbers of cells, including haemopoietic cells, of each parental phenotype with the ratio of phenotypes between progenitors of different lineages being similar. In untransplanted cats the percentage of G6PD isotypes remained relatively constant for 6 years suggesting that haemopoiesis was polyclonal and stable. When these cats are transplanted with bone marrow after lethal irradiation, the G6PD phenotype fluctuated considerably for 1 to 4.5 years after transplantation and the pattern of clonal contribution was unique for each cat. Computer simulations of a stochastic differentiation model, based on probabilities of selfrenewal, differentiation or apoptosis, gave similar results. The authors concluded that whilst the computer simulations did not prove that stem cell ‘decisions’ to differentiate in vivo are stochastic, stochastic differentiation was sufficient to explain the patterns of clonal contributions to haemopoiesis seen in experimental animals and that clonal dominance can occur by chance. Other workers, using in vitro culture systems, found that replating of erythroid colonies (Humphries et al., 1981), granulocyte–macrophage (CFU-GM) colonies (Gordon et al., 1998) or blast cell colonies (Nakahata and Ogawa, 1982), which is analogous to retransplanting CFU-S in vivo and self-renewal is revealed by the presence of secondary colonies, found a skewed distribution in secondary colony numbers similar to that seen in CFU-S by Till et al. (Siminovitch et al., 1963). Paulus et al. (1995) used commitment to polyploidization to investigate differentiation in megakaryocytes. They found that distributions of

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numbers of doublings revealed that colony-forming units megakaryocyte (CFU-Mk) exit randomly from the progenitor compartment, but that there is a constant rate of exit per generation which is related to a polyploidization probability. In an analogous fashion, analysis of burst-forming units-erythroid (BFU-E) subcolony number serves as a model of self-renewal (Kurnit et al., 1985; Lewis et al., 1998). BFU-E when plated in semisolid culture with erythropoietin (Epo) form characteristic haemoglobinised bursts consisting of variable numbers of subcolonies. BFU-E that form bursts that consist of more than one subcolony may be considered to have undergone several rounds of self-renewal prior to commitment to subcolony formation, whilst BFU-E that commit immediately to subcolony formation will form single bursts. In all of these experimental systems, the endpoints measured (megakaryocyte ploidy, numbers of secondary colonies per primary CFU-GM colony and number of subcolonies per BFU-E burst) followed skewed distributions similar to those described by Till et al. (1964). Thus, there is a considerable amount of evidence that stochastic events are widespread in haemopoiesis (Gordon and Amos, 1994). The stochastic regulation of stem cell self-renewal was challenged by the observation that the CFU-S forming colonies in the splenic red pulp consisted predominantly of red cells, whilst those colonies forming just under the splenic capsule and along the trabeculae consisted mainly of white cells. This indicated that the response of multipotent cells was determined by the nature of its environment. A fierce debate ensued which is surprising in view of the fact that one side of the argument related to self-renewal and differentiation, whilst the other side related to lineage selection. These two aspects should be distinguished from one another. Nevertheless, other evidence shows that the probability of stem/progenitor cell renewal can be regulated extrinsically by changes in their environment, including cytokines (see below). The deterministic model proposes that decisions to self-renew or differentiate are predetermined and that once a response is initiated it will continue to completion. Morrison and Weissmann (1994) proposed a model based on the purification of three multipotent progenitor populations that had different selfrenewal capacities. They suggested that these populations represented a lineage of progenitors from most

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primitive to most differentiated. The progenitors from the most primitive population exhibited a greater selfrenewal potential than those from less primitive progenitors. They concluded that self-renewal is deterministic based on the fact that cell separation by phenotypic markers could predict self-renewal potential (Morrison and Weissman, 1994). However, they did not investigate the distribution of selfrenewal probabilities within each subpopulation. One criticism of the deterministic model is that it is inflexible and does not allow the haemopoietic system to respond to situations, such as haemorrhage or infection, where large numbers of cells need to be produced relatively quickly.

Lineage selection/commitment Stochastic effects may also apply to lineage commitment and selection. This concept is based on work originally done by Suda et al. (1983) using murine blast cell colonies. In these experiments single progenitors were plated and the resulting multilineage colonies were analysed for the cell types present. They found a variety of lineage combinations which was consistent with the idea that commitment to a particular lineage may be stochastic. This was further confirmed by analysis of paired progenitors derived from a single parent cell. Colonies grown from these cells contained cells of different lineages and also different numbers of lineages (Suda et al., 1984). The same group showed similar results for human blast colonyforming cells (Leary et al., 1985). More recently Mayani et al. (1993) investigated primitive progenitor cells derived from human umbilical cord blood grown in various combinations of cytokines. They found that cells divided asymmetrically (i.e. daughter cells produced colonies of different types) and whilst the presence of cytokines was necessary for growth, they appeared to be permissive rather than instructive. Consequently the results supported the concept that lineage commitment and selection is stochastic. An example of the deterministic concept assumes that cells at a particular stage are restricted to particular differentiation options and that all cells will go through the same predetermined number of divisions (Novak and Stewart, 1991). Nicola and Johnson (1982) proposed the concept of sequential loss of lineage potential. They analysed the colony-forming cells that

appeared after different times in liquid culture. These indicated that there was a predetermined differentiation sequence of primitive multipotential cells to committed colony-forming cells. The order of appearance of committed colony-forming cells and concomitant loss of potential was macrophage, eosinophil, granulocyte–macrophage, granulocyte megakaryocyte and erythroid. This model indicates that a stem cell would have to perform six cell cycles, which could take 3 days with a cell cycle time of 12 h, before producing an erythroid progenitor. The implications for haemorrhage are obvious.

Intrinsic versus extrinsic regulation As pointed out above, the issue of intrinsic versus extrinsic regulation is separate from that of stochastic and deterministic regulation. Thus, stochastic events may be intrinsically and/or extrinsically influenced as may deterministic regulation. In this context the term deterministic seems to take on a slightly different meaning, that is, that cell fate may be determined by extrinsic factors which play an instructive role.

Stem/progenitor cell renewal The idea that specific genes might be responsible for regulating haemopoietic stem cell renewal has found support in studies of C57Bl/6 and DBA2 mouse strains which differ in stem cell numbers and cell cycling activity (de Haan et al., 1997). Van Zant and colleagues bred B6 D2 F1 hybrids, then transplanted them with B6 cells, D2 cells or mixtures of B6 and D2 cells (Kamminga et al., 2000). They found that the behaviour of the cells post transplant reflected their genetic origin, rather than the common environment they were transplanted into, supporting an intrinsic genetically determined element of stem cell regulation. Using the same strains of mice, MullerSieberg et al. (2000) made aggregation chimeras and genotyped individual long-term culture-initiating cells (LTCIC) as surrogate stem cells. They fitted their results to models corresponding to the outcomes expected from intrinsic and extrinsic regulation. Although they did not fit either model precisely, the results favoured the intrinsic model, so that 84% of stem cell regulation was attributable to intrinsic factors and 16% to extrinsic factors.

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The stem cell regulatory genes have been mapped to chromosome 1 (Muller Sieburg and Riblet, 1996) and chromosome 18 (de Haan and Van Zant, 1997). However, other genes associated with different functions may influence the probability of progenitor/ stem cell self-renewal. In particular, we have studied the influence of cell cycle regulating genes, p16, p21 and p27. These are cyclin-dependent kinase inhibitors which, classically, act on the cell cycle machinery by inhibiting complex formation between CDK 4/6 h and cyclin D resulting in reduced phosphorylation of retinoblastoma protein (pRb). The hypophosphorylated pRb binds the transcription factor E2F reducing its availability to induce the transcription of genes necessary for cell cycle progression (Weinberg, 1995). However, studies in p16 knockout mice identified a novel function for cyclin-dependent kinase inhibitors in regulating haemopoietic progenitor cell self-renewal. CFU-GM from the bone marrow of p16 knockout mice had an increased self-renewal capacity, whilst human normal bone marrow CFU-GM transduced with cyclindependent kinase 4 (CDK4), which sequesters p16, and also exhibited an increased probability of renewal compared with non-transduced controls (Lewis et al., 2001). Similarly, progenitor cell renewal is increased in mice with deleted p21 or p27 (Lewis, 2000). Another way in which gene expression may regulate stem/progenitor renewal is by suppressing differentiation and/or apoptosis. This is because the probabilities of the three cell fates add up to one, so that reducing the probability of one fate will increase the probabilities of the others (see below). In this regard, we have found that progenitor cells taken from mice with mutated Fas or Fas ligand have an increased probability of renewal, presumably reflecting a reduction in the probability of cells dying by apoptosis. In spite of being crucial for the success of stem cell expansion and gene therapy protocols, there are few formal studies of the effects of extrinsic factors on the probability of stem cell renewal. The first studies were those of Till and colleagues (Siminovitch et al., 1963; Till et al., 1964) who reported that the probability of renewal of murine stem cells transplanted into irradiated recipients was 0.6, thereby implicating the irradiated bone marrow microenvironment in

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causing an increase in renewal probability above the steady-state value of 0.5. Metcalf and colleagues studied the murine cell lines WEHI-3B and M1 which grow autonomously as undifferentiated blast cells with a high clonogenic capacity and a self-renewal probability of essentially 100% (P  1.0). However, when cultured in the presence of cytokines (G-CSF for WEHI-3B cells and interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) or oncostatin M for M1 cells), self-renewal is markedly suppressed and the clonogenic cell progeny exhibit features of macrophage or granulocytic lineage maturation (Metcalf, 1982; Smith et al., 1997). Lewis et al. (2000) used the in vitro clonogenic models provided by CFU-GM colony replating and BFU-E subcolony formation. These models were used to investigate the effects of cytokines on the self-renewal capabilities of progenitor cells and whether any common effects could be attributed to the type of receptor bound. It was found that cytokines that bind to tyrosine kinase receptors including stem cell factor (SCF) and flt-3 ligand (FL) do not change the self-renewal capacity of the progenitor cells, whilst IL-3 or GM-CSF, which bind specific a chains but signal through common b subunits, increased the self-renewal of the progenitor cells. This work provides evidence that in two lineages cytokines can alter the self-renewal capabilities of progenitor cells and that this in part may be a result of the type of receptor bound, and presumably of the intracellular signalling pathways that are activated (Lewis et al., 1998; 2000).

Lineage selection/commitment Much of the evidence for intrinsic regulation of lineage commitment and selection rests on negative evidence for a role of cytokines in this process. Thus, examination of mice with knocked out cytokines or cytokine receptors have not shown that cytokines are essential for lineage determination. Similarly, manipulation of cytokine and receptor expression, largely through retrovirus-mediated gene transfer, has not revealed marked changes in lineage representation (Enver et al., 1998 and references therein). The case for extrinsic regulation of lineage commitment and selection has been supported by the results

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of recloning daughter cells of individual clonogenic cells under different conditions of cytokine exposure and observing the lineage phenotype generated. Some, but not all, of these studies indicate that cytokines may play a role in lineage selection (Metcalf, 1998 and references therein), but are subject to the criticism that the results may equally be explained by cytokines selectively permitting the survival of cells that had already undergone lineage selection induced by a cell-intrinsic mechanism (Enver et al., 1998).

COMPONENTS OF HAEMOPOIETIC PROGENITOR CELL RESPONSES TO CYTOKINES The essential components of the progenitor cell response to cytokines are the cytokines themselves and the target cells expressing the correct cell surface receptor. The cytokines are glycoproteins with little primary amino acid sequence homology but computerassisted molecular modelling, based on predictions of secondary structure, suggest similar structural features among the various cytokines. Typically, they consist or bundles of anti-parallel a helices joined by loops and b sheets. Intermolecular disulphide bonds stabilise the structure. Deletional mutagenesis has been used to test predicted structures and identify sequences that are essential for secretion, biological activity and receptor binding. These aspects of cytokine biology are covered in detail elsewhere throughout this volume, as are the features and activities of different cytokines. Cytokines exert their effects by binding to receptors expressed on the cell surface. There are two major families of receptors. The first are those belonging to the haematopoietin family, which can be further divided according to the common subunits required for receptor activity. In other words, the receptor consists of specific ligand-binding subunits, which are associated with a common signalling subunit. Second, are those belonging to the tyrosine kinase family which consist of a single glycoprotein with an intrinsic tyrosine kinase domain.

The haematopoietin receptor superfamily The members of this family are type 1 transmembrane glycoproteins. The extracellular domains consist of immunoglobulin, fibronectin and haematopoietininterferon receptor domains which occur in varying numbers depending on the receptor. There are conserved regions within the haematopoietin domains, including four conserved cysteine residues and the five amino acid motif WSXWS (tryptophan-serineXaa-tryptophan-serine) (Figure 55.2). All haematopoietin receptors undergo multimerization. They form either homodimers, for example G-CSF and Epo receptors, where the individual subunits have responsibility for both cytokine binding and signalling or they can form heterodimers or trimers. The latter often involve a subunit responsible for ligand binding dimerizing with other single or multiple subunits. These are often shared between receptor groups and this provides a basis for describing three groups of receptors based on shared subunits

IL-3, IL-5 and GM-CSF receptors IL-3, IL-5 and GM-CSF bind to the a subunits of their particular receptors with low affinity. The formation of a high affinity receptor requires the dimerization of the a subunit with a b subunit which is common to all three receptors, but is unable to bind cytokine on its own (Kitamura et al., 1991; Tavernier et al., 1991). The b subunit is essential for signal transduction and regions have been identified by deletions in the cytoplasmic domain. Two domains in the membrane proximal portion appear to be critical for signalling (Sakamaki et al., 1992).

The IL-2 receptor family The IL-2 receptor consists of three subunits. The a subunit of the IL-2 receptor is a member of the immunoglobulin superfamily (Nikaido et al., 1984; Leonard et al., 1984). It binds IL-2 with low affinity and is not required for signal transduction. The b subunit was shown to be a member of the cytokine receptor superfamily and is essential for signal transduction. The b subunit when expressed in fibroblasts

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GM-CSF

G-CSF

b

a

lg-like domain

Transmembrane domain

Haematopoietin domain

Cytoplasmic domain

Fibronectin domain

FIGURE 55.2 Diagrammatic representation of haematopoietin receptors, showing the modular nature of the receptor structure. The two examples shown are the receptor for GM-CSF consisting of a and b subunits and the receptor for G-CSF which forms a homodimer. The conserved cysteines (lines) and the conserved WSXWS motif (thick line) are shown in the haematopoietin domain.

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The gp130 receptor family This family includes the receptors for IL-6, IL-11, ciliary neurotropic factor (CNTF), LIF, oncostatin M (OSM) and cardiotrophin (CT-1). As with the other two families, these bind to specific a subunits with low affinity which alone do not transduce a signal. For cells to respond they require the presence of an additional transmembrane glycoprotein, gp130. gp130 is a member of the haematopoietin family and is similar to the G-CSF receptor (Hibi et al., 1990). Expression of gp130 in BaF3 cells showed that it was essential for the formation of a high affinity IL-6 receptors and for the proliferation of the BaF3 cells (Murakami et al., 1993). The receptor for LIF also shows sequence similarity to gp130 and G-CSF receptor. Like IL-6, binding of LIF to its receptor does not result in a proliferative signal (Gearing et al., 1994). However, coexpression with gp130 results in a high affinity receptor capable of transducing a signal (Gearing et al., 1992). Both gp130 and LIF receptor are components of the oncostatin M (OSM) and CNTF receptor complexes. OSM binds to gp130 with low affinity and requires coexpression of the LIF receptor for the generation of a high affinity receptor and signal transduction (Gearing et al., 1992; Gearing and Bruce, 1992). CNTF binds an a subunit (Davis et al., 1991) followed by association with both gp130 and the LIF receptor (Davis et al., 1993).

The tyrosine kinase receptor superfamily cannot bind IL-2, but it can when it is expressed in lymphoid cells suggesting that a third subunit may be involved (Hatakeyama et al., 1989). Subsequently, the third (c) subunit was cloned (Takeshita et al., 1992). The importance of the c subunit was suggested by work showing that mutations in the human IL-2 c subunit resulted in X linked agammaglobulinemia (Noguchi et al., 1993). As mice which are null mutants for IL-2 have a mild phenotype, it suggested that the c subunit also functioned as a part of other receptors. It was subsequently shown that the c subunit is a component of other cytokine receptors including the receptors for IL-4, IL-7, IL-9 and IL-15 acting in both signal transduction and in the formation of a high affinity receptor (Kondo et al., 1993, 1994; Giri et al., 1994).

This grouping includes receptors for SCF, macrophage colony-stimulating factor (M-CSF), FL and hepatocyte growth factor (HGF). The receptor for M-CSF was identified from work investigating the oncogene v-fms from the McDonough strain of the feline sarcoma viruses. Both v-fms and its cellular form c-fms were shown to contain a tyrosine kinase domain (Hampe et al., 1984) with homology to the platelet-derived growth factor (PDGF) receptor. It was subsequently shown that c-fms was the receptor for M-CSF (Sherr et al., 1985). The discovery of SCF receptor and its ligand initially arose from research into mice carrying mutations in the white spotting (W) and steel (SL) loci. c-kit is the cellular homologue of the transforming oncogene v-kit. It was shown to be a tyrosine kinase receptor

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with homology to both the PDGF and M-CSF receptors. The c-kit oncogene was then found to be in the white spotting locus, whilst the gene involved in the steel locus defect was found to encode a cytokine capable of binding to and activating the c-kit receptor, now often known as stem cell factor (SCF) (Witte, 1990). The flt-3/flk-2 receptor tyrosine kinase was discovered independently by two groups using different strategies. Rosnet et al. (1991) used a probe constructed from the DNA of the M-CSF receptor, which was used to isolate a related DNA sequence. The second group made degenerate oligonucleotides based on a conserved sequence of the kinase domain of tyrosine kinase receptors (Matthews et al., 1991). Unlike c-kit and c-fms which are expressed on both primitive and committed progenitors, the flt-3/flk-2 receptor tyrosine kinase appears to be preferentially expressed on primitive haemopoietic cells (Matthews et al., 1991). This group also contains the receptor for HGF – c-met (Bottaro et al., 1991). It has been found to be expressed by RT-PCR on cells from CFU-GM and BFU-E colonies and normal bone marrow mononuclear cells (Ratajczak et al., 1997).

NATURE OF HAEMOPOIETIC PROGENITOR CELL RESPONSES TO CYTOKINES Haemopoietic progenitor cells responding to cytokine stimulation may proliferate (i.e. self-renew), differentiate along one or another blood cell lineage, or die by apoptosis. It is the balance between these three options which determines the size of the progenitor cell population and the numbers of mature functioning cells that will be produced (Figure 55.3). Several inferences may be drawn from Figure 55.3. First the probability (P) of any one outcome will affect the probabilities of the others since their individual probabilities must add up to unity. Second, a maximum of only two of the three outcomes can be expressed on cell division. Haemopoietic progenitor cells require the presence of growth factors for survival and continued viability. Leary et al. (1992) isolated CD34, HLA DR progenitor cells and measured their colony-forming capacity

in the presence of IL-3 alone or IL-3 plus a number of other ‘synergistic’ cytokines including G-CSF, IL-11 and SCF and found that in order for the cells to proliferate and form colonies they required the presence of the other growth factors. Interestingly experiments involving the delayed addition of cytokines showed that the survival of the quiescent CD34-positive HLADR cells required the presence of IL-3 or GM-CSF. The other cytokines could not replace these factors in promoting the survival of progenitor cells. Subsequent studies have highlighted the fact that SCF can act as a survival factor. Katayama et al. (1993) enriched primitive progenitors from mouse bone marrow by treating the mice with 5-fluorouracil which kills cycling cells and, therefore, enriches for the quiescent primitive progenitors. These workers found that these primitive cells could survive in liquid culture for a week in either IL-3 or SCF and still retain their capacity to form colonies. Other cytokines could also support the survival of these cells, but not to the same extent as SCF and IL-3. These results have been supported by work that has shown primitive progenitors isolated by another method (counter centrifugal elutriation) also require SCF and to a lesser extent IL-3 for survival (Keller et al., 1995). As mentioned earlier there is some controversy about whether cytokines initiate specific differentiation programs or are simply permissive, allowing intrinsic differentiation programs to proceed. This model of cytokines simply acting as survival factors is

Input

Differentiation Renewal

Apoptosis

FIGURE 55.3 Representation of the factors contributing to the size of a cell compartment. The size of the compartment is a product of input and renewal minus output (differentiation and apoptosis). The stem cell compartment does not have any input from previous compartments.

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supported by work which has shown that overexpression of bcl-2 in a cell line suppresses apoptosis and makes the cells cytokine independent. Despite the lack of cytokines the cells were able to differentiate into a number of different lineages sometimes without cell division indicating that cytokines were not necessary for initiation of differentiation. However, others have shown that cytokines can induce differentiation (Fairbairn et al., 1993). Iwatsuki et al. (1997) used a murine cell line that is responsive to Epo and introduce chimeric receptors that have substitutions for critical tyrosine residues. These failed to activate the transcription factor STAT5 and could not induce erythroid differentiation suggesting erythroid differentiation could be regulated by STAT5.

MODULATION OF HAEMOPOIETIC PROGENITOR CELL RESPONSES TO CYTOKINES The simple model of a progenitor cell response to a cytokine involves expression by the cell of the appropriate cytokine receptor and its exposure to soluble cytokine. However, the multiplicity of cytokines with the ability to regulate haemopoiesis, and the fact that many target cells express receptors for more than one cytokine, means that cytokine-mediated regulation could not easily be controlled. An alternative hypothesis holds that the distribution and concentrations of cytokines are controlled and is supported by studies of cytokine-binding properties and modes of expression. Similarly, the binding properties of cytokine target cells have been implicated in the regulation of their response to cytokines (Gordon, 1991, 1997). Other ways in which cytokine responses could be modulated in vivo include the actions of inhibitors, agonists and suppressors of cytokine signalling (SOCS). All of these mechanisms may influence the outcome of the clinical administration of haemopoietic growth factors and cytokines.

Modulation by extracellular matrix molecules Cytokines and interleukins are mainly secreted for localised activity but, characteristically, are soluble

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molecules (Najjam et al., 1997). This dichotomy led to the idea that these soluble molecules might be immobilised by binding interactions with the extracellular matrix in the vicinity of the cytokine-secreting cell and could consequently be presented to an appropriate target cell (Gordon, 1991). The proteoglycans are the most versatile group of extracellular matrix constituents with respect to the cytokine modulating abilities. Proteoglycans consist of a core protein with associated polysaccharide glycosaminoglycan side chains. Heparan sulphate is the glycosaminoglycan that has aroused most interest in this context, together with the free chain heparin. Both of these sulphated polysaccharides consist of highly variable sequences differing in uronic acid epimerization, N-deacetylation and sulphation patterns. Therefore, it is possible that sequence variation at the microstructural level provides different binding sites for different cytokines. Heparan sulphate proteoglycans and heparin have been shown to influence interactions between cytokines and target cells in numerous ways.

Immobilisation of cytokines Studies using GM-CSF and IL-3 showed that they could be adsorbed by the heparan sulphate in the extracellular matrix produced by bone marrowderived stromal cells in vitro (Gordon et al., 1987; Roberts et al., 1988). These studies, together with studies of progenitor cell binding to extracellular matrix (see below) led to the concept of cytokine localisation in the haemopoietic microenvironment and presentation of cytokine to its receptor on appropriate target cells (Gordon and Greaves, 1989).

Immobilisation of target cells Heparan sulphate has been shown to act as a cell adhesion molecule for haemopoietic progenitor cells (Siczkowski et al., 1992), so that it could immobilise target cells in the vicinity of appropriate cytokine producing cells or cytokines binding to other sites on the heparan sulphate. In this way, cytokines could be presented to appropriate target cells in a controlled manner (Gordon and Greaves, 1989). In fact, binding to heparan sulphate has been implicated in the long-term maintenance of primitive

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haemopoietic progenitor cells in culture (Gupta et al., 1996).

Modulation of receptor activity Heparin/heparan sulphate may act as a co-receptor to facilitate cytokine binding. For example, fibroblast growth factor does not bind to its high affinity receptor unless cell surface heparan sulphate is also expressed (Yayon et al., 1991). The type III TGF beta receptor is a heparan-sulphate-containing proteoglycan, betaglycan, which presents transforming growth factor b (TGF b) directly to the kinase subunit of the TGF b receptor to form a high affinity complex (Lopez Casillas et al., 1993). Analysis of pro-B cells lacking cell surface heparan sulphate suggests that it is required for efficient receptor binding and cell responsiveness to IL-7 (Borghesi et al., 1999).

Changes in cytokine conformation Balasubramanian and Ramanathan (2000) investigated the effects of heparin on the structure of the pro-inflammatory cytokine, interferon gamma, using circular dichroism and fluorescence spectroscopy. They found loss of alpha helical content and alterations in the tertiary structure of the cytokine, although quaternary structure was not affected. Changes at the dimeric interface between interferon gamma monomers suggested that interaction with heparin reduced dimerization and could therefore affect cytokine activity (Korlipara et al., 1994; Daubener et al., 1995).

Localisation and storage The binding of cytokines to heparan sulphate in extracellular matrix raises the possibility that cytokines might be stored thereby providing a pool of activity for later use. This has been suggested to be the case for interferon gamma (Lortat-Jacob et al., 1991) and several haemopoietic cytokines. Presumably, the cytokines would have to be released from storage by lytic heparinase enzymes when their activity was required. Such enzymes are, in fact, secreted by normal neutrophils and lymphocytes (Matzner et al., 1985; Fridman et al., 1987).

Protection from proteolysis Studies on fibroblast growth factor revealed that binding to heparin or heparan sulphate protected it from proteolytic degradation (Gospodarowicz and Cheng, 1986; Saksela et al., 1988). Later, Clarke et al. (1995) demonstrated that binding to heparin significantly protected IL-7 from the proteolytic activity of plasmin. This mechanism may be responsible for protecting bound cytokines during storage or transport.

Blocking of cytokine activity Heparin and heparan sulphate have been shown to inhibit proliferation in response to several cytokines. They include B cell responses to IL-7 (Clarke et al., 1995), T cell responses to stroma-derived T cell growth factor (Kimura et al., 1991) and fibroblast responses to fibroblast growth factor (FGF) (Westergren Thorsson et al., 1991). Again, this could function to prevent inappropriate target cell stimulation during cytokine transport or storage.

Transport Heparin is a constituent of normal serum (Israels et al., 1960) and could therefore act as a carrier molecule for cytokines in the circulation. This would allow a cytokine to be carried from its site of production to its site of action in an inactive form and protected from proteolytic degradation.

Inhibitors, agonists and suppression of cytokine responses Complex formation between administered cytokines and other soluble molecules could theoretically influence the efficacy of cytokine therapy. Examples exist of cytokine binding with soluble versions of their cognate receptor, growth factor autoantibodies and a2macroglobulin (Gordon, 1991). The recent discovery of a family of cytokine-inducible suppressors of cytokine signalling (SOCS) (Starr et al., 1997; Endo et al., 1997; Naka et al., 1997; Hilton et al., 1998) may also be relevant to the outcome of cytokine therapy. These molecules appear to act by suppressing differentiation so that growth is increased. This is consistent with the observation that cells overexpressing

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SOCS-1 exhibit increased colony size in vitro when stimulated with IL-6 (Starr et al., 1997).

CLINICAL USE OF HAEMOPOIETIC CYTOKINES The effects of cytokines on the proliferation and survival of haemopoietic progenitors, and the production of mature functional blood cells, makes them attractive for therapeutic use in cytopenic patients. Thus, they have been used to enhance red cell, white cell and platelet production in individuals who are anaemic, neutropenic or thrombocytopenic for a variety of reasons. The causes of cytopenia may be constitutional or idiopathic (e.g. bone marrow failure in aplastic anaemia) or iatrogenic (e.g. chemotherapyinduced bone marrow suppression in cancer patients). Cytokines have also been used to mobilise stem cells into the peripheral circulation for autologous or allogeneic stem cell transplantation, and form an essential part of so-called ‘ex vivo expansion protocols’, which are designed to amplify the numbers of stem cells available for transplantation in situations where the number of stem cells is a limiting factor (e.g. use of cord blood transplantation into adult recipients). A few cytokines have already been licensed for clinical use. G-CSF and GM-CSF are probably the most widely used because they stimulate the production of neutrophils to combat life-threatening infections in patients with suppressed bone marrow function. Also, G-CSF is the most effective cytokine in terms of stem cell mobilisation into the periphery. However, the first cytokine to be licensed for clinical use was Epo.

Stimulation of red cell production As Epo stimulates the production of mature red blood cells it has been used in the treatment of patients with anaemia of chronic disease. For example, it has been used in order to maintain haemoglobin levels in renal failure patients who can develop anaemia as a result of reduced erythropoietin production (Eschbach et al., 1989). Epo can stimulate the production of red blood cells prior to surgery which are harvested and subsequently used for autologous transfusion. This avoids the need and potential hazards of peri-

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operative transfusions of donated blood. It may also be valuable in the management of patients with aplastic anaemia, b-thalassaemia or sickle cell disease. Ninety per cent of patients with HIV infection develop anaemia, which can be treated with Epo (Armstrong and Kazanjian, 2001). Epo has also been administered to patients who have anaemia due to treatment for malignancy and has been particularly successful in the treatment of anaemia associated with myeloma and lymphoma, as well as with some solid tumours. Of interest is the use of Epo to treat patients that have a religious objection to blood transfusion, particularly Jehovah’s Witnesses. A recent report found that administration of Epo to four Jehovah’s Witness patients, who had refused blood transfusions, increased their haemoglobin and haematocrit levels to such an extent that heart surgery was able to proceed without blood transfusion (Podesta and Carmagnini, 1999). Others have reported that a Jehovah’s Witness boy with acute promyelocytic leukaemia treated with all-trans retinoic acid (ATRA) and Epo achieved complete remission (Menendez et al., 1998). Epo was also part of a successful treatment for a Jehovah’s Witness with acute myeloid leukaemia (AML) (Kaito et al., 1998). These studies further support the use of Epo as an alternative to blood transfusion. A more controversial issue has been the misuse of Epo by athletes. Administration of Epo, by increasing haemoglobin and haematocrit levels, increases the oxygen carrying capacity of the blood, thereby improving the athlete’s endurance. The use of Epo in this manner can be dangerous, increasing the risk of heart failure, strokes and thrombosis. A number of high profile cases have been reported in the press, including in 1998 the arrest on drugs charges of the doctors of the Tour de France cycling team, Festina. One of the Festina riders, Richard Virenque, has only recently returned to competition after a ban after admitting to regularly taking Epo. More recently there has been controversy surrounding the Russian runner Olga Yegorova at the World Athletics Championships in Edmonton in 2001, who tested positive for Epo but was allowed to continue competing on a technicality.

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Stimulation of neutrophil production G-CSF has been licensed since the early 1990s. It has been used to treat patients with neutropenia after myelosuppressive therapy where there is a risk of infection. Phase I/II trials with G-CSF have shown that it can reduce the time to recovery of neutrophils after myelosuppressive chemotherapy (Gabrilove et al., 1988b; Trillet-Lenoir et al., 1992). Some studies have indicated that G-CSF allows the dose of chemotherapy to be increased safely when used in conjunction with G-CSF (Bronchud et al., 1989; Ardizzoni et al., 1996; Budd et al., 1999). A phase III trial comparing G-CSF against placebo after myelosuppressive treatment for small lung carcinoma showed that the administration of G-CSF could reduce the period of neutropenia after chemotherapy and reduced the risk of infection (Crawford et al., 1991). G-CSF can also be used to treat cyclic or congenital neutropenia (Hammond et al., 1989; Dale et al., 1993; Heussner et al., 1995). Patients with severe chronic neutropenia suffer from often lifethreatening infections. Treatment with G-CSF reduces the frequency of these infections. GM-CSF has also been available for treatment of patients since the early 1990s. Like administration of G-CSF, administration of GM-CSF increases the neutrophil count in patients who have received myeloablative chemotherapy. It does however have an associated increased toxicity compared with G-CSF. In one study it was shown that GM-CSF increased the recovery from neutropenia, but its effectiveness in preventing infection was counteracted by almost 25% of patients having to discontinue treatment because of side-effects (Gerhartz et al., 1993). Both G-CSF and GM-CSF have been used to improve neutrophil recovery after autologous bone marrow transplantation (Brandt et al., 1988; Sheridan et al., 1989; Nemunaitis et al., 1991; Advani et al., 1992).

Stimulation of platelet production Whilst a number of cytokines have been shown to stimulate megakaryocyte production including IL-3 and SCF, it was not until the identification of ligand for c-mpl, thrombopoietin (Tpo), that the regulator of megakaryopoiesis was discovered (de Sauvage et al.,

1994; Foster et al., 1994; Wendling et al., 1994). A number of in vitro studies showed that Tpo in conjunction with other cytokines could stimulate megakaryocyte colony formation (Kaushansky et al., 1994; Broudy et al., 1995; Debili et al., 1995). In animal studies, administration of recombinant megakaryocyte growth and development factor (MGDF), a truncated, pegylated molecule related to Tpo, has been shown to increase megakaryocyte number in the bone marrow followed by an increase in the numbers of platelets in peripheral blood (Ulich et al., 1996; Harker et al., 1996). A murine model where thrombocytopenia has occurred after treatment with carboplatin revealed that MGDF accelerated platelet recovery in a dosedependent fashion (Ulich et al., 1995). Another investigated the effect of MDGF in mice that had received bone marrow transplants. Mice treated with MDGF had an accelerated platelet recovery compared with controls. Animals were also treated with G-CSF and showed the previously reported enhanced recovery of white blood cells. Combination of G-CSF with MDGF showed simultaneous enhanced recovery of leucocytes and platelets (Molineux et al., 1996). These studies suggested that MGDF or Tpo could be used for patients that have reduced platelet levels after chemotherapy. Clinical trials have now taken place. One study investigated the effect of MGDF on patients who were receiving chemotherapy for nonsmall lung cancer (Fanucchi et al., 1997). The platelet level recovered to base-line levels in 14 days in the treated patients compared with more than 21 days for patients receiving placebo. However, a number of groups have reported that MGDF has no effect on platelet recovery. A multicentre trial where MGDF was administered to patients with de novo acute myeloid leukaemia who had received myelosuppressive therapy showed that there was no difference in the median time for peak platelet recovery in patients that received MGDF compared with placebo controls (Archimbaud et al., 1999). Similarly Bolwell et al. (2000) reported on the use of MDGF in the treatment of breast cancer patients who had received autologous peripheral blood progenitor cell transplants. There was no difference in the median time to platelet recovery compared with placebo. Fields et al. (2000) also reported that MGDF had no effect on platelet recovery in patients with advanced malignancies who had received autologous stem cell transplantation. It

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is clear that if MDGF or Tpo are to be used in a clinical setting for patients who have a delayed platelet recovery after treatment, then further studies on doses and timing need to be carried out.

Effects of cytokines on malignant cells It has been suggested that GM-CSF can be used as a priming agent for leukaemia cells. Some leukaemic cells are resting and therefore resistant to chemotherapy agents. Myeloid leukaemic cells can be induced to proliferate and differentiate in vitro in response to GM-CSF. Therefore GM-CSF could bring these cells into cycle and thereby increase their sensitivity to chemotherapy. In a trial of newly diagnosed patients with AML, GM-CSF was administered to patients receiving chemotherapy and the results compared with patients receiving chemotherapy alone. Seventynine per cent of GM-CSF-treated patients achieved disease remission compared with 84% of patients receiving chemotherapy alone, whilst persistent leukaemia was seen in 4% compared with 18% of patients, respectively. When patients younger than 60 years old were analysed, there were fewer relapses in the GM-CSF-treated patients compared with controls in the first 6 months (3% versus 22%) (Buchner et al., 1997). However, there remains some doubt about the efficacy of cytokine priming of patients as there is no correlation with overall survival. It has been suggested that subgroups of patients would benefit more than others. One study has suggested that priming with cytokines be reserved for patients with a low baseline S phase activity. Using cytokine combinations, patients with high levels of S phase activity were less responsive to cytokines than those with low S phase activity and were less likely to achieve complete remission (Smith et al., 1999). Others have shown priming with GM-CSF or G-CSF increased the proliferative activity of AML blasts and also the incorporation of cytosine arabinoside (AraC) into DNA (Braess et al., 2000). GM-CSF may also be used as an anti-tumour therapy. It has been noted in animal models to have antitumour effects and in vitro to enhance the cytotoxic activity of peripheral blood monocytes and lymphocytes (Masucci et al., 1989). Dranoff et al. (1993) using a B16 melanoma model where irradiated tumour cells alone do not induce significant anti-tumour immu-

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nity in mice, found that irradiated tumour cells engineered to express murine GM-CSF produced significant immunoprotection. In the clinic, Leong et al. (1999) injected 20 patients with autologous melanoma vaccine plus GM-CSF. Two patients showed a complete response, whilst others showed a partial response indicating that GM-CSF may have an immunotherapeutic effect.

Stem cell mobilisation During trials of G-CSF and GM-CSF to accelerate neutrophil recovery after autologous bone marrow transplantation it was noted that they resulted in the release of progenitor cells into the peripheral blood of patients. This finding resulted in the use of G-CSF and GM-CSF for the mobilisation of haemopoietic stem and progenitor cells into the bloodstream (referred to as peripheral blood progenitor cells or PBPC) and the subsequent harvest by leukapheresis of PBPC for autologous stem cell transplantation. Mobilisation of stem cells from normal volunteers for allogeneic transplantation to matched unrelated recipients was introduced later. Provision of PBPC rather than bone marrow has the advantage that general anaesthesia is not required, and although leukapheresis takes some time the procedure is preferable to bone marrow donation to some donors. The mechanisms by which progenitor cells are mobilised into the peripheral blood remain elusive. However, as primitive haemopoietic cells express a number of cell adhesion molecules (CAMs) one explanation is that mobilisation affects the binding between haemopoietic cells and bone marrow stromal cells resulting in a release of progenitors into the peripheral blood. It has been shown that CD34positive cells that are released into the periphery have lower expression of certain cell adhesion molecules compared with CD34-positive cells from the bone marrow, particularly VLA-4, LFA-1 and LFA-3, whilst the expression of others is unchanged (Mohle et al., 1993; Dercksen et al., 1995). It is also possible that mobilisation may induce conformational changes in the CAMs without changes in expression levels. Bone marrow CD34-positive cells express the integrins VLA4 and VLA-5 in low affinity states (Kerst et al., 1993; Kovach et al., 1995). Exposure to a number of cytokines results in a transient dose-dependent

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increase in binding properties of VLA-4 and VLA-5 (Kovach et al., 1995; Levesque et al., 1995, 1996). However, it is as yet unknown whether the binding properties of other adhesion molecules, whose expression is unchanged, was affected by exposure to cytokines. Protocols for the mobilisation of progenitor cells involve chemotherapy, cytokine administration or a combination of both. Initially the only method available was myelosuppressive chemotherapy. It was noted that after chemotherapy that there were large increases in the peripheral blood CFU-GM numbers. However, the use of chemotherapy for the mobilisation of progenitor cells was limited by associated problems of infection due to neutropenia and the fact that the timing of apheresis was often unpredictable. Also, it is not appropriate to expose normal allogeneic PBPC donors to cytotoxic agents. The main cytokines that have been used clinically are GM-CSF and G-CSF. One study using the combinations of chemotherapy (high-dose cyclophosphamide (HDC), HDC plus etoposide (HDCE)) found that there was a higher CD34-positive yield when G-CSF was added to the chemotherapy (Schwartzberg et al., 1992). There was a doubling of mononuclear yield and a five-fold increase in CD34-positive yield compared with chemotherapy alone. More recently, a study using docetaxel with G-CSF in patients with breast cancer found it to be effective in mobilising PBPCs with apheresis taking place 8 days after administration (Prince et al., 2000). Studies of mobilisation in chronic myeloid leukaemia (CML) patients have shown an interesting effect. Chronic myeloid leukaemia is characterised by a translocation (t9:22), which results in the Philadelphia (Ph) chromosome. It was noted that in CML patients that had been treated with a chemotherapy regimen of idarubicin, AraC and etoposide, that there was a preferential release of Ph-negative cells. In almost half the patients all metaphases were Ph-negative. The collected Ph-negative cells were used for autotransplants with over 70% showing sustained engraftment. One patient was Ph-negative 19 months after transplant and remarkably also negative as measured by a more sensitive PCR-based technique (Carella et al., 1993). This has led to a number of efforts to preferentially mobilise Ph-negative using cytokine-based regimens. Pratt et al. (1998) used a combination of hydroxyurea and G-CSF and found,

whilst all the patients were 10% Ph-positive prior to treatment, a third of patients had a 100% Ph-negative collections and half had less than 50% Ph-positive collections in the early stages of regeneration. After autografting seven patients, five remained alive and well 13 to 25 months after transplant, whilst two had only 18 and 34% Ph-positive cells in the bone marrow after transplant. In another study, 29 patients who did not respond to treatment with a-interferon were treated with a combination of idarubicin, cytarabine and G-CSF. Nine patients had 100% Ph-negative cells in the PBPC collection whilst a further 15 had greater than 95% Ph-negative cells. Fifteen patients were transplanted with PBPCs. Cytogenetic analysis showed that seven patients became partially Phnegative, whilst one showed a complete cytogenetic response (McBride et al., 2000). Other protocols have used cytokines alone to mobilise progenitor cells. G-CSF is mainly used, although there are reports of it being used in conjunction with other cytokines such as SCF and IL-3. In 1992, Sheridan et al. (1992) reported on the use of G-CSF to mobilise PBPCs and assessed their ability to restore neutrophil and platelet counts in patients after they had received chemotherapy. They found there was a 58-fold increase in the number of CFUGM in the peripheral blood and a 24-fold increase in erythroid progenitors. Platelet recovery was significantly faster in these patients compared with those that were treated in the same way, with the exception of the infusion of PBPCs (15 days versus 39 days). Bensinger et al. (1993) collected PBPC from patients with a range of malignancies, including nonHodgkin’s lymphoma, breast cancer and testicular cancer, who had bone or bone marrow disease or marrow hypoplasia. The time to neutrophil and platelet recovery was more rapid compared with historical controls that had received autologous bone marrow with or without post-transplant administration of G-CSF or GM-CSF.

Ex vivo expansion Haemopoietic cells from normal bone marrow, mobilised peripheral blood and umbilical cord blood have been used transplant patients. However, there are cases where there are insufficient cell numbers for a successful transplant. For example where a low

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number of cells have been mobilised or in the case of cord blood where there are too few primitive stem cells to transplant adults. Therefore there is a need for primitive haemopoietic cells to be expanded ex vivo in order to achieve the required number of cells for transplantation. In addition, the possibility of gene therapy also requires the expansion of cells not just for transplant, but also because many protocols use retroviruses as a gene delivery system and these require dividing cells. The first question that needs to be addressed is what are the candidate cells to be expanded? Obviously the target cells must be the pluripotent haemopoietic stem cells which are capable of selfrenewal. Cell-surface markers have been used in an attempt to define and isolate candidate stem cell populations for expansion. CD34 is an obvious candidate, but the presence or absence of other markers is also used to define phenotypically primitive populations. Thus stem cell immunophenotypes include expression of Thy-1 and the absence of CD38 and lineagespecific markers (Lin). Whilst no direct assays for stem cells are available, some surrogate assays have been described which can be used to indicate changes in primitive haemopoietic cell populations after expansion. In the long-term culture-initiating cell (LTC-IC) assay, haemopoietic cells are added to a preformed stroma which has been irradiated to remove any residual haemopoiesis. Using limiting dilution, Eaves et al. (1992) showed the numbers of cells used to seed the stromal layer have a linear relationship with the output of CFU-GM at 5 weeks of culture. The cells responsible for the production of CFU-GM at 5 weeks were the LTCIC. For the cobblestone area-forming cell (CAFC) assay, haemopoietic cells are also incubated with a stromal cell layer. The presence of primitive haemopoietic cells is indicated by foci of phase dark cells (cobblestone areas). The frequency of primitive progenitors can be calculated by setting up this assay at limiting dilution. (Ploemacher et al., 1989, 1991). Recently an in vivo functional assay has been developed which utilises the ability of human primitive cells to repopulate the bone marrow of severe combined immunodeficient (SCID) and non-obese diabetic/SCID (NOD/SCID) mice. Transplantation of bone marrow or umbilical cord blood cells results in

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engraftment of primitive cells that give rise to multiple lineages. The cell capable of this engraftment is called the SCID repopulating cell (SRC) (Lapidot et al., 1992; Vormoor et al., 1994). Using retroviral gene transfer, it was found that CFC and LTCIC were gene marked, but that these cells did not contribute to repopulation. The SRC were not gene marked implying that they were probably quiescent during the transduction period (Larochelle et al., 1996). A large number of ex vivo expansion protocols have been tested, but to date no consensus of the requirements for a practical method has emerged. The aim of an ex vivo expansion protocol is to increase the numbers of transplantable cells without depleting, by differentiation, the primitive cells required for a transplant. Most studies have used cytokines in order to achieve the necessary number of cell divisions, but it is possible that whilst there may be an increase in cell numbers there is a likelihood that cells will also be induced to differentiate. Early attempts at ex vivo expansion protocols have used a number of cytokines including SCF, IL-3, G-CSF, GM-CSF, etc. However, the most successful recent attempts to increase primitive haemopoietic cell numbers in vitro have included Tpo and FL in their protocols. Piacebello et al. (1997) combined FL and Tpo to expand CD34-positive cells isolated from cord blood. They found a prolonged maintenance and massive expansion of primitive cells. After 6 months of culture there was a 2 000 000-fold increase in the numbers of CFU-GM compared with numbers present at the beginning of the culture and a 200 000-fold increase in LTC-IC after 20 weeks of culture. This latter population suggest that extensive self-renewal had taken place without significant differentiation. They later investigated the effect of 18 different cytokine combinations on the growth and expansion of cord blood CD34-positive cells (Piacibello et al., 1998). After 7 and 14 days of incubation, several combinations could expand the LTC-IC pool, but for extended periods only combinations containing FL, Tpo, SCF and IL-6 could support further expansion (270 000fold of initial values). Colony-forming cell production also expanded for over 7 months to levels of 1 000 000fold that of the initial value. Both FL and Tpo were required for the LTC-IC expansion, but not SCF or IL-6, which however were required for the expansion in colony-forming cells.

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Luens et al. (1998) also used FL and Tpo along with SCF in an attempt to expand CD34-positive, Thy1, Lin cells from human bone marrow. In a 6-day stroma-free system, more than 95% of cells divided by day 6. The post-division CD34-positive cells retained a high CAFC frequency and a capacity for in vivo marrow repopulation similar to that of freshly isolated CD34, Thy1, Lin cells. More recently, Yamaguchi et al. (2001) used a combination of Tpo, FL and SCF to grow cord blood CD34-positive cells on a primary bone marrow stroma. They found that there was a 100- and 1000fold increase in the number of CD34-positive cells and colony-forming cells after 2 and 4 weeks of incubation, respectively. They also found that primitive progenitor cell numbers were expanded by 18- and 60-fold over the same time period, whilst SCID repopulating assays showed an increased engraftment of the cultured cells. Some clinical trials of ex vivo expanded cells for transplantation have been performed. One of the earliest reports was from Naparstek and colleagues (Naparstek et al., 1992), who used a short in vitro preincubation of GM-CSF and IL-3. Patients who received these cells had a significantly faster platelet recovery compared with historical controls. Brugger et al. (1995) used a starting population of 11 million CD34-positive cells corresponding to less than 10% of the typical CD34-positive dose used after leukapheresis. They used a combination of SCF, IL-1b, IL-3, IL-6 and Epo. The cells resulted in a rapid and sustained haemopoietic recovery with a pattern that was identical to that of historical controls treated with either unseparated mononuclear cells or CD34-selected cells.

OVERVIEW From crude beginnings based on soups of undefined growth factors, the study of haemopoietic cytokines has evolved into sophisticated biochemical and molecular biological investigations. The clinical use of cytokines is a remarkable story of successful translational research, but more work remains to be done. The true expansion of stem cells has not been documented adequately and optimum expansion conditions remain poorly defined. This challenge needs to

be met before the best clinical use of expanded stem cells and gene therapy can be made.

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56 Antiangiogenesis Andrew L. Feldman, and Steven K. Libutti National Cancer Institute, Bethesda, MD, USA

The difference between perseverance and obstinacy is that one comes from a strong will, and the other from a strong won’t. Henry Ward

INTRODUCTION Angiogenesis refers to the growth of new capillaries from pre-existing blood vessels. This process is required for a variety of physiologic processes, including normal growth and development, reproduction and wound healing (O’Reilly, 2000). Recently, intense focus has been placed on the strategy of inhibiting angiogenesis to treat patients with cancer, based on the demonstration that tumor growth is angiogenesisdependent (Libutti and Pluda, 2000). In addition, a number of non-neoplastic diseases are characterized by excessive angiogenesis, including ocular, rheumatologic and vascular diseases (Folkman, 1995a). Because angiogenesis is mediated by cytokines that promote the ability of endothelial and supporting cells to form neovessels, the ability to regulate this process has emerged as a key area in cytokine biology. The formal study of angiogenesis spans the last century (Steiner, 1992). Goldman (1907) began the formal study of the phenomenon of tumor hyperThe Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

vascularity in the early 1900s, noting ‘marked capillary budding and new vessel formation, particularly at the advancing border’ of the developing tumor. Clark et al., (1931) developed the first true experimental assay for studying angiogenesis: the transparent ear chamber technique in the rabbit. The term ‘angiogenesis’ was first coined by Hirtig (1935) to describe blood vessel formation in the placenta. Use of this model led Ide (1939) to hypothesize the production of vascular growth factors by tumors. In the 1940s and 1950s, Algire and his co-workers at the National Cancer Institute recognized that tumor-associated vasculature was derived from the host, and was required for tumor growth (Algire et al., 1945; Merwin and Algire, 1956). Further studies by Folkman, published in the early 1970s, demonstrated unequivocally that tumor growth is angiogenesis-dependent (Folkman, 1971, 1990). This led to intensive investigations into the tumor-derived cytokines that promoted this process (Desai and Libutti, 1999; Beckner, 1999). Numerous angiogenic factors have been identified,

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the most clinically relevant of which belong to the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) families. These families of cytokines subsequently have been shown to have a fundamental role in a wide variety of physiologic and pathologic processes. In addition, Folkman’s work sparked a search for endogenous antiangiogenic molecules that might be used clinically (O’Reilly et al., 1994, 1997), as well as synthetic inhibitors (Sebti and Hamilton, 2000). Early clinical trials of synthetic angiogenesis inhibitors for the treatment of cancer began in the 1980s. The first clinical trial using a recombinant endogenous inhibitor (endostatin) began in 2000 (Herbst et al., 2001). Meanwhile, research continues into the basic biology of angiogenesis (O’Reilly, 2000) and the differences in blood vessels in normal and pathologic states (St. Croix et al., 2000) to develop more specific and effective angiogenesis inhibitors.

BIOLOGY OF ANGIOGENESIS Overview Central to the strategy of antiangiogenesis for treating human disease is the concept that physiologic angiogenesis represents a balance between stimulators and inhibitors of angiogenesis (Folkman, 1995a) (Table 56.1). Overexpression of stimulators (proangiogenic cytokines) can lead to pathologic neovascularization,

TABLE 56.1 Angiogenesis in health and disease Physiologic angiogenesis Female estrus cycle Pregnancy Wound healing Pathologic angiogenesis in malignant diseases Solid tumors Hematologic malignancies Vascular neoplasms Pathologic angiogenesis in non-neoplastic diseases Macular degeneration Diabetic retinopathy Rheumatoid arthritis Psoriasis Atherosclerosis

while relative overexpression of inhibitors can lead to vascular quiescence. The delicate balance between stimulators and inhibitors of angiogenesis has been demonstrated in transgenic animal models (Bergers et al., 1999), and the shift of this balance toward a state of pathologic angiogenesis (e.g. cancer) has been termed the ‘angiogenic switch.’ Angiogenesis, whether physiologic or pathologic, requires several fundamental events. First, an initiating event, caused by proangiogenic cytokines, causes endothelial budding from pre-existing, neighboring capillaries (Noden, 1989; O’Reilly, 2000). These endothelial cells must proliferate in order to provide coverage of the lumen of the newly forming capillary structure. In addition, the endothelial cells must migrate in the direction of neovessel formation (Desai and Libutti, 1999). This process depends on adhesion of the endothelial cells to some stromal elements in the extracellular matrix, and proteolytic digestion of other stromal elements (such as basement membrane collagen) (Stetler-Stevenson, 2001). Finally, neovessel formation involves the recruitment of supporting cells (pericytes), whose role has not been fully elucidated (Hirschi and D’Amore, 1997). Angiogenesis in pathologic situations does not mirror physiologic angiogenesis identically (St. Croix et al., 2000; O’Reilly, 2000). The best data come from studies of tumor-associated angiogenesis, in which tumor-associated neovessels tend to lack pericytes (Orlidge and D’Amore, 1987), and sometimes even endothelial cells (Folberg et al., 2000). This process, in which vascular channels are formed which are lined by tumor cells themselves, has been termed ‘vasculogenic mimicry’. Clearly, identifying differences between normal and abnormal angiogenesis may be critically important in developing strategies to inhibit angiogenesis-dependent diseases like cancer without affecting normal vessels or physiologic processes like tissue repair.

Physiologic angiogenesis Angiogenesis in the female reproductive tract Although most organs in the adult do not need to form new blood vessels, angiogenesis is a critical component in the normal functioning of the female reproductive tract (Fraser and Lunn, 2000). In the

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BIOLOGY OF ANGIOGENESIS

ovary, angiogenesis is involved in the processes of folliculogenesis, ovulation and corpus luteum function. In the uterus, endometrial angiogenesis regulates menstruation and implantation. During pregnancy, angiogenesis is necessary for normal development of the placental villous tree and thus for fetal well-being (Kingdom et al., 2000). Endogenous inhibitors of angiogenesis have been identified in the female reproductive tract, including angiopoietin-2 (Maisonpierre et al., 1997), which may play a role in the degeneration of the corpus luteum (Goede et al., 1998). In addition to the importance of such events in reproductive health, the tight, cyclic regulation of angiogenesis in the female reproductive tract may lend insight into how endogenous inhibitors might be employed in diseases like cancer.

Angiogenesis in tissue repair Angiogenesis is an important part of the body’s response to tissue injury, including the inflammatory response, wound healing after surgery or trauma, fracture healing and tissue repair after burn injuries (Glowacki, 1998; Tonnesen et al., 2000; Gibran and Heimbach, 2000). Normal tissue repair requires a coordinated series of interactions involving endothelial cells, supporting cells and the extracellular matrix. For example, endothelial cell expression of the integrin avb3 appears necessary for wound angiogenesis (Tonnesen et al., 2000). As mentioned above, a deeper understanding of the regulation of the physiologic angiogenesis that occurs in tissue repair may lead to improved strategies for inhibiting pathologic angiogenesis. In addition, suppression of normal wound healing could be a serious side-effect in patients undergoing antiangiogenic therapy. Thus, evaluation of angiogenesis inhibitors for therapeutic clinical use has involved studies of their effects on wound healing (Berger et al., 2000).

Cancer Angiogenesis in solid tumors Folkman (1995a) demonstrated that tumors up to a volume of 2–3 mm3 can derive nutrients from their environment by simple diffusion (prevascular stage). To grow beyond this size, however, tumors require the

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ingrowth of new vessels. It should be noted, however, that tumor cells can remain dormant, but viable, for long periods of time in the prevascular stage (Demicheli et al., 1994; Holmgren et al., 1995). One strategy for antiangiogenesis, therefore, is to maintain a state of tumor dormancy, for example in cases of micrometastases. Interestingly, it has been shown that primary murine tumors can release endogenous angiogenesis inhibitors into the circulation and maintain microscopic tumor dormancy at a distant site (Holmgren et al., 1995). These distant metastases grow rapidly when the primary tumor is removed. This phenomenon has been hypothesized to exist in humans with cancer as well (Prehn, 1993), although this has not been rigorously proven. Folkman’s group has shown that in addition to preventing further angiogenesis-dependent growth, antiangiogenic treatment actually can regress established, vascularized tumors in mice (O’Reilly et al., 1996). Thus it appears that even established tumor-associated blood vessels remain under the influence of a balance between stimulators and inhibitors of angiogenesis, and that treatment strategies that target these vessels can lead to tumor regression.

Angiogenesis and hematologic malignancies Initial investigations in the use of angiogenesis inhibitors in the treatment of cancer focused on solid tumor histologies. However, it has been shown that antiangiogenic agents also affect hematologic malignancies (Mangi and Newland, 2000), and it is clear that angiogenesis plays a key role in the bone marrow (Padro et al., 2000). The response rates of patients with refractory multiple myeloma to thalidomide represent one of the most promising clinical results in the field (Singhal et al., 1999).

Angiogenesis and vascular neoplasms Finally, the use of antiangiogenic agents for the treatment of vascular neoplasms deserves special mention. These neoplasms include hemangiomas and their malignant counterparts, and HIV-associated Kaposi’s sarcoma (see interferon-a, below). Because the cells of origin in these diseases are endothelial in nature, antiangiogenesis might be a particularly effective strategy (Dezube, 2000; Paller, 2000).

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Angiogenesis in non-neoplastic diseases Although the majority of antiangiogenesis research has focused on cancer, a number of non-neoplastic diseases are characterized by pathologic angiogenesis. These include ocular diseases, which may cause blindness; rheumatologic diseases, including rheumatoid arthritis; and possibly atheromatous plaque rupture.

Angiogenesis in ocular diseases Pathologic ocular angiogenesis can originate from the choroid circulation and cause macular degeneration, or from the retinal vasculature causing retinopathy. Abnormal retinal vessels are stimulated by ischemia, a phenomenon that appears to be due to increased vitreous levels of VEGF. The normal vascular quiescence of the vitreous and cornea appears due to the presence of endogenous inhibitors of angiogenesis, such as pigment epithelium-derived factor (PEDF) (Dawson et al., 1999). The therapeutic use of PEDF has been shown to prevent ischemia-induced retinopathy in experimental models (Stellmach et al., 2001). Inhibition of choroidal neovascularization has also been demonstrated in preclinical models, including a recent report of a gene therapy approach utilizing an adenovirus carrying the endostatin gene (Mori et al., 2001).

Angiogenesis in rheumatologic diseases Because rheumatologic diseases generally involve inflammation, which in turn causes shifts in the cytokine balance and promotes angiogenesis, antiangiogenic strategies have been proposed to treat these conditions. Rheumatoid arthritis (RA) is characterized by angiogenesis accompanying leukocyte migration to synovial tissues (Koch, 2000). One angiogenic mediator which appears prominent in RA is endoglin. Endoglin is an endothelial glycoprotein adhesion molecule that acts as a receptor for transforming growth factor-b (see below) and is upregulated on synovial endothelial cells from patients with RA (Szekanecz et al., 1995). VEGF also appears to play a role in the pathogenesis of RA, and arthritis can be attenuated through the local administration of a

soluble form of the VEGF receptor, flt-1 (Miotla et al., 2000). The role of endogenous inhibitors of angiogenesis in the synovium is not understood. Cartilage, however, is relatively avascular and tumor-resistant, and contains multiple inhibitors of angiogenesis. In fact, the first tissue inhibitor of metalloproteinases (TIMP-1, see below) was originally isolated from cartilage (Moses et al., 1990). Thus, it has been speculated that inhibitors in cartilage juxtaposing synovial surfaces may play a role in regulating angiogenesis as it relates to RA (Koch, 2000). In any case, results in experimental models suggest that antiangiogenic strategies may be useful in the treatment of patients with RA.

Angiogenesis in cardiovascular diseases The role of angiogenesis in cardiovascular diseases has been an area of intense interest for two reasons. First, the strategy of therapeutic angiogenesis for ischemic cardiac, cerebral and peripheral vascular disease has been the subject of extensive investigation (Freedman and Isner, 2001). Although beyond the scope of this review of antiangiogenesis, this strategy deserves mention because of its clinical promise and because of common lessons to be learned from the study of regulating angiogenesis, and in particular the use of gene therapy (Nikol and Huehns, 2001). Second, it has been suggested that intimal neovascularization leads to atherosclerotic plaque growth in blood vessels, and that angiogenesis inhibitors might slow or reverse this process (Moulton, 2001). In apolipoprotein E-deficient mice, which develop advanced atherosclerotic lesions, plaque growth has been related to intimal angiogenesis, which was inhibited by the angiogenesis inhibitors, endostatin and TNP-470 (Moulton et al., 1999).

ENDOGENOUS INHIBITORS OF ANGIOGENESIS More than 40 endogenous substances that inhibit angiogenesis have been identified (Feldman and Libutti, 2000) (Table 56.2). A number of these are cleavage fragments of larger proteins, including endostatin and angiostatin (O’Reilly et al., 1994, 1997). There are also a number of cytokines originally

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TABLE 56.2 Endogenous inhibitors of angiogenesis Inhibitor Proteolytic fragments Angiostatin Antithrombin Arresten Canstatin Endostatin Fibronectin PEX Prolactin Prothrombin Restin Vasostatin Interferons (IFNs) IFN-a

Molecular and physiologic characteristics

Reference(s)

38 kDa internal plasminogen fragment (kringles 1–4); other kringle combinations also active 53–55 kDa serpin; cleavage of C-terminal loop 26 kDa C-terminal fragment of a1 chain of type IV collagen 24 kDa C-terminal fragment of a2 chain of type IV collagen 20 kDa C-terminal fragment of type XVIII collagen 29 kDa N-terminal and 40 kDa C-terminal heparin-binding fragments C-terminal hemopexin-like domain of matrix metalloproteinase-2 16 kDa N-terminal fragment

O’Reilly et al. (1994)

22 kDa fragment (kringle 2) 22 kDa C-terminal fragment of type XV collagen N-terminal fragment of calreticulin 8 to 20 kDa glycoproteins secreted by lymphocytes and phagocytes

IFN-b IFN-c Interleukins (ILs) IL-1 IL-4 IL-10 IL-12

23 kDa glycoprotein secreted by fibroblasts and epithelial cells 20 to 25 kDa glycoprotein secreted by T cells and natural killer cells

IL-18 Other molecules Angiopoietin-2 EMAP-II

19 kDa IFN-c-inducing cytokine

17 kDa b-isoform involved in autocrine antiangiogenic pathway 13 kDa lymphokine Inhibitor of synthesis of multiple cytokines 75 kDa glycoprotein with antiangiogenic effects mediated by IFN-c and IP-10

Inhibitor of angiopoietin-1-mediated activation of Tie2 20 kDa (34 kDa pro-form) tumor-derived inflammatory cytokine

Gro-b IP-10

8 kDa C-X-C chemokine 8.6 kDa C-X-C chemokine induced by IFN-c

2-Methoxyestradiol MIG PEDF Platelet factor-4 TGF-b1 TIMP-1 to -4

0.3 kDa estrogen metabolite 11.7 kDa C-X-C chemokine induced by IFN-c 50 kDa inhibitor expressed by retinal pigment epithelial cells 28 kDa heparin-binding factor derived from platelets 25 kDa pleuripotential growth regulator Family of naturally occurring collagenase inhibitors

TSP-1 and -2

450 kDa trimeric glycoproteins derived from platelets and fibroblasts

O’Reilly et al. (1999) Colorado et al. (2000) Kamphaus, et al, (2000) O’Reilly et al. (1997) Homandberg et al. (1985) Brooks et al. (1998) Ferrara et al. (1991); Clapp et al. (1993) Lee et al. (1998) Ramchandran et al. (1999) Pike et al. (1998) Singh et al. (1995); Ribatti et al. (1996) Singh et al. (1995) Friesel et al. (1987) Cozzolino et al. (1990) Volpert et al. (1998) Huang et al. (1996) Voest et al. (1995); Sgadari et al. (1996) Cao et al. (1999a) Maisonpierre et al. (1997) Schwarz, et al, (1999); Berger et al. (2000) Cao et al. (1995) Angiolillo et al. (1995); Strieter et al. (1995) Fotsis et al. (1994) Strieter et al. (1995) Dawson et al. (1999) Maione et al. (1990) RayChaudhury (1991) Moses et al. (1990); Takigawa et al. (1990); Anand-Apte et al. (1997); Wang et al. (1997) Good et al. (1990); Volpert et al. (1995)

EMAP-II, endothelial monocyte-activating polypeptide-II; IFN, interferon; IP-10, interferon-inducible protein-10; MIG, monokine induced by interferon-c; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TSP, thrombospondin.

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characterized for their role in other processes (mostly immune mediators), which subsequently have been found to have antiangiogenic properties. Many of these have been described elsewhere in this book, but their role in angiogenesis will be briefly delineated here. Understanding endogenous inhibitors of angiogenesis is critical because (1) their presence in normal, avascular tissues such as cartilage may help elucidate natural mechanisms by which the body prevents pathologic angiogenesis; (2) recombinant forms of these proteins may be therapeutic in patients with cancer and other diseases; and (3) delivery of genes which encode these inhibitors may offer advantages over other antiangiogenic strategies (see below).

Proteolytic fragments Endostatin Endostatin is a 20 kDa C-terminal fragment of the noncollagenous domain of collagen XVIII first isolated from conditioned media from the murine hemangioendothelioma cell line, EOMA (O’Reilly, et al., 1997). In mice, recombinant endostatin has shown pronounced inhibitory activity against a wide range of tumors. Repeated administration to mice bearing Lewis lung carcinomas did not induce resistance to these effects, and, in fact, ultimately led to the disappearance of cancer in these animals even after the discontinuation of therapy (Boehm et al., 1997). As mentioned above, murine endostatin has also shown activity in reducing intimal neovascularization and atherosclerotic plaque growth in apolipoprotein E-deficient mice (Moulton et al., 1999). The human form of endostatin has been cloned and produced in a recombinant form (rhEndo) that also exhibits antiangiogenic activity (Dhanabal et al., 1999a). rhEndo does not inhibit wound healing in mice (Berger et al., 2000). Phase I trials of rhEndo have recently commenced for patients with advanced solid malignancies (Herbst et al., 2001) (Table 56.3). Preliminary data suggest no serious adverse events and a suggestion of minor or partial responses (Zogakis and Libutti, 2001), but definitive toxicity and efficacy data are pending. The mechanism of action of endostatin has not been fully elucidated. In vitro, endostatin appears to

specifically inhibit endothelial cell proliferation, without having antiproliferative effects on other cell types, such as smooth muscle cells, fibroblasts or tumor cells (O’Reilly et al., 1997). This effect may be due in part to cell cycle effects, resulting in G1 arrest (Dhanabal et al., 1999a). In addition, endostatin induces endothelial cell-specific apoptosis (Dhanabal et al., 1999b). Some of these cellular effects may be due to alterations in intracellular calcium signaling pathways (Jiang et al., 2001). In addition, endostatin may function in part by inhibiting endothelial cell migration. Endostatin has been shown to inhibit the activity of matrix metalloproteinases (Kim et al., 2000), and to interact with a5 and av integrins on endothelial cell surfaces (Rehn et al., 2001). Recently, a wide variety of genes have been shown to be down-regulated in proliferating endothelial cells treated with endostatin, lending insight into some of the mechanisms of the aforementioned effects (Shichiri and Hirata, 2001). Endogenous endostatin can be detected in the circulation of healthy human subjects (Standker et al., 1997), and increased levels are present in patients with various cancer histologies (Feldman et al., 2000, 2001a, 2001b) and systemic sclerosis (Hebbar et al., 2000). The mechanism for generation of endogenous human endostatin has not been demonstrated, but based on experimental models may involve cleavage from collagen XVIII by elastase (Wen et al., 1999) or cathepsin L (Felbor et al., 2000). Elucidating the mechanisms by which endogenous angiogenesis inhibitors such as endostatin are cleaved from their parent proteins in vivo may suggest new treatment strategies that shift the angiogenic balance toward a more quiescent phenotype.

Angiostatin Angiostatin is a 38 kDa internal fragment (kringles 1–4) of plasminogen with pronounced antiangiogenic and antitumor properties in mice (O’Reilly et al., 1994). Other plasminogen fragments also have antiangiogenic activity, including kringles 1–5, kringles 1–3, and kringle 5 alone (Cao et al., 1997, 1999b; Lu et al., 1999; Joe et al., 1999). Human endostatin has been cloned and the recombinant form retains antitumor activity in experimental models (Sim et al., 1997). Angiostatin has not been associated with

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

TABLE 56.3 Angiogenesis inhibitors in clinical trials Inhibitor

Class of agent

Naturally occurring inhibitors Endostatin Direct EC inhibitor Interferon-a Interferon Interleukin-12 Interleukin Squalamine Neovastat Synthetic inhibitors Marimastat COL-3 BMS-275291

Sponsor

I II/III I/II

Advanced solid tumors Multiple solid tumors Kaposi’s sarcoma

I, II

Non-small cell lung, ovarian, other advanced cancers Non-small cell lung, kidney cancers

EntreMed, Rockville, MD, USA Commercially available Genetics Institute, Cambridge, MA, USA Magainin Pharmaceuticals, Plymouth Meeting, PA, USA Aeterna, Sainte-Foy, Quebec, Canada

III

MMP inhibitor MMP inhibitor MMP inhibitor

III I/II II/III

Small cell lung, breast cancers Brain malignancies Non-small cell lung cancer

I/II, II, III

Melanoma, multiple myeloma, Kaposi’s sarcoma, CLL, prostate, ovarian, colorectal, liver, kidney, and non-small cell lung cancers, gynecologic sarcomas Kaposi’s sarcoma, anaplastic glioma Ovarian and colorectal cancers, Kaposi’s sarcoma Ovarian, kidney cancers; other solid tumors

Integrin inhibitor

IM862 CAI

Indication

Dogfish shark liver extract MMP inhibitor

Thalidomide

EMD121974

Trial phase

I, I/II I,II,II

Calcium influx Inhibitor

I, II

Anti-VEGF strategies SU5416 Tyrosine kinase I, I/II, II, III inhibitor (VEGF receptor) SU6668 Tyrosine kinase I inhibitor (VEGF, FGF, and PDGF receptors) Anti-VEGF Monoclonal antibody I, II, III antibody

Multiple solid tumors, AML, multiple myeloma Advanced solid tumors Kidney, breast, colorectal cancers, other refractory solid tumors

British Biotech, Annapolis, MD, USA Collagenex, Newtown, PA, USA Bristol-Myers Squibb, Wallingford, CT, USA Celgene, Warren, NJ, USA

Merck KcgaA, Darmstadt, Germany Cytran, Kirkland, WA, USA National Cancer Institute, Bethesda, MD, USA Sugen, South San Francisco, CA, USA Sugen, South San Francisco, CA, USA National Cancer Institute, Bethesda, MD, USA; Genentech, South San Francisco, CA, USA

AML, acute myelocytic leukemia; CAI, carboxyamido-triazole; CLL, chronic lymphocytic leukemia; FGF, fibroblast growth factor; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor.

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significant toxicity (Zetter, 1998), and is being readied for phase I clinical trials. The mechanisms of angiostatin’s antiangiogenic effects is not well understood, but may involve inhibiting endothelial cell proliferation by binding the alpha subunit of adenosine triphosphate synthase, thus inhibiting enzyme activity (Moser et al., 1999). Angiostatin also induces endothelial cell apoptosis (Lucas et al., 1998). Interestingly, angiostatin is generated endogenously by subcutaneous Lewis lung carcinomas in mice, inhibiting the growth of spontaneously occurring pulmonary metastases (O’Reilly et al., 1994). This conversion of plasminogen to angiostatin is catalyzed by macrophage-derived metalloelastase (Dong et al., 1997). In other models involving human cell lines, different molecules have been implicated in the conversion of plasminogen to angiostatin, including urokinase, tissue plasminogen activator and free sulfhydryl donors (Gately et al., 1997; Westphal et al., 2000). As with endostatin, the enzymes that catalyze this conversion might be useful therapeutic agents to generate antiangiogenic proteolytic fragments in vivo. This strategy already has shown efficacy in preclinical gene therapy models (Matsuda et al., 2000; Gorrin-Rivas et al., 2000).

1987; Singh et al., 1995; Ribatti et al., 1996) (Table 55.2).

Interferon-a and -b Interferon-a and -b share components of the same receptor, and together comprise the type I IFNs (see Chapter 23). IFN-a represents a group of 8 to 20 kDa glycoproteins secreted by lymphocytes and phagocytes, and has multiple properties, including antiviral and tumor suppressor activities (Gutterman, 1994). IFN-b is a 23 kDa glycoprotein secreted by fibroblasts and epithelial cells. Clinical data suggested that chronic administration of IFN-a could lead to the regression of vascular neoplasms, including hemangiomas (Ezekowitz et al., 1992), Kaposi’s sarcoma (Real et al., 1986), and pulmonary hemangiomatosis (White et al., 1989). These observations led to a search for antiangiogenic properties, and it was found that both IFN-a and IFN-b could down-regulate bFGF expression in human tumors (Singh, et al., 1995). IFN-a currently is in an ongoing phase II/III trial for patients with advanced solid tumors of various histologies (Table 55.3).

Interferon-c Other proteolytic fragments Although not as thoroughly developed for use in clinical trials, a number of other proteolytic fragments with antiangiogenic activity have been discovered (Table 56.2). Like endostatin, several of these are fragments of collagen molecules. These include arresten and canstatin, which are C-terminal fragments of the a1 and a2 chains, respectively, of collagen IV (Colorado et al., 2000; Kamphaus et al., 2000); and restin, which is a C-terminal fragment of collagen XV (Ramchandran et al., 1999). All have demonstrated antitumor activity in experimental models.

Interferons The interferons (IFNs), including IFN-a, IFN-b, and IFN-c, represent a family of endogenous glycoproteins initially identified by their antiviral properties (Baron and Dianzani, 1994). Subsequent studies have revealed that all three IFNs have antiangiogenic, as well as immunologic, activity (Friesel et al.,

Interferon-c represents a group of 20 to 25 kDa glycoproteins secreted by T cells and natural killer cells; it uses a different receptor than IFN-a and IFN-b, and is referred to as a type II IFN (see Chapter 24). Friesel et al., (1987) demonstrated that IFN-c inhibited endothelial cell proliferation in a dose-dependent fashion. IFN-c has antitumor properties in a number of experimental models, and it has not always been possible to dissect its immunologic effects from its antiangiogenic properties. However, recent studies have shown that the antiangiogenic effects of IFN-c in vivo are primarily due to its ability to induce expression of the downstream mediators, interferoninducible protein-10 (IP-10) (Angiolillo et al., 1995; Strieter et al., 1995) and monokine induced by interferon-c (MIG) (Strieter et al., 1995).

Interleukins The interleukins (ILs) are a family of leukocyte-derived proteins with broad-ranging effects on multiple

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

ANTIANGIOGENIC AGENTS CURRENTLY IN CLINICAL TRIALS

biologic properties. Recently, a number of ILs have been shown to have either stimulatory or inhibitory effects on angiogenesis. ILs with an N-terminal gluleu-arg (ELR) motif, such as IL-8, have been found to be angiogenic; those lacking an ELR motif generally inhibit angiogenesis (Strieter et al., 1995) (Table 56.2). Interleukin-4 and the b-chain of IL-1 both have been reported to inhibit FGF-stimulated angiogenesis (Cozzolino et al., 1990; Volpert et al., 1998). IL-10 has been shown to inhibit angiogenesis and experimental tumors, in part by down-regulating VEGF expression (Huang et al., 1996). IL-12 and IL-18 both have marked antitumor effects which are due in part to their ability to inhibit angiogenesis. This activity appears related to their ability to induce expression of IFN-c and its downstream antiangiogenic mediators, IP-10 and MIG (see above) (Voest et al., 1995; Sgadari et al., 1996; Cao et al., 1999a). Several phase I trials using recombinant IL-12 have been performed, most finding a maximum tolerated dose of 500 ng kg1 (Libutti and Pluda, 2000). Some responses have been noted, although it is difficult to discern whether these might be immunologic responses rather than due to antiangiogenic effects. Correlation of response with circulating IFN-c levels suggest the possibility that downstream mediators, such as IP-10 and MIG might be involved. A phase I/II trial in patients with Kaposi’s sarcoma is ongoing.

Other molecules Numerous other endogenous substances have been found to have antiangiogenic properties (Table 56.2). Of these, 2-methoxyestradiol (2-ME) deserves mention because of planned clinical trials using this agent. 2-ME is a 0.3 kDa estrogen metabolite which potently inhibits endothelial cell proliferation and invasion (Fotsis et al., 1994). Oral administration of 2-ME to mice bearing Meth-A sarcoma or B16 melanoma inhibited tumor growth without causing toxicity. In another study (Klauber et al., 1997), 2-ME inhibited both bFGF- and VEGF-induced neovascularization in a murine corneal assay, and inhibited the growth of human breast cancer xenografts in severe combined immunodeficient (SCID) mice without significant toxicity. In a recent human pancreatic cancer model (Schumacher et al., 1999), 2-ME was also found to prolong S-phase and induce apoptosis in the tumor

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cells. This was associated with an inhibition of lung metastases in nude mice. These data, along with the low toxicity profile and oral administration, make 2-ME an attractive candidate for clinical trials in patients with cancer.

ANTIANGIOGENIC AGENTS CURRENTLY IN CLINICAL TRIALS The number of clinical trials evaluating angiogenesis inhibitors has recently increased dramatically (Table 56.3). This increase has been accompanied by the significant public attention received by the clinical testing of endostatin, as well as the development of more specific, second-generation, synthetic inhibitors (e.g. SU6668, see below). Because of the tremendous flux in the field, this list may not be complete, but should demonstrate the variety of agents currently being tested in the clinic. Although the agents listed here are being tested in patients with cancer, favorable results would be expected to be applicable to a variety of non-neoplastic diseases as discussed above.

Naturally occurring inhibitors The endogenous inhibitors of angiogenesis, endostatin, IFN-a and IL-12 (Tables 56.2 and 56.3) have been discussed above. A number of other angiogenesis inhibitors, not known to be endogenously present in humans, are naturally occurring compounds isolated from plants or animals (Paper, 1998).

Squalamine Squalamine lactate (squalamine; MSI-1256F) is an amino sterol isolated from the liver of the dogfish shark. Preclinical studies demonstrated inhibition of endothelial cell proliferation, with a mechanism of action apparently related to inhibition of the sodiumhydrogen exchanger NHE3. In an initial, phase I trial, patients received up to 255 mg m2 day1 by continuous infusion without dose-limiting toxicity. In a second continuous-infusion trial, dose-limiting toxicity consisting of grade 3 transaminase elevations were encountered at a dose of 538 mg m2 day1 (Libutti

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and Pluda, 2000). Phase I and II trials are ongoing for patients with a variety of advanced cancers.

patients with a wide variety of solid and hematologic malignancies (Table 56.3).

Synthetic inhibitors

Matrix metalloproteinase inhibitors

A wide variety of synthetic inhibitors of angiogenesis have been tested in experimental models, with a number proceeding to clinical trials. These include thalidomide, matrix metalloproteinase (MMP) inhibitors and other agents.

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent enzymes which degrade basement membranes and other extracellular matrix components (Zogakis and Libutti, 2001). These enzymes have been shown to be produced by tumors and to correlate with tumor aggressiveness and metastasis. In addition, MMPs facilitate angiogenesis by degrading the capillary basement membrane and allowing endothelial cell migration through the extracellular matrix. Thus, inhibiting MMPs is an attractive antitumor strategy for multiple reasons. Endogenous tissue inhibitors of metalloproteinases (TIMPs) exist (TIMP-1 to -4) and have been shown to have antiangiogenic and antitumor properties in experimental models (Moses et al., 1990; Takigawa et al., 1990; Anand-Apte et al., 1997; Wang et al., 1997) (Table 56.2). Because of the sub-optimal molecular characteristics of the TIMPs, synthetic MMP inhibitors have been designed for clinical use. These inhibitors function similarly to TIMPs in their ability to bind the active zinc-binding site on MMPs (Nelson et al., 2000). Marimastat is a synthetic MMP inhibitor which selectively inhibits MMP-1, MMP-2, MMP-7 and MMP-9 (Primrose et al., 1999). In a preliminary clinical study in patients with recurrent colorectal cancer, serum carcinoembryonic antigen (CEA) levels were chosen as a serologic endpoint (Primrose et al., 1999). Median rates of CEA rise decreased significantly with twice daily, but not once daily, dosing. Toxicities were mainly musculoskeletal. Similarly, a reduced rate of CA 19–9 rise was noted in a study of marimastat in patients with pancreatic cancer (Rosemurgy et al., 1999). Marimastat has been used in combination with paclitaxel and carboplatin in patients with non-small cell lung cancer, yielding a 50% response rate (Libutti and Pluda, 2000). Currently, marimastat is being evaluated in phase III trials for patients with small cell lung cancer or breast cancer (Table 56.3). COL-3 is a synthetic MMP inhibitor derived from tetracycline that decreases MMP-2 and MMP-9 expression (McGarvey et al., 1998). COL-3 inhibited bony metastases in an experimental prostate cancer model (Selzer et al., 1999). Phase I/II clinical trials are underway.

Thalidomide Thalidomide was developed in the 1950s as an oral sedative after extensive safety testing in experimental animal models (Gradishar, 1997). Subsequently, an association was made between maternal thalidomide use and infants born with dysmelia (stunted limb growth) (Mellin and Katsenstein, 1962), and the drug was removed from the market. Subsequent studies revealed that mice lacked the enzymes necessary to produce the active metabolites of thalidomide responsible for this effect. Based on evidence that the teratogenic effects were associated with anomalous blood vessel growth in the developing fetal limb buds, D’Amato and colleagues (1994) tested thalidomide in the rabbit corneal neovascularization assay and found it to have antiangiogenic properties. Although the mechanism of this antiangiogenic activity is not known, it may relate to the ability of thalidomide to inhibit TNF-a production (Ching et al., 1998) or to down-regulate expression of b integrin subunits, which play an important role in endothelial cell adhesion and migration (McCarty, 1997). Based on the antiangiogenic properties of thalidomide, its antitumor activity in preclinical models (Ching et al., 1998), and the ease of oral dosing, a phase I trial was conducted in patients with AIDSrelated Kaposi’s sarcoma (Libutti and Pluda, 2000). The maximum tolerated dose was 600 mg day1, with the dose-dependent toxicity being excessive somnolence. In a phase II trial in 66 patients with melanoma, renal, breast or ovarian cancers, a lower dose of 100 mg day1 was used, and no grade 3 or 4 toxicity was encountered (Eisen et al., 2000). Three of 18 patients with renal cancer experienced partial responses, and another three patients had stabilization of disease. Phase II and III trials are ongoing for

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

ANTIANGIOGENIC AGENTS CURRENTLY IN CLINICAL TRIALS

Other agents Among the other synthetic antiangiogenic agents in clinical trials (Table 56.3), carboxyamido-triazole (CAI) deserves mention. CAI blocks invasion and angiogenesis by inhibiting non-voltage-gated calcium influx in endothelial cells (Alessandro et al., 1996). CAI entered clinical trials in an oral form consisting of a polyethylene glycol (PEG-400) solution (Kohn et al., 1996). Dose-limiting toxicities of sensory axonal neuropathy and neutropenia were encountered at 330 mg m2 every other day. A phase II study of patients with stage D2 androgen-independent prostate cancer did not yield clinical responses (Bauer et al., 1999). Phase I and II clinical trials for patients with a variety of other cancer histologies are ongoing (Table 56.3).

Anti-VEGF strategies Vascular endothelial growth factor (VEGF) was first identified as a vascular permeability factor (VPF) secreted by tumor cells (Senger et al., 1983). It has since become recognized as one of the most important proangiogenic cytokines in cancer and other diseases characterized by pathologic neovascularization. Thus, inhibiting the angiogenic properties of VEGF is an attractive strategy for treating these conditions. Two approaches will be discussed: inhibiting VEGF receptors on endothelial cells and neutralizing VEGF directly using a monoclonal antibody.

VEGF receptor inhibitors VEGF exerts its effects on endothelial cells via two membrane-associated receptor tyrosine kinases, VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR/Flk-1) (Zhu and Witte, 1999). A number of synthetic inhibitors of VEGF receptors have been studied, including two currently in clinical trials: SU5416 and SU6668 (Table 56.3). Both consist of an indolin-2-one core structure that occupies the receptor ATP binding site, inhibiting receptor phosphorylation and subsequent intracellular signal transduction (Mendel et al., 2000). SU5416 was introduced as a selective inhibitor of KDR, as shown by its ability to inhibit VEGF-, but not FGF-stimulated endothelial cell proliferation in vitro (Fong et al., 1999). In vivo, SU5416 inhibited a variety

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of tumor types at nontoxic doses in murine models (Fong et al., 1999; Angelov et al., 1999), and also inhibited the growth of experimental liver metastases (Shaheen, 1999). These promising findings prompted a phase I dose-escalation study in patients with AIDSrelated Kaposi’s sarcoma, which demonstrated minimal toxicity and a number of patients whose lesions regressed during therapy (Zhu and Witte, 1999). A subsequent phase I/II trial in combination with 5-fluorouracil (5-FU) and leucovorin in patients with metastatic colon cancer yielded complete or partial responses in 37% of patients and a median time to progression of 9 months, longer than would be expected with 5-FU and leucovorin alone (Zogakis and Libutti, 2001). Based on these clinical results, SU5416 is in ongoing phase I–III clinical trials for a variety of tumor histologies (Zhu and Witte, 1999) (Table 56.3). SU6668 was introduced as a modified form of SU5416 whose inhibition of receptor tyrosine kinases extended to the receptors for FGF and plateletderived growth factor (PDGF), as well as KDR (Laird et al., 2000). This was evidenced by its ability to inhibit both VEGF- and FGF-stimulated endothelial cell proliferation in vitro. In addition, the drug had better oral bioavailability than SU5416. As with SU5416, SU6668 was able to inhibit a variety of tumor types in mice (Laird et al., 2000), as well as experimental liver metastases (Shaheen et al., 1999). SU6668 represents the ability to respond to our increasing understanding of the biology of angiogenesis in the development of new, more specific therapeutic drugs. Phase I trials of SU6668 are underway (Table 56.3).

Anti-VEGF antibody Early work in the development of neutralizing antibodies to VEGF led to the development of a murine monoclonal antibody called A.4.6.1, which demonstrated the ability to recognize the multiple isoforms, VEGF121, VEGF165, and VEGF189 (Kim et al., 1992). A.4.6.1 was shown to inhibit angiogenesis and growth of a variety of human tumors in vivo in experimental models (Borgstrom et al., 1996, 1998). To minimize the human anti-globulin response to mouse IgG1 in clinical trials, the antibody was humanized by sitedirected mutagenesis of a human framework. This humanized antibody demonstrated preserved VEGF

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binding efficiency, inhibited endothelial cell proliferation in vitro, and inhibited human rhabdomyosarcoma tumor growth in nude mice (Presta et al., 1997). A phase I trial of anti-VEGF antibody suggested safety at doses of up to 10 mg kg1. Three of 24 patients developed tumor-related hemorrhage during treatment (Zogakis and Libutti, 2001). A phase II trial combined anti-VEGF antibody with 5-FU and leucovorin in patients with metastatic colon cancer and demonstrated improved response rates compared with 5-FU and leucovorin alone, and a phase III trial has been initiated. A phase II trial of anti-VEGF antibody in combination with carboplatin and paclitaxel in patients with non-small cell lung cancer did not demonstrate results as favorable as those seen in patients with colon cancer (Zogakis and Libutti, 2001). In addition, six patients developed severe pulmonary hemorrhage that was fatal in four patients, suggesting anti-VEGF antibody may not be safe in patients with lung cancer. Phase I–III trials in a variety of other tumor histologies are currently under way (Table 56.3).

ANTIANGIOGENIC GENE THERAPY As the strategy of inhibiting angiogenesis for the treatment of human diseases (particularly cancer) has matured to the point of clinical trials, several potential limitations have been cited that might be ameliorated by gene therapy approaches (Feldman and Libutti, 2000; Libutti and Feldman, 2001). First, because most antiangiogenic agents are not cytotoxic to tumor cells themselves (O’Reilly et al., 1994, 1997), and because small, prevascular micrometastases may remain dormant for a prolonged period of time (Demicheli et al., 1994; Holmgren et al., 1995), antiangiogenic therapy may need to be administered chronically (Folkman, 1995b). Also, the recombinant forms of some endogenous angiogenesis inhibitors (such as endostatin) have demonstrated relative instability in vitro and have required very high doses for efficacy in vivo (O’Reilly et al., 1997). These factors may impose economic and manufacturing constraints should such agents appear suitable for widespread clinical use (Tomlinson, 1992; Crystal, 1999). Finally, continuous infusion of recombinant agents

may be inconvenient, while data suggest that peak/ trough pharmacokinetics associated with bolus dosing may have a suboptimal antiangiogenic effect (Hahnfeldt et al., 1999; Drixler et al., 2000). Gene therapy might obviate these problems by providing prolonged, endogenous production of antiangiogenic proteins in patients with a variety of diseases characterized by pathologic angiogenesis. Using endostatin as an example, gene therapy approaches have demonstrated antitumor responses using a variety of gene delivery vectors, including adenoviruses, retroviruses, liposomes and plasmid DNA (Blezinger et al., 1999; Chen et al., 1999; Feldman et al., 2000b, 2001c). Endostatin gene therapy recently has been applied to an experimental model of choroidal neovascularization, demonstrating that this approach is not limited to the treatment of cancer (Mori et al., 2001). A further potential of antiangiogenic gene therapy is that normal tissues, such as the liver, might be used as a ‘factory’ for the systemic release of therapeutic protein (Feldman and Libutti, 2000; Feldman et al., 2000b). This approach takes advantage of the low toxicity profile of many antiangiogenic agents, may obviate difficulties in developing tumor-specific gene delivery vectors, and might allow diffusion of circulating protein to sites of micrometastatic disease that might not be accessible to blood-borne vectors (Feldman and Libutti, 2000). Based on accumulating data on the safety of antiangiogenic proteins in humans, and efficacy data from numerous experimental studies with a variety of vectors and genes, it appears likely that clinical trials of antiangiogenic gene therapy will be initiated in the near future.

SUMMARY The finding that tumors depend on angiogenesis for sustained growth has prompted intense investigation into the biology of angiogenesis and the pharmacology of antiangiogenic agents. These efforts have brought antiangiogenesis to clinical trials for patients with cancer, and offer promise for non-neoplastic diseases, including arthritis and several causes of blindness. Exponential progress in the field of molecular analysis and diagnostics is likely to enhance understanding of the basic biology of angiogenesis

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and its inhibition. Meanwhile, the results of the ongoing clinical trials discussed in this chapter are eagerly anticipated. In addition, further study into the use of antiangiogenic agents as a part of multimodality therapy, the administration of adjuvant antiangiogenic therapy, and the clinical development of antiangiogenic gene therapy, will help define the role antiangiogenesis will have in the armamentarium of the clinician.

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Volpert, O.V., Fong, T., Koch, A.E. et al. (1998). Inhibition of angiogenesis by interleukin 4. J. Exp. Med. 188, 1039–1046. Wang, M., Liu, Y.E., Greene, J. et al. (1997). Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4. Oncogene 14, 2767–2774. Wen, W., Moses, M.A., Wiederschain, D. et al. (1999). The generation of endostatin is mediated by elastase. Cancer Res. 59, 6052–6056. Westphal, J.R., Van’t Hullenaar, R., Geurts-Moespot, A. et al. (2000). Angiostatin generation by human tumor cell lines: involvement of plasminogen activators. Int. J. Cancer 86, 760–767. White, C.W., Sondheimer, H.M., Crouch, E.C. et al. (1989). Treatment of pulmonary hemangiomatosis with recombinant interferon alfa-2a. N. Engl. J. Med. 320, 1197–1200. Zetter, B.R. (1998). Angiogenesis and tumor metastasis. Annu. Rev. Med. 49, 407–424. Zhu, Z. and Witte, L. (1999). Inhibition of tumor growth and metastasis by targeting tumor-associated angiogenesis with antagonists to the receptors of vascular endothelial growth factor. Invest. New Drugs 17, 195–212. Zogakis, T.G. and Libutti, S.K. (2001). General aspects of anti-angiogenesis and cancer therapy. Exp. Opin. Biol. Ther. 1, 253–275.

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57 Cytokines and transplantation Geetha Chalasani and Fadi G. Lakkis Yale University School of Medicine, New Haven, CT, USA

‘If the model does not succeed in transforming reality, reality must succeed in transforming the model . . . The model of models Mr. Palomar dreams of must serve to achieve transparent models, diaphanous, fine as cobwebs, or perhaps even to dissolve models, or indeed to dissolve itself.’ Mr. Palomar, Italo Calvino

INTRODUCTION

ANIMAL STUDIES

The role of cytokines in allograft rejection and tolerance has been largely inferred from their in vitro actions and from in vivo experiments in which cytokine expression is correlated with graft rejection or acceptance (Dallman, 1993). These studies, however, have failed to provide cause–effect data because of the complexity and redundancy of the cytokine cascades triggered after transplantation. More recently, cytokine gene-knockout mice and cytokine-neutralizing agents have been utilized to dissect cytokine functions in vivo. In this chapter, we will summarize how these experiments have led to novel insights into the role of cytokines in the acute and chronic rejection of solid organ allografts and into their role in mediating immunologic tolerance and long-term allograft acceptance. In addition, we will summarize clinical studies that shed light on how cytokine expression influences the fate of a transplanted organ in humans.

The role of cytokines in acute rejection

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

Mitogenic cytokines (Interleukin (IL)-2, IL-4, and IL-15) A study by Strom and colleagues in 1995 was the first to underscore the redundancy of mitogenic cytokines in the acute rejection process (Steiger et al., 1995). These authors demonstrated that IL-2 gene-knockout (IL-2/) mice are capable of rejecting fully allogeneic pancreatic islet cells. This suggested that IL-2, a potent T cell mitogen in vitro, is not critical for allograft rejection. Dai et al. confirmed these findings using a murine vascularized cardiac allograft model (Dai et al., 1998). Despite experimental evidence indicating that IL-2 promotes T cell proliferation and cytotoxic T lymphocyte (CTL) generation in vitro, IL-2/ mice were found to mount effective proliferative and cytotoxic responses to allogeneic splenocytes Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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and tumor cells in vivo (Steiger et al., 1995; Dai et al., 1998). Although these studies appear to contradict those in which allograft rejection was prevented in mice by blocking IL-2 receptors with monoclonal antibodies, it is important to remember that the mechanism of action of these antibodies is unclear (Kupiec-Weglinski et al., 1986; Vincenti et al., 1998). In addition to inhibiting the binding of IL-2 to its receptor, IL-2 receptor antibodies may induce the deletion or modulate the function of activated T cells (Nickerson et al., 1997). Furthermore, the administration of IL-2-neutralizing antibodies does not delay cardiac allograft rejection in mice (Dai et al., 1998), suggesting that IL-2 receptor antibodies exert their actions independent of IL-2. Since it has been shown that IL-4 stimulates T cell proliferation in vitro and that the administration of soluble IL-4 receptors delays cardiac allograft rejection in vivo (Fanslow et al., 1991), it has been proposed that IL-4 may mediate allograft rejection in the absence of IL-2. However, IL-4-deficient mice (IL-4/) and double gene-knockout mice deficient in both IL-2 and IL-4 reject pancreatic islet cell and cardiac allografts with the same vigor as wild-type recipients (Nickerson et al., 1996; Lakkis et al., 1997; Li et al., 1998a). These findings raise the possibility that other cytokines which signal through the -chain of the IL-2 receptor may compensate for the absence of IL-2 and/or IL-4. The -chain, also known as the common cytokine receptor -chain (c), is shared by the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and is responsible for the transduction of mitogenic signals following binding of these cytokines to their receptors (Sugamura et al., 1996). Smith et al. provided evidence that another c-binding cytokine, IL-15, may contribute to acute rejection (Smith et al., 2000). These investigators found that the administration of soluble IL-15 receptor a-chain (IL-15Ra), which selectively neutralizes IL-15 activity in vivo, prolongs vascularized cardiac allograft survival in mice that received minorhistocompatibility-mismatchedhearts(approximately 70% of the grafts in the treated group were accepted indefinitely). Soluble IL-15Ra, however, prolonged graft survival only marginally in mice transplanted with fully allogeneic hearts (Smith et al., 2000). Similar results were shown by Ferrari-Lacraz et al. using an IL-15 mutant/Fc2a fusion protein in semiallogenic and fully allogenic murine pancreatic

islet cell transplantation (Ferrari-Lacraz et al., 2001). IL-15 mutant/Fc2a treatment alone prolonged graft survival marginally, while in combination with CTLA4/Fc significant prolongation of graft survival was observed (all of semiallogenic and 70% of fully allogenic graft recipients accepted their grafts more than 120 days). In addition to blocking the action of IL-15 by binding to the high affinity IL-15Ra site, the IL-15 mutant/Fc2a could act by depleting IL-15Ra cells due to the cytolytic nature of the Fc fusion protein moiety.

Proinflammatory cytokines (IFN, TNFa and IL-12) Among pro-inflammatory cytokines, IFN is postulated to be crucial for acute allograft rejection because it up-regulates MHC class II expression on antigen presenting cells (APC) and activates natural killer (NK) cells, CTL and macrophages in vitro (Billiau, 1996). Despite the complete absence of IFN, however, fully allogeneic vascularized cardiac graft survival is not prolonged in IFN gene-knockout (IFN/) mice (Saleem et al., 1996; Konieczny et al., 1998). Furthermore, Steiger et al. demonstrated that acute cellular rejection of fully allogeneic pancreatic islet cells is not delayed in IFN receptor knock-out (IFNR/) recipients (Steiger et al., 1998). A compensatory increase in the expression of proinflammatory cytokines such as tumor necrosis factor-a (TNFa) was not detected in IFN/ or IFNR/ recipients (Saleem et al., 1996; Konieczny et al., 1998; Steiger et al., 1998). In addition, IFNdeficient T lymphocytes mount effective proliferative and cytotoxic responses to alloantigen in vitro and in vivo (Dalton et al., 1993; Konieczny et al., 1998), confirming that the pro-inflammatory actions of IFN in alloimmunity are dispensable. A study in which IFNneutralizing antibodies failed to prolong skin allograft survival in Rhesus monkeys also suggests that IFN activity is not critical for the acute rejection of fully allogeneic transplants (Stevens et al., 1990). In the same study (Stevens et al., 1990), TNFa neutralization also failed to prolong graft survival. Unlike its redundant role in the rejection of fully allogeneic transplants, IFN is essential for the acute rejection of MHC class II-incompatible allografts. IFN/ recipient mice reject fully allogeneic and

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MHC class I-incompatible skin grafts at the same rate as wild-type mice, but are not capable of rejecting MHC class II-incompatible skin (Ring et al., 1999b). This is consistent with work showing that IFNneutralizing antibody prolongs the survival of MHC class II-incompatible, but not MHC class I-incompatible skin grafts in mice (Rosenberg et al., 1990). It is postulated that IFN is essential for initiating the alloimmune response to MHC class II antigens by inducing or up-regulating MHC class II expression on skin APC (Goes et al., 1995, 1996; Ring et al., 1999b). IL-12 is a cytokine which promotes the generation of pro-inflammatory T helper 1 (TH1) lymphocytes (see below) partly by inducing IFN production and thus, is presumed to play a critical role in allograft rejection. Studies by Bishop and colleagues, however, have repeatedly shown that IL-12, like IFN, is not an essential requirement for the acute rejection of fully allogeneic grafts. For example, IL-12 antagonism in mice that received heart transplants does not delay rejection but, instead, accelerates it (Picotti et al., 1996, 1997a). Similarly, cardiac allograft survival in IL-12 p40/ or IL-12 p35/ (p40 and p35 are the subunits of IL-12) is not longer but, instead, slightly shorter than that in wild-type recipients (Picotti et al., 1998). Furthermore, recombinant IL-12 administration does not accelerate allograft rejection despite increased IFN production (Picotti et al., 1999b). Like IFN, IL-12 may play a less redundant role in the rejection of semiallogeneic grafts or those mismatched with the recipient at minor histocompatibility loci only. Li et al. found that anti-IL-12 administration delays the rejection of partially MHCmismatched murine pancreatic islet cell grafts modestly (median graft survival increased from 17 to 25 days), and completely prevents the rejection of islets mismatched at multiple minor histocompatibility loci in certain mouse strain combinations (Li et al., 1998c).

Summary The studies described in this section indicate that individual cytokines (for example, IL-2, IL-12 and IFN) may contribute to acute rejection, but that none of these cytokines is critical for rejecting fully allogeneic grafts. They also suggest that for a cytokine-based immunosuppressive strategy to be

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successful, it should target multiple cytokines or block a common pathway of cytokine action, such as the IL-2 receptor -chain (c). In fact, antibodies which target c significantly prolong pancreatic islet allograft survival in mice and, in some cases, induce transplantation tolerance (Li et al., 2000). It is now clear that the redundancy of cytokine functions in vivo guarantees that acute rejection proceeds uninterrupted in the absence of one cytokine or another. Exceptions to the redundancy rule emerge when studying less powerful alloimmune responses, such as the rejection of minor mismatched organs, where IFN, IL-12 and IL-15 appear to play important roles.

The role of cytokines in chronic rejection Chronic rejection of a vascularized organ transplant is characterized by obliterative changes in small and medium-sized arteries (obliterative vasculopathy) resulting from intimal arteritis and intimal thickening (Russell et al., 1998). The vasculopathy is associated with varying degrees of interstitial fibrosis. The extent of chronic rejection is usually quantitated by measuring the frequency and degree of vascular obliteration.

IFN and IL-12 The contribution of the pro-inflammatory cytokines IFN and IL-12 to chronic rejection has been carefully analyzed in a murine transplantation model. In this model, heart allografts transplanted to wild-type recipients that are transiently depleted of CD4 and CD8 T lymphocytes develop obliterative vasculopathy consistent with chronic rejection. IFN/ recipients, mice treated with IFN-neutralizing antibody, or mice that lack the STAT4 signaling pathway (which mediates IL-12’s cellular actions), develop significantly less frequent and less severe vascular lesions (Nagano et al., 1997; Raisanen-Sokolowski et al., 1998a; Koglin et al., 2000), indicating that these cytokines contribute to the pathogenesis of chronic allograft rejection. A similar conclusion was reached by Mitchell and colleagues who demonstrated that heart allografts undergoing transient rejection do not develop chronic obliterative vasculopathy if IFN is absent (Nagano et al., 1998). In contrast to IFN, TNFa

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does not appear to have a dominant role in chronic cardiac allograft rejection (Nagano et al., 1999). That IFN and IL-12 play a dominant role in chronic allograft vasculopathy, however, is not without controversy. Bishop and colleagues found that IL-12-deficient (IL-12p40/) mice develop chronic rejection at the same rate and with the same severity as wild-type cardiac allograft recipients and that T cells deficient in IFN production are capable of transferring full-blown chronic rejection to scid mice (Picotti et al., 1999a). In these models (Picotti et al., 1999a), chronic rejection is induced by transient depletion of CD4 T cells only. Further controversy has been generated by the finding that IFN may protect the allograft endothelium from acute immune-mediated injury. Halloran and colleagues demonstrated that IFN receptor (IFNR)/- hearts transplanted into wild-type recipients, or wild-type hearts transplanted to IFN/ recipients, are more susceptible to a form of acute rejection characterized by endothelial damage and vascular thromboses (Halloran et al., 2000). Though it is not known how IFN exerts its protective action against acute endothelial injury, nitric oxide could be one potential mediator.

IL-4 and IL-10 Despite their in vitro anti-inflammatory actions, neither IL-4 nor IL-10 prevents chronic rejection in murine models of cardiac transplantation. Hearts transplanted to IL-4/ mice have similar survival and develop the same degree of obliterative vasculopathy as those transplanted to wild-type recipients (Mottram et al., 1998; Nagano et al., 1999). Likewise, the administration of IL-10-neutralizing antibodies does not alter the chronic rejection of murine cardiac allografts (Furukawa et al., 1999). The administration of recombinant murine IL-10 on the other hand exacerbates both acute and chronic rejection (Furukawa et al., 1999), suggesting that mammalian IL-10 exerts pro-inflammatory actions. One mechanism by which IL-10 can exacerbate rejection is by enhancing CTL differentiation (Chen and Zlotnik, 1991; Berman et al., 1996). Using IL-10/ mice, Russell and colleagues arrived at somewhat different conclusions (RaisanenSokolowski et al., 1997, 1998b). In these studies, immunosuppressed IL-10/ cardiac allograft recip-

ients were found to reject slightly faster than wildtype recipients (median survival time of 57 days versus 99 days, respectively). Moreover, when heart tissue was analyzed at 50 days after transplantation, a distinct form of late allograft rejection characterized by increased vascular occlusion and myocyte destruction was observed in IL-10/ recipients. Enhanced vascular occlusion in these mice was not due to the obliterative vasculopathy associated with chronic rejection but was caused by T cell-rich arteritis and medial necrosis (Raisanen-Sokolowski et al., 1998b). These findings indicate that isolated IL-10 deficiency leads to a qualitative alteration of the histologic manifestations of rejection rather than a significant acceleration of either acute or chronic rejection.

Tumor growth factor-beta (TGF b) It is suspected that TGFb contributes to chronic rejection by virtue of its pro-fibrotic effects (Shihab et al., 1996). However, TGFb is also an immunomodulatory cytokine (Palladino et al., 1991), making it extremely difficult to discern which action of TGFb predominates in the rejection process. This difficulty becomes apparent when attempts to correlate TGFb expression with chronic allograft rejection in murine models result in discordant conclusions (Shihab et al., 1996; Mannon et al., 1999). One study has addressed the role of TGFb in chronic rejection by using TGFb1/ mice. Koglin et al. found that cardiac allografts in TGFb1/ recipients have increased concentric intimal thickening and luminal occlusion compared with wild-type recipients (Koglin et al., 1998). Expression of STAT4, IFN and IL-2 mRNA was also enhanced in TGFb1/ mice suggesting that immune dysregulation in the absence of TGFb1 is responsible for the accelerated obliterative vasculopathy. This conclusion is consistent with the original reports describing the TGFb1/ mouse phenotype which is characterized by multifocal inflammation and polyclonal T lymphocyte expansion (Shull et al., 1992; Kulkarni et al., 1993).

Summary The data summarized in this section indicate that pro-inflammatory cytokines, such as IFN and IL-12, are important contributors to the pathogenesis of

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chronic allograft vasculopathy in mice although some controversy remains. On the other hand, antiinflammatory cytokines such as IL-4 and IL-10 do not diminish chronic rejection. One study using TFGb/ mice strongly suggests that the dominant in vivo role of TGFb is an immunomodulatory one which leads to suppression of chronic rejection.

The role of cytokines in allograft acceptance and tolerance induction Transplantation tolerance can be defined as indefinite allograft survival in the absence of continuous immunosuppressive therapy (Charlton et al., 1994). Implicit to this definition is that tolerant recipients are unresponsive to donor antigens, but maintain reactivity to other (third-party) antigens (Charlton et al., 1994; Kruisbeek and Amsen, 1996; Lechler and Bluestone, 1997). Allograft acceptance, on the other hand, can be defined as a state of indefinite allograft survival in the experimental animal in the absence of donor-specific immunologic unresponsiveness. The immunologic pathways that lead to tolerance are traditionally divided into central and peripheral (Charlton et al., 1994; Kruisbeek and Amsen, 1996; Lechler and Bluestone, 1997). Central tolerance occurs in the thymus where antigen-specific lymphocytes are eliminated. Peripheral tolerance, on the other hand, occurs extrathymically and may be crucial for achieving antigen-specific unresponsiveness in adult animals in whom the thymus has involuted. Mechanisms proposed to mediate peripheral tolerance include deletion, anergy, suppression/ regulation, and immune deviation of alloantigenspecific, mature T lymphocytes (Charlton et al., 1994; Kruisbeek and Amsen, 1996; Lechler and Bluestone, 1997). The clonal deletion hypothesis presumes that tolerance results from apoptosis of the alloreactive T cell population. Alternatively, a tolerance-inducing strategy could inactivate alloreactive T lymphocytes without causing their death (anergy), generate regulatory cells which suppress T lymphocyte activation and function (suppression), or induce differentiation of alloreactive T lymphocytes into a non-harmful phenotype (immune deviation). These mechanisms are not mutually exclusive, and their relative contributions to tolerance depend on the organ being transplanted, the tolerance-inducing regimen used, and

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the degree of donor–recipient histo-incompatibility. In this section, we will focus on the role of cytokines in mediating peripheral tolerance to transplanted organs.

The T helper 1 (TH1 )/T helper 2 (TH2 ) paradigm The role of cytokines in the induction of allograft acceptance and peripheral transplantation tolerance has been largely studied in the context of the TH1/TH2 paradigm. This paradigm was launched more than 15 years ago with the discovery of CD4 T helper lymphocyte subsets (TH1 and TH2), that differ in their cytokine secretion patterns (Abbas et al., 1996; Mosmann and Sad, 1996; O’Garra, 1998) Studies in mice, rats, and to some extent in humans have shown that TH1 lymphocytes produce IL-2, IFN, and lymphotoxin (LT) (also known as TNFb), while TH2 lymphocytes produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Both cell types originate from the same precursor lymphocyte (TH0), whose differentiation is largely influenced by the cytokines present at the time of antigen recognition. IL-12 and IFN favor the TH1 phenotype, while IL-4 and IL-13 promote the generation of TH2 lymphocytes (McKenzie et al., 1999a, 1999b). Differentiation of TH0 cells into either a TH1 or TH2 phenotype is also governed by the antigen dose, the strength of interaction between the T cell receptor for antigen (TCR) and the major histocompatibility molecule (MHC)/peptide complex, and the nature of costimulatory molecules expressed on the APC (O’Garra, 1998). TH1 and TH2 subsets are reciprocally regulated. IFN inhibits the differentiation and proliferation of TH2 cells resulting in a dominant TH1 response. On the other hand, IL-4 and IL-10 inhibit IFN production by TH1 lymphocytes. In addition to the presence of TH1 and TH2 lymphocytes, there is evidence to suggest that similar lymphocyte subsets exist in the CD8 T cell population (Tc1 and Tc2) (Carter and Dutton, 1996). Moreover, the existence of a TH3 subset has been proposed based on data showing that a subgroup of CD4 T cells regulate immune responses by secreting TGFb (O’Garra, 1998). More recently, a subset of CD4 T cells that express the IL-2Ra chain (CD25) have been shown to mediate peripheral tolerance to selfantigens (Shevach, 2000). The common feature among all of these regulatory cells, irrespective of

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their nomenclature, is that they suppress immune responses partly by secreting ‘inhibitory’ cytokines. TH1-type cytokines (IL-2 and IFN) are postulated to promote allograft rejection and hinder tolerance by mediating delayed-type hypersensitivity (DTH) reactions, CTL generation, macrophage activation and production of complement-fixing antibodies which also facilitate antibody-dependent cellular cytotoxicity (Nickerson et al., 1994; Dallman, 1995). In contrast, TH2-derived cytokines are postulated to protect against rejection and to promote tolerance by suppressing DTH reactions and counteracting IFN’s actions on macrophages. They also deviate antibody production towards IgE and non-cytotoxic subclasses of IgG. Intragraft expression of TH1-type cytokines is often associated with acute rejection while expression of TH2-type cytokines has been shown to correlate with allograft acceptance (Takeuchi et al., 1992; O’Connell et al., 1993; Kupiec-Weglinski et al., 1993; Sayegh et al., 1995). This led to the proposal that a balance between TH1 and TH2 lymphocytes determines the fate of a transplanted organ (Nickerson et al., 1994; Dallman, 1995; Field et al., 1997) Much of the evidence that supports this hypothesis is based on assessing intragraft cytokine expression by reverse transcriptase-polymerase chain reaction (RT-PCR) or by identifying graft-infiltrating cells that stain for cytokine protein by immunohistochemistry (Picotti et al., 1997b). Although important, these data do not establish cause–effect relationships between cytokines and allograft rejection or acceptance. Recent studies using cytokine gene-knockout mice, cytokine transgenic mice, and cytokine-neutralizing antibodies have raised doubts about the applicability of the TH1/TH2 paradigm to the induction of allograft acceptance and peripheral transplantation tolerance. These studies will be summarized below with the intention to highlight three evidence-based conclusions: (1) TH2-type cytokines are neither necessary nor sufficient for the acceptance of fully allogeneic grafts; (2) TH2-type cytokines may be necessary and sufficient for the acceptance of minor-histocompatibilityantigen mismatched grafts; and (3) TH1-type cytokines (IL-2 and IFN) which activate T cells, are in themselves regulatory cytokines that are necessary for the induction of peripheral tolerance to transplanted organs.

TH2-type cytokines are neither necessary nor sufficient for the acceptance of fully allogeneic grafts Evidence that TH2-type cytokines are not sufficient for inducing allograft acceptance is provided by studies showing that the transgenic overexpression of IL-4 by murine pancreatic islet cell or cardiac grafts does not delay allograft rejection in wild-type recipients (Mueller et al., 1997; Takeuchi et al., 1997). Similarly, systemic administration of mammalian IL-10 does not prolong islet graft survival, but instead accelerates their rejection, possibly due to increased CTL generation (Zheng et al., 1995; Qian et al., 1996). Matesic et al. demonstrated that, unlike predictions based on the TH1/TH2 paradigm, the adoptive transfer of TH2-type CD4 or CD8 lymphocytes results in rapid rejection of skin allografts in T cell-deficient scid mice (Matesic et al., 1998). Picotti et al. also found that the induction of TH2 immunity in mice by antagonizing IL-12 at the time of transplantation exacerbates rather than delays cardiac allograft rejection (Picotti et al., 1996). Evidence that TH2-type cytokines are not necessary for the induction of tolerance or allograft acceptance is provided by several studies. Blocking B7–CD28 T cell costimulation induces indefinite acceptance of fully allogeneic pancreatic islet cell and cardiac grafts in IL-4/ recipients despite the preponderance of TH1 cytokine expression in these mice (Nickerson et al., 1996; Lakkis et al., 1997). Similarly, B7–CD28 blockade induces tolerance to fully allogeneic cardiac grafts in STAT6/ mice which are characterized by a severe impairment in TH2 immunity (Kishimoto et al., 2000). Further evidence that TH2-type cytokines are not necessary for tolerance induction is provided by studies showing that peripheral transplantation tolerance to fully allogeneic cardiac allografts can be readily achieved in IL-4/ mice treated transiently with anti-CD4 plus anti-CD8 antibodies (Mottram et al., 1998), and that IL-4 neutralization does not prevent transplantation tolerance induced by anti-CD4 monoclonal antibodies given alone or in conjunction with donor-specific transfusion (Hall et al., 1998; Bushell et al., 1999). There are examples in the literature, however, of transplantation tolerance in mice which appears to be dependent on IL-4 production or on the inhibition of

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TH1 responses characteristic of TH2 immune deviation. They include neonatal tolerance to fully allogeneic grafts (Gao et al., 1996; Chen et al., 1996; Inoue et al., 2001), tolerance induced by treatment regimens consisting of anti-CD3 plus anti-CD2 monoclonal antibodies (Punch et al., 1998), tolerance induced by gallium nitrate immunosuppression (Sirak et al., 1998), and tolerance to minor histocompatibility antigenmismatched grafts (discussed below). Whether infectious tolerance to fully MHC-incompatible grafts is IL-4-dependent remains controversial. One recent study demonstrated that IL-4 is necessary for acquiring infectious tolerance in mice pretreated with antiCD4 (Bushell et al., 1999). In this study, neutralizing IL-4 at the time of tolerance induction had no effect on cardiac allograft outcome in primary recipients, while neutralizing IL-4 in adoptive transfer donors at the time of tolerance induction prevented long-term allograft acceptance in the majority of secondary recipients. In contrast, a different group of investigators showed that anti-CD4-induced infectious tolerance in rats is not blocked by IL-4-neutralizing antibodies (Hall et al., 1998). The ability of CD4 T cells to transfer tolerance in this model was not associated with preferential induction of TH2 cytokines, the need for IL-4, or the inhibition of TH1 cytokines (Hall et al., 1998). The studies described in this paragraph serve as a reminder that multiple tolerance mechanisms exist and that certain experimental models may be dependent on one particular mechanism or another. For example, immune regulation may play an important role in experimental models where the number of alloreactive T cells has been significantly reduced by treatment with anti-T cell antibodies.

TH2-type cytokines may be necessary and sufficient for the acceptance of minor histocompatibiliy antigen mismatched grafts Several studies provide evidence that TH2 immune deviation may be necessary and sufficient for the acceptance of organs transplanted across minor histocompatibility differences. For example, antiCD4-induced infectious tolerance to multiple minor histocompatibility antigen mismatched skin grafts is partially blocked by administering IL-4-neutralizing antibodies at the time of adoptive transfer (Davies

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et al., 1996). Similarly, adult mice treated with antiIL-12 antibody, such that the alloimmune response is deviated toward the TH2 phenotype, were able to accept minor antigen-mismatched, but not fully MHC-mismatched pancreatic islet grafts (Li et al., 1998c). Intragraft IL-4 gene transfer also synergizes with regulatory CD4 T cells to prolong the survival of rat hearts transplanted across incomplete MHC barriers (Ke et al., 2000). Using gene-knockout mice, neonatal tolerance to minor-mismatched skin grafts has been shown to require the combined actions of IL-4 and IL-13, and not IL-4 alone (Inoue et al., 2001). Failure to suppress IFN production was responsible for the lack of neonatal tolerance in mice deficient in both IL-4 and IL-13 (Inoue et al., 2001). Taken together, these studies suggest that TH2-mediated regulatory phenomena play a dominant role in situations where the number of responding T cells is smaller than that observed in fully allogeneic transplantation.

TH1-type cytokines (IL-2 and IFN) facilitate allograft acceptance by regulating the alloimmune response IFN and IL-2 are produced by T lymphocytes shortly after alloantigenic stimulation (Smith, 1988; Boehm et al., 1997). Based on their immunostimulatory actions in vitro, it is postulated that IFN and IL-2 impede the induction of transplantation tolerance. Recent studies in IFN/ and IL-2/ mice provide evidence that IFN and IL-2 have essential immunoregulatory functions in vivo which facilitate rather than impede the induction of allograft acceptance. For example, the postoperative blockade of the CD28 and/or CD40 ligand (CD40L) T cell costimulation pathways induces long-term survival of fully allogeneic cardiac and skin grafts in wild-type mice, but fails to do so in IFN/ recipients or in wildtype mice treated with IFN-neutralizing antibodies at the time of transplantation (Konieczny et al., 1998; Markees et al., 1998). Similarly, T cell costimulation blockade (inhibition of B7-CD28 alone or both B7-CD28 and CD40-CD40L) failed to induce longterm cardiac allograft acceptance or tolerance to pancreatic islet cell allografts in IL-2/ mice or in wild-type recipients injected with IL-2-neutralizing antibodies (Dai et al., 1998; Wells et al., 1999). These findings are supported by studies which demonstrate

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that tolerance to protein antigens cannot be induced in IFN/ or IL-2/ mice (Kweon et al., 1998; Van-Parijs et al., 1997; Kneitz et al., 1995). What are the mechanisms by which IFN and IL-2 facilitate induction of long-term allograft survival? Compared to wild-type T cells, IFN/ T lymphocytes display exaggerated proliferation and CTL activity upon allostimulation in vitro (Dalton et al., 1993; Konieczny et al., 1998). Addition of recombinant mouse IFN reduces proliferation of IFN/ T cells, while addition of IFN-neutralizing antibody increases proliferation of wild-type T cells in mixed lymphocyte cultures (Dalton et al., 1993; Konieczny et al., 1998). Exaggerated T lymphocyte proliferation is also observed in vivo after stimulating IFN/ mice with either a bacterial superantigen or allogeneic splenocytes (Konieczny et al., 1998; Hassan et al., 1999). The cutaneous delayed-type hypersensitivity (DTH) response to allogeneic splenocytes is also increased in the absence of IFN (Ring et al., 1999b; Hassan et al., 1999). CTLA4Ig which inhibits B7–CD28 T cell costimulation blocks alloantigen-induced T cell proliferation in wild-type mice, but fails to do so in IFN/ mice (Hassan et al., 1999). The latter observation could explain why allograft acceptance can not be achieved in IFN-deficient mice treated with CTLA4Ig (Konieczny et al., 1998). IFN did not influence activation-induced T cell apoptosis in any of these studies. Taken together, these findings indicate that IFN is crucial for down-regulating alloimmune responses by limiting the proliferation of activated T cells. Immunosuppressive properties of endogenous IFN have been demonstrated in other murine models such as collagen-induced arthritis (ManourySchwartz et al., 1997; Vermeire et al., 1997), experimental allergic encephalitis (Ferber et al., 1996; Willenborg et al., 1996), anti-CD3-induced cytokine release syndrome (Matthys et al., 1995), murine graftversus-host disease (Wall et al., 1988), and accelerated anti-glomerular basement membrane nephritis (Ring et al., 1999a). Although IL-2 is a potent T cell mitogen in vitro, IL-2/ mice or those that lack high affinity IL-2 receptors (IL-2Ra/ and IL-2Rb/) are immunocompetent (Horak et al., 1995; Theze et al., 1996). When housed in a non-germ-free environment, these mice display lymphoid hyperplasia due to increased proliferation of mature T and B cells and develop

autoimmunity characterized by hemolytic anemia or inflammatory bowel disease (Sadlack et al., 1993, 1995; Suzuki et al., 1995; Willerford et al., 1995). Uncontrolled lymphocyte proliferation and autoimmunity in IL-2/ and IL-2R/ mice is in part attributed to impaired apoptosis of activated T lymphocytes. Lenardo and coworkers demonstrated that IL-2 predisposes mature CD4 and CD8 T lymphocytes to apoptosis following antigenic restimulation and that antibody blockade of IL-2 inhibits antigeninduced deletion of T cells in vivo (Lenardo, 1991; Boehme and Lenardo, 1993; Critchfield et al., 1994). Other studies have confirmed these findings by demonstrating that activation-induced T cell death is impaired in IL-2/ and IL-2Ra/ mice (Kneitz et al., 1995; Van-Parijs et al., 1997). Exaggerated T cell proliferation and impaired T cell apoptosis is also observed in IL-2/ mice repeatedly stimulated with allogeneic splenocytes (Dai et al., 1998; Wells et al., 1999). The mechanisms by which IL-2 programs activated T cells for antigen-induced apoptosis include the up-regulation of FasL in conjunction with the down-regulation of FLIP, a protein which inhibits Fas-mediated cell death (Refaeli et al., 1998). These data demonstrate that IL-2 plays a dual role in the immune system (Van-Parijs et al., 1997). On one hand, it has a redundant mitogenic role that can be replaced by other lymphocyte growth factors such as IL-4, IL-7, IL-9 and IL-15. On the other hand, it is critical for terminating immune responses by predisposing activated T cells to apoptotic signals. IL-2 can also trigger additional feedback mechanisms which inhibit T lymphocyte proliferation. For example, CTLA4 expression on the cell membrane is up-regulated following T cell activation (Linsley et al., 1992). Subsequently, high affinity B7–CTLA4 interactions inhibit cell cycle progression and contribute to tolerance induction (Krummel and Allison, 1996; Walunas et al., 1996; Perez et al., 1997; Zheng et al., 1999). The dual role of IL-2 in the immune response is further emphasized by a recent study showing that IL-2 increases the size of the CD8 memory pool if present at the time of initial T cell activation but reduces the size of the pool if present during memory maintenance by inhibiting the proliferation of CD8 memory T cells (Dai et al., 2000). Thus, to ensure long-term allograft acceptance IL-2-targeting strategies must take into account these subtleties of IL-2 immunobiology.

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HUMAN STUDIES

Summary The experimental findings described in this section raise doubts about the universality of the TH1/TH2 paradigm and its applicability to the induction of allograft acceptance and peripheral transplantation tolerance. First, TH2-type cytokines are neither necessary nor sufficient for the acceptance of fully allogeneic grafts, but may be necessary and sufficient for the acceptance of minor histocompatibility antigen mismatched grafts. Second, TH1-type cytokines, such as IL-2 and IFN, are important regulatory cytokines that are necessary for the induction of transplantation tolerance by limiting the alloimmune response. IL-2 is crucial for the apoptosis of activated T cells while IFN inhibits their proliferation. Since IL-2 and IFN are produced only after the immune response is initiated, it is reasonable to propose that T cell activation is a prerequisite for peripheral tolerance. This is clearly elucidated in spontaneously tolerant liver transplant models in rodents where the use of agents interfering with T cell activation result in abrogation of graft acceptance (Sharland et al., 1999). In fact, mounting evidence indicates that traditional immunosuppressive agents which suppress T cell activation by blocking signal 1 also hinder the induction of transplantation tolerance (Larsen et al., 1996; Li et al., 1999).

HUMAN STUDIES Cytokine gene expression studies Acute rejection Several investigators have used the semiquantitative RT-PCR method to correlate intragraft cytokine gene expression with acute rejection. Although the findings tend to be conflicting, two common themes have emerged: (1) multiple cytokine genes are coexpressed during acute rejection which underscores their redundancy; and (2) functional dichotomy of cytokines according to the TH1/TH2 paradigm does not necessarily hold true in human allograft rejection. The majority of T cell mitogenic cytokines which bind the common cytokine receptor c are coexpressed in human renal allografts undergoing acute

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rejection (Li et al., 1998b; Strehlau et al., 1997; Lipman et al., 2000). These cytokines include IL-2, IL-4, IL-7 and IL-15. One notable exception is IL-9 which binds c but is not expressed in renal biopsy samples (Li et al., 1998b). Absence of IL-9 during acute rejection could be related to the observation that IL-9 is not a mitogen for freshly activated T cells but, instead, is an important growth factor for transformed T cells (Schmitt et al., 1989). Among the T cell mitogens, IL-15 was detected most often and at the highest intensity in acutely rejecting grafts, while IL-2 and IL-4 expression correlated poorly with rejection (Pavlakis et al., 1996; Strehlau et al., 1997). For example, analysis of 45 renal biopsy samples revealed IL-15 expression in all and IL-2 expression in only three. The expression of IL-15, but not IL-2, increased significantly with acute rejection (Pavlakis et al., 1996). Further evidence that IL-15 may play a role in the acute rejection of human allografts is provided by a study in heart transplant recipients, who experienced acute rejection despite induction therapy with anti-CD25 (anti-IL-2Ra) antibody (Baan et al., 2000). IL-15responsive T cells were detected in the blood of these patients even after in vitro addition of anti-CD25 antibody, indicating that IL-15 stimulates T cell proliferation in the absence of IL-2 signaling. Among the pro-inflammatory cytokines, both IFN and TNFa mRNA expression have been detected in renal allograft biopsy samples, but their expression did not consistently correlate with acute rejection (Suthanthiran, 1997; Lipman et al., 2000). Interestingly, pentoxiphylline administration inhibited TNFa production and improved 1-year graft survival in renal transplant recipients who had experienced acute rejection, suggesting that TNFa may play a role in determining the severity of rejection (Noel et al., 1998, 2000). Although IL-10 is generally recognized as a TH2type anti-inflammatory cytokine, Suthanthiran and coworkers found that IL-10 is the most prevalent intragraft cytokine during acute renal rejection (Suthanthiran, 1997). Strong correlation between intragraft IL-10 expression and acute rejection was confirmed by others (Strehlau et al., 1997; Lipman et al., 2000). This unexpected finding can be explained by the fact that mammalian IL-10 is a potent stimulus for CTL differentiation and antibody production (Go et al., 1990; Chen and Zlotnik, 1991).

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Chronic rejection TGFb has been implicated as a mediator of chronic allograft rejection in humans because of its profibrotic actions. Shihab et al. found that intragraft TGFb, detected by immunofluorescence staining, was significantly increased in kidneys with evidence of chronic rejection compared with normal kidney biopsies or to biopsies from patients with native kidney disease (Shihab et al., 1996). Similarly, intragraft TGFb1 mRNA correlates with chronic rejection in renal transplant recipients. Chronic rejection was found in 38% of grafts that were positive and in only 17% of grafts that were negative for TGFb1 mRNA expression (Suthanthiran, 1997). Quantitative RT-PCR also showed that patients with higher chronic allograft nephropathy scores and higher proteinuria levels have higher intragraft TGFb mRNA expression (Mas et al., 2000).

Gene expression studies using high-density oligonucleotide arrays The RT-PCR method of quantitating gene expression is limited in that only a small number of genes can be screened simultaneously. Recent technologic advances have made possible the detection of thousands of human gene transcripts using a very small tissue sample. One such advance is high-density oligonucleotide micorarrays (Lockhardt et al., 1996; Lockhardt and Winzeler, 2000). This technology involves the determination of the relative concentrations of mRNA based on hybridization of entire mRNA populations to high-density arrays of synthetic oligonucleotides. The arrays commonly used contain more than 65 000 different 20-mer oligonucleotides of defined sequence in an area of 1.6 cm2. The surfacebound oligonucleotides serve to probe more than 7000 different human gene transcripts simultaneously. The relative abundance of bound mRNA species is then quantitated by fluorescence techniques as mRNA extracted from tissue samples is labeled with fluorescein-tagged nucleotides prior to hybridization to the microarrays. In addition to allowing for the simultaneous monitoring of thousands of genes, the oligonucleotide microarray method provides a less biased view of the cellular response as it does not rely on a priori knowledge of

the mechanisms and genes involved in the process under investigation. We have recently conducted a study of seven acute rejection and three normal renal allograft biopsy samples using high density oligoarrays that probe the expression of 6800 different human genes (Akalin et al., 2001). Surprisingly, we did not detect a significant increase in the expression of cytokine or cytokine receptor genes during acute rejection. In contrast, two chemokine genes were consistently expressed in acute rejection biopsy samples. These were HuMig, the human counterpart of monokine induced by IFN (Mig), and macrophage inflammatory protein 3-beta (MIP-3b). Both chemokines mediate the early recruitment of inflammatory cells into the transplanted organ. Another gene whose expression was universally induced in acute rejection samples was IL-2stimulated phosphoprotein. This 65 kDa protein is present in human T cells and is phosphorylated upon binding of IL-2 to its receptor (Zu et al., 1990). After transition of an activated T cell from an IL-2dependent to an IL-2-independent state this protein remains constitutively phosphorylated. Therefore, it is possible that despite inhibition of IL-2 production by currently available immunosuppressive agents, initial IL-2 production may be sufficient to stimulate this protein and lead to persistent T cell activation.

Gene polymorphism studies The level of cytokine production has been associated with polymorphisms in cytokine gene promoters. For example, G to A polymorphism at position 308 of the TNFa gene promoter increases TNFa production by six- to seven-fold (Wilson et al., 1997), while a similar polymorphism at position 1082 of the IL-10 gene promoters is associated with lower IL-10 production (Turner et al., 1997b). Sankaran et al. found that the high TNFa or high IL-10 producer genotype is associated with multiple acute rejection episodes in HLA-DR-mismatched renal transplant recipients (Sankaran et al., 1999). Moreover, the TNFa high producer genotype was associated with steroid-resistant rejection. When genotypes were analyzed together, the TNFa high/IL-10 high producer genotype had the worst prognosis, whereas TNFa low/IL-10 low producer genotype was protective. These findings were confirmed in heart and simultaneous kidney–

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REFERENCES

pancreas allograft recipients (Turner et al., 1997a; Pelletier et al., 2000). Other cytokines, such as IFN and TGFb gene polymorphisms do not appear to influence the incidence or severity of acute rejection (Pelletier et al., 2000). TNFa and IL-10 polymorphisms do not correlate with chronic allograft nephropathy or allograft survival at 5 years post transplantation (Sankaran et al., 1999). In contrast, TGFb gene polymorphisms predict coronary vasculopathy following cardiac transplantation. In a study of 129 heart transplant recipients, patients homozygous for the TGFb1 high-producer polymorphism (G instead of C at position 915 of the TGFb1 gene) developed coronary vasculopathy in a significantly shorter time than patients who carried the low-producer C allele (1240 versus 2266 days, respectively) (Densem et al., 2000). Similarly, TGFb1 high-producer lung transplant recipients are at risk of developing allograft fibrosis (Awad et al., 1998). Pulmonary fibrosis after lung transplantation also correlates with IFN gene polymorphism, suggesting that IFN plays a role in the chronic rejection of human allografts (Awad et al., 1999). Overall, gene polymorphism studies may eventually provide the clinician with the means to stratify patients according to risk of rejection and perhaps open the door to customizing immunosuppression therapy.

CONCLUDING REMARKS Some take-home lessons that can be learned from the experimental work summarized in this chapter and pertain to the treatment of transplant recipients are the following: (1) Cytokine actions are redundant. Therefore, blocking one cytokine at a time is not sufficient for preventing rejection or inducing tolerance. Instead, strategies that target multiple cytokine actions simultaneously, for example blocking the common cytokine receptor -chain, are required to achieve these goals. (2) Immunostimulatory cytokines, such as IL-2 and IFN, are also regulatory and are essential for turning off the immune response. Therefore, judicial timing of therapies that suppress the actions of these cytokines must be applied in order to achieve long-term allograft acceptance or tolerance in transplant recipients. (3) Emerging tools, such as gene polymorphism analysis and

microarray technology, deserve further exploration to develop methods that predict and accurately diagnose rejection.

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Lechler, R. and Bluestone, J.A. (1997). Transplantation tolerance – putting the pieces together. Curr. Opin. Immunol. 9, 631–633. Lenardo, M.J. (1991). Interleukin-2 programs mouse alpha/beta T lymphocytes for apoptosis. Nature 353, 858–861. Li, X.C., Roy-Chaudhury, P., Hancock, W.W. et al. (1998a). IL-2 and IL-4 double knockout mice reject islet allografts: a role for novel T cell growth factors in allograft rejection. J. Immunol. 160, 890–896. Li, X.C., Schachter, A.D., Zand, M.S. et al. (1998b). Differential expression of T-cell growth factors in rejecting murine islet and human renal allografts. Transplantation 66, 265–271. Li, X.C., Zand, M.S., Li, Y. et al. (1998c). On histocompatibility barriers, TH1 to TH2 immune deviation, and the nature of the allograft responses. J. Immunol. 161, 2241–2247. Li, X.C., Ima, A., Li, Y. et al. (2000). Blocking the common -chain of cytokine receptors induces T cell apoptosis and long-term islet allograft survival. J. Immunol. 164, 1193–1199. Li, Y., Li, X. C., Zheng, X.X. et al. (1999). Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral tolerance. Nature Medicine, 5, 1298–1302. Linsley, P.S., Greene, J.L., Tan, P. et al. (1992). Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J. Exp. Med. 176, 1595–1604. Lipman, M., Shen, Y., John, J.R. et al. (2000). Immuneactivation gene expression in clinically stable renal allograft biopsies: molecular evidence for subclinical rejection. Transplantation 66, 1673–1681. Lockhardt, D.J., Dong, H., Byrne, M.C. et al. (1996). Expression monitoring by hybridization to highdensity oligonucleotide arrays. Nat. Biotech. 14, 1675–1680. Lockhardt, D.J. and Winzeler, E.A. (2000). Genomics, gene expression and DNA arrays. Nature. 405, 827–836. Mannon, R.B., Kopp, J.B., Ruiz, P. et al. (1999). Chronic rejection of mouse kidney allografts. Kidney Int. 55, 1935–1944. Manoury-Schwartz, B., Chiocchia, G., Bessis, N. et al. (1997). High susceptibility to collagen-induced arthritis in mice lacking IFN- receptors. J. Immunol. 158, 5501–5506. Markees, T.G., Phillips, N.E., Gordon, E.J. et al. (1998). Longterm survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4 T cells, interferon-, and CTLA4. J. Clin. Invest. 101, 2446–2455. Mas, V., Diller, A., Albano, S. et al. (2000). Intragraft expression of transforming growth factor-beta 1 by a novel quantitative reverse transcription polymerase chain reaction ELISA in long lasting kidney recipients. Transplantation 70, 612–616. Matesic, D., Valujskikh, A., Pearlman, E. et al. (1998). Type 2 immune deviation has differential effects on alloreactive CD4 and CD8 T cells. J. Immunol. 161, 5236–5244. Matthys, P., Froyen, G., Verdot, L. et al. (1995). IFN- receptordeficient mice are hypersensitive to the anti-CD3induced cytokine release syndrome and thymocyte apoptosis: protective role of endogenous nitric oxide. J. Immunol. 155, 3823–3829.

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McKenzie, G.J., Emson, C.L., Bell, S.E. et al. (1999a). Impaired development of TH2 cells in IL-13-deficient mice. Immunity 9, 423–432. McKenzie, G.J., Fallon, P.G., Emson, C.L. et al. (1999b). Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses. J. Exp. Med. 189, 1565–1572. Mosmann, T.R. and Sad, S. (1996). The expanding universe of T-cell subsets: TH1, TH2 and more. Immunol. Today 17, 138–146. Mottram, P.L., Raisanen-Sokolowski, A., Glysing-Jensen, T. et al. (1998). Cardiac allografts from IL-4 knockout recipients: assessment of transplant arteriosclerosis and peripheral tolerance. J. Immunol. 161, 602–609. Mueller, R., Davies, J.D., Krahl, T. and Sarvetnick, N. (1997). IL-4 expression by grafts from transgenic mice fails to prevent allograft rejection. J. Immunol. 159, 1599–1603. Nagano, H., Libby, P., Taylor, M.K. et al. (1998). Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-gamma. Am. J. Pathol. 152, 1187–1197. Nagano, H., Mitchell, R.N., Taylor, M.K. et al. (1997). Interferon-gamma deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts. J. Clin. Invest. 100, 550–557. Nagano, H., Tilney, N.L., Stinn et al. (1999). Deficiencies of IL-4 or TNF-alpha receptor-1 do not diminish graft arteriosclerosis in cardiac allografts. Transplant. Proc. 31, 152. Nickerson, P., Steurer, W., Steiger, J. et al. (1994). Cytokines and TH1/TH2 paradigm in transplantation. Curr. Opin. Immunol. 6, 757–764. Nickerson, P., Zheng, X., Steiger, J. et al. (1996). Prolonged islet allograft acceptance in the absence of interleukin 4 expression. Transplant Immunol. 4, 81–85. Nickerson, P., Steiger, J., Zheng, X.X. et al. (1997). Manipulation of cytokine networks in transplantation. False hope or realistic opportunity for tolerance? Transplantation 63, 489–494. Noel, C., Hazzan, M., Labalette, M. et al. (1998). Improvement in the outcome of rejection with pentoxiphylline in renal transplantation. Transplantation 65, 385–389. Noel, C., Copin, M.-C., Hazzan, M. et al. (2000). Immunomodulatory effect of pentoxifylline during human allograft rejection: involvement of tumor necrosis factoralpha and adhesion molecules. Transplantation 69, 1102–1107. O’Connell, P.J., Pacheco-Silva, A., Nickerson, P.W. et al. (1993). Unmodified pancreatic islet allograft rejection results in the preferential expression of certain T cell activation transcripts. J. Immunol. 150, 1093–1104. O’Garra, A. (1998). Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8, 275–283. Palladino, M., Morris, R., Fletcher Starnes, H. and Levinson, A. (1991). The transforming growth factor betas. A new family of immunomodulatory molecules. Ann. NY Acad. Sci. 593, 181–187. Pavlakis, M., Strehlau, J., Lipman, M. et al. (1996). Intragraft IL-15 transcripts are increased in human renal allograft rejection. Transplantation 62, 543–545. Pelletier, R., Pravica, V., Perrey, C. et al. (2000). Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation. Transplantation 70, 674–680.

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Perez, V.L., Van Parijs, L., Biuckians, A. et al. (1997). Induction of peripheral tolerance in vivo requires CTLA-4 engagement. Immunity 6, 411–417. Picotti, J.R., Chan, S.Y., Goodman, R.E. et al. (1996). IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection. Evidence against a dominant protective role for T helper 2 cytokines in alloimmunity. J. Immunol. 157, 1951–1957. Picotti, J.R., Chan, S.Y., Li, K. et al. (1997a). Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4 and CD8 IFNgamma-producing cells. J. Immunol. 158, 643–648. Picotti, J.R., Chan, S.Y., VanBuskirk, A.M. et al. (1997b). Are TH2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting allograft survival? Transplantation 63, 619–624. Picotti, J.R., Li, K., Chan, S.Y. et al. (1998). Alloantigenreactive TH1 development in IL-12-deficient mice. J. Immunol. 160, 1132–1138. Picotti, J.R., Li, K., Chan, S.Y. et al. (1999a). Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for TH1 in the disease process. Transplantation 67, 1548–1555. Picotti, J.R., Li, K., Chan, S.Y. et al. (1999b). Interleukin-12 (IL-12)-driven alloimmune responses in vitro and in vivo: requirement for beta1 subunit of the IL-12 receptor. Transplantation 67, 1453–1460. Punch, J.D., Tono, T., Qin, L. et al. (1998). Tolerance induction by anti-CD2 plus anti-CD3 monoclonal antibodies: evidence for an IL-4 requirement. J. Immunol. 161, 1156–1162. Qian, S., Li, W., Fu, F. et al. (1996). Systemic administration of cellular interleukin-10 can exacerbate cardiac allograft rejection in mice. Transplantation 62, 1709–1714. Raisanen-Sokolowski, A., Mottram, P.L., Glysing-Jensen, T. et al. (1997). Heart transplants in interferon-, interleukin-4, and interleukin-10 knockout mice. Recipient environment alters graft rejection. J. Clin. Invest. 100, 2449–2456. Raisanen-Sokolowski, A., Glysing-Jensen, T., Koglin, J. and Russell, M.E. (1998a). Reduced transplant arteriosclerosis in murine cardiac allografts placed in interferon-gamma knockout recipients. Am. J. Pathol. 152, 359–365. Raisanen-Sokolowski, A., Glysing-Jensen, T. and Russell, M.E. (1998b). Leukocyte-suppressing influences of interleukin (IL)-10 in cardiac allografts: insights from IL-10 knockout mice. Am. J. Pathol. 153, 1491–1500. Refaeli, Y., Van Parijs, L., London, C.A. et al. (1998). Biochemical mechanisms of IL-2-regulated Fasmediated T cell apoptosis. Immunity 8, 615–623. Ring, G.H., Dai, Z., Saleem, S. et al. (1999a). Increased susceptibility to immunologically mediated glomerulonephritis in IFN--deficient mice. J. Immunol. 163, 2243–2248. Ring, G.H., Saleem, S., Dai, Z et al. (1999b). Interferon- is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts. Transplantation 67, 1362–1365. Rosenberg, A.S., Finbloom, D.S., Maniero, T.G. et al. (1990). Specific prolongation of MHC class 2 disparate skin allografts by in vivo administration of anti-IFN- monoclonal antibody. J. Immunol. 144, 4648–4650. Russell, M.E., Raisanen-Sokolowski, A., Koglin, J. and Glysing-Jensen, T. (1998). Immunobiology of chronic

rejection: effector pathways that regulate vascular thickening. Graft 1, 7–10. Sadlack, B., Merz, H., Schorle, H. et al. (1993). Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75, 253–261. Sadlack, B., Lohler, J., Schorle, H. et al. (1995). Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4 T cells. Eur. J. Immunol. 25, 3053–3059. Saleem, S., Konieczny, B.T., Lowry, R.P. et al. (1996). Acute rejection of vascularized heart allografts in the absence of IFN Transplantation 62, 1908–1911. Sankaran, D., Asderakis, A., Ashraf, S. et al. (1999). Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int. 56, 281–288. Sayegh, M.H., Akalin, E., Hancock, W.W. et al. (1995). CD28–B7 blockade after alloantigenic challenge in vivo inhibits TH1 cytokines but spares TH2. J. Exp. Med. 181, 1869–1874. Schmitt, M., Van Brandwijk, R., Van Snick, J. et al. (1989). TCGFIII/P40 is produced by naive murine CD4 T cells but is not a general T cell growth factor. Eur. J. Immunol. 19, 2167–2170. Sharland, A., Wang, C., Bowen, D.G. et al. (1999). Evidence that apoptosis of activated T cells occurs in spontaneous tolerance of liver allografts and is blocked by manipulations which break tolerance. Transplantation 68, 1736–1745. Shevach, E.M. (2000). Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18, 423–449. Shihab, F.S., Tanner, A.M., Shao, Y. and Weffer, M.I. (1996). Expression of TGF-beta 1 and matrix proteins is elevated in rats with chronic rejection. Kidney Int. 50, 1904–1913. Shull, M.M., Ormsby, I., Kier, A.B. et al. (1992). Targeted disruption of the mouse transforming growth factor-b1 gene results in multifocal inflammatory disease. Nature 359, 693–699. Sirak, J.H., Orosz, C.G., Roopenian, D.C. et al. (1998). Cardiac allograft tolerance: failure to develop in interleukin-4deficient mice correlates with unusual allosensitization patterns. Transplantation 65, 1352–1356. Smith, K.A. (1988). Interleukin-2: inception, impact, and implications. Science 240, 1169–1176. Smith, X.G., Bolton, E.M., Ruchatz, H. et al. (2000). Selective blockade of IL-15 by soluble IL-15 receptor a-chain enhances cardiac allograft survival. J. Immunol. 165, 3444–3450. Steiger, B., Nickerson, P.W., Steurer, W. et al. (1995). IL-2 knockout recipient mice reject islet cell allografts. J. Immunol. 155, 489–498. Steiger, J.U., Nickerson, P.W., Hermle, M. et al. (1998). IFNc receptor signaling is not required in the effector phase of the alloimmune response. Transplantation 65, 1649–1652. Stevens, H.P.J.D., Van der Kwast, T.H., Van der Meide, P et al. (1990). Synergistic immunosuppression effects of monoclonal antibodies specific for interferon- and tumor necrosis factor alpha. Transplantation 50, 856–861. Strehlau, J., Pavlakis, M., Lipman. et al. (1997). Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation. Proc. Natl Acad. Sci. USA 94, 695–700.

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58 Cytokines and asthma Timothy B. Oriss and Anuradha Ray University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Asthma is a slight ailment that promotes longevity. Oliver Wendell Holmes Asthma n’est pas fatale; Asthma is not fatal. Armand Trousseau

INTRODUCTION These statements from the nineteenth and early twentieth centuries speak to the basic misunderstanding of asthma pathogenesis in those days, but also to the relatively low prevalence of disease at that time. Much has been learned about how asthma occurs and progresses, and the knowledge gained in recent years has resulted in the realization that it is a complex immunologic disease that can have significant impact in terms of morbidity and mortality. Combined with the fact that the incidence of asthma appears to be rising in modern societies, it is a condition worthy of a great deal of scientific attention. Once thought to be merely a defect in airway contraction, asthma is now known to be a chronic disorder of the lung characterized by inflammation of the airways leading to airway hyperresponsiveness (AHR) to specific and non-specific agents, mucus hypersecretion by goblet cells, and remodeling of the airway walls. The inflammatory cells found in asthThe Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

matic lungs include predominantly eosinophils and lymphocytes. Other cell types present in fewer numbers, such as mast cells and neutrophils, also contribute to disease pathogenesis. Asthma is considered to be a ‘TH2-type disease’ since CD4 TH2-derived cytokines are involved in the maturation and trafficking of eosinophils, and in promoting B cell isotype switching towards IgE, the hallmark isotype of atopic disease. In this chapter, we will present a description of the scope of the asthma problem, a summary of the pathogenesis of the disease, an account of the individual cytokines that have been implicated in various stages of disease pathogenesis, and finally a discussion of cytokine-based therapies.

DESCRIPTION OF THE PROBLEM Although asthma occurs throughout the world in countries where socioeconomic status can differ

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widely, the incidence of the disease appears to be rising in developed countries. The belief is that changes that have occurred in high-technology societies, including changes in immunization practices have resulted in increased incidence of the disease. These changes include quantitative and qualitative differences in environmental factors such as pollutants, infectious agents and allergens. Unfortunately, an accurate description of the scope of the problem on a world-wide basis is lacking due to the fact that uniform standards for diagnosing asthma do not exist in all areas of the world. Therefore, statistics describing asthma prevalence and incidence can vary considerably from country to country, and from study to study. Generally speaking, the diagnosis of asthma is made under clinical conditions consisting of wheezing, coughing and tightness in the chest accompanied by hyperresponsiveness to inhalation of irritants, such as methacholine or histamine. Estimates of incidence based upon this diagnosis range from 0 to 30%, with the higher values generally being found in developed countries, such as Australia and New Zealand, and lower numbers in undeveloped countries such as Papua New Guinea, Indonesia and China. According to statistics published by the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health in the United States, approximately 15 million people have asthma in the USA and this generates about half a million hospital admissions and 5000 deaths per year for an economic impact estimated to be over $11 billion per year. This number of people with asthma translates to a prevalence rate of about 30 cases per 1000 of population, a number which has increased by about 75% over the past 20 years. The prevalence rate is highest in children in their late preschool years through the early teenage years at about 74 cases per 1000, and that value has also risen at a similar rate over the same time span. The prevalence rate for adults over the age of 18 remains at about the level of the overall prevalence until age 65, when it declines slightly. Therefore, although asthma does not represent a major source of mortality in the USA, it is a very significant problem in terms of morbidity and economic impact.

PATHOGENESIS OF ATOPIC ASTHMA Asthma often begins during infancy, although it may be difficult to discern the etiology of wheezing and coughing as being indicative of asthma versus a whole host of respiratory infections to which infants are susceptible. Further complicating the issue is that although allergens are the principal inducers of asthma, respiratory viral infections have also been implicated as risk factors in the initial stages of the disease. The diagnosis is more easily arrived at in early childhood when symptoms of AHR become apparent, usually by 8 years of age. A number of studies suggest that asthma disappears in 30–50% of cases as children reach puberty, which is also reflected in the declining prevalence rates after 18 years of age published by the National Institutes of Health. However, the general notion that many children ‘grow out of it’ belies the fact that many do not, and that recurrence in later adult life can and does occur. Of those children whose symptoms continue into their adult years, there is the general perception that the severity of childhood symptoms is prognostic for severity of adult disease. Adult-onset asthma also occurs, potentially in response to environmental triggers, such as occupational exposures and viral infections. In all stages of asthma, there can be significant impact on lung function and quality of life, and clearly severe exacerbation can result in acute attacks of airway constriction leading to death. Therefore, asthma represents a significant, continuing health problem deserving a great deal more clinical and basic research. A considerable amount of basic science research has been already been conducted in the field of asthma. Extensive studies in mice and rats have demonstrated many parallels with human asthma. Hence, much of what is know about asthma pathogenesis comes from data generated in these animal models. It is thought that the asthma disease process is initiated by environmental factors (such as dust mite allergen or other immunologic irritants) that lead to the initial inflammation. The T cell response appears to be biased towards TH2 based upon finding TH2-type cytokines including IL-3, IL-4, IL-5, IL-9 and IL-13 in BAL fluid from human asthmatics or animals with induced AHR. This TH2-biased response is thought to drive isotype switching in B cells towards

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ROLE OF CYTOKINES IN ATOPIC ALLERGIC ASTHMA

production of IgE, which in turn, sensitizes mast cells to respond to the environmental stimuli (Figure 58.1). IgE-mediated degranulation of mast cells further drives inflammation by stimulation of the production of various inflammatory mediators from a variety of cells. Later in the disease process, there is a massive infiltration of eosinophils into the airways via TH2 production of IL-5 and the secretion of eosinophil chemoattractants (Figure 58.1). Ultimately, there is desquamation of epithelial cells from airway linings, hyperplasia of goblet cells leading to increased mucus production, damage to the basement membrane through the deposition of collagen, a heightened sensitivity of smooth muscle cells to endogenous and exogenous stimuli resulting in severe episodic bronchoconstriction, formation of new blood vessels, and aberrant regeneration of the airway walls. Indi-

viduals with asthma therefore, are predisposed to shortness of breath, wheezing, coughing and episodic exacerbation of these symptoms which can be severe and potentially fatal in the worst cases.

ROLE OF CYTOKINES IN ATOPIC ALLERGIC ASTHMA The most striking pathology in asthma is denudation of the bronchial epithelium, infiltration of eosinophils and lymphocytes into the mucosa and submucosa of the airways and mucus plugging of bronchi (Gleich and Kita 1997; Holgate 1997; Busse 1998). Each of these features of asthma results from effects of the different TH2-derived cytokines IL-4, IL-5, IL-9, IL-10 and IL-13 and other pro-inflammatory cytokines,

Antigen Airborne Allergen

IgE Secretion

B-cell

Mast Cell degranulation

Mast Cell

Mast Cell growth and differentiation

Airway DC

IL-4, IL-13, IL-9

EXACERBATION OF ASTHMA

Th2 IL-9

Eosinophil accumulation in the lung

IL-5

Eosinophil differentiation, growth, trafficking

FIGURE 58.1 TH2 cytokine-mediated effects on asthma pathogenesis. Two of the main effects of TH2 in asthma are the production of IL-5 leading to eosinophilia and the production of IgE leading to mast cell degranulation. CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

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such as TNF-a that regulate the expression of cell adhesion molecules and chemokines that play important roles in the net inflammatory response that is characteristic of this disease. Collectively, the effects of the TH2 cytokines range in function from stimulation of TH2 development through various effector functions involving target cells in the lungs. Interestingly, the human chromosomal locus 5q31–33 that has been linked to asthma in genetic studies, includes the cluster of genes for the cytokines IL-4, IL-5, IL-9 and IL-13 (Marsh et al., 1994; Postma et al., 1995). In this review we will restrict our discussion to the TH2derived cytokines which are central to the initiation and progression of atopic asthma (Table 58.1).

IL-4 Increased levels of IL-4, IL-5 and IL-13 have been detected in the serum, broncholaveolar lavage (BAL) fluid and biopsies of patients with asthma. These cytokines are secreted in the airways of patients with mild or asymptomatic disease and the numbers of TH2 lymphocytes increase upon antigen challenge in allergic asthmatics (Walker et al., 1991, 1992; Robinson et al., 1992). Since TH2 cells have been identified in the airways of asthmatics, and because TH2 cytokines are required for the development of airway eosinophilia and IgE, it has been proposed that TH2 cells stimulate an inflammatory response that results in asthma. IL-4 is thought to be the most important cytokine driving T-helper cell differentiation towards the TH2 subtype and therefore it can be considered to play a much more complex role than simple induction of IgE synthesis. In vitro studies in both mice and humans clearly demonstrate the powerful influence of IL-4 in overriding TH1-promoting stimuli and promoting TH2 differentiation (Tanaka et al., 1993; Seder and Paul, 1994). In vivo, the picture is not so clear in regard to an absolute dependence on IL-4 for TH2 differentiation. Dendritic cells initiate immune responses by presenting antigens to naive T cells, and whereas they make IL-12, the main stimulus for TH1 differentiation, they have not been described as making IL-4 (Rissoan et al., 1999). However, other sources of IL-4, including mast cells (Plaut et al., 1989), NKT 1.1 cells (Yoshimoto et al., 1995) and T cells themselves

(Schmitz et al., 1994) have been identified that could provide the TH2-promoting stimulus (Figure 58.2). DC populations have been described that promote TH2 differentiation in an IL-4 independent manner, but there is no mistaking the impact IL-4 can have in driving TH2 differentiation when it is present. In a surprising finding, it was shown by adoptive transfer experiments in mice that T cell differentiation along either lineage was not dependent on IL-4 or IL-12 production by DCs, but rather on recipientderived cytokines although IL-12 production by DCs enhanced TH1 responses (MacDonald and Pearce, 2002). IL-4 and IL-13 have been shown to have overlapping properties in asthma. This stems from common receptor subunit usage; IL-4 utilizes two distinct receptor sets consisting of the high-affinity IL-4 receptor a chain (IL-4Ra) and either the common c chain (cc), the common signaling subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, or the IL-13Ra1 chain which binds IL-13 (Jiang et al., 2000). The effects of IL-4 result from activation of STAT6. The signal transducer and activator of transcription (STAT) proteins play an important role in the selective response of cells to particular cytokines (Kaplan and Grusby, 1998). After stimulation with cytokines, STAT proteins undergo phosphorylation by the Janus family of kinases (Jak). This causes dimerization and nuclear translocation of the proteins where they induce transcription of target genes. IL-4 engagement with its receptor leads to phosphorylation of STAT6 by Jak1 and Jak3 and results in the activation of IL-4 regulated genes such as IL-4R, IgE, FcR and class II MHC molecules (Kaplan and Grusby, 1998). An alternate pathway for STAT6 activation in human T cells is through IL-13. The IL-13-binding subunit IL-13Ra1 is not expressed on murine T cells and thus differentiation of murine T cells along the TH2 lineage can only be induced by IL-4 and not IL-13 as evident in in vitro studies. However, cells expressing the IL-4Ra-IL-13Ra1 heterodimer can respond to both IL-4 and IL-13. While IL-4 is undoubtedly the TH2-differentiating cytokine, IL-4-independent production of IL-4 has been observed using IL-4Ra mice and NK T cells have been implicated as the source of IL-4 in these studies (Noben-Trauth et al., 1997). Again, while IL-4 mice do display residual TH2 responses (Kopf

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

TABLE 58.1 Cytokines in asthma pathogenesis Cytokine

Produced by

Role(s) In asthma

References

IL-1

macrophage

IL-2

TH1

IL-3

TH1/TH2/mast cells

Borish 1992; Sousa 1996 Godding 1995 Rosenwasser 1998 Reviewed in Morel 1998 Broide 1992; Robinson 1992; Walker 1992 Robinson 1992; Kotsimbos 1996

IL-4

TH2/mast cells/eosinophils

IL-5

TH2/Eosinophil

Increased in asthma BAL/general inflammation Increases cellular adhesion molecules Promotes production of other inflammatory cytokines Generalized T cell proliferation Increased in asthma BAL Increased in asthma BAL Eosinophil growth and maturation Major promoter of TH2 differentiation Increased in asthma BAL Blocking IL-4R inhibits AHR, goblet metaplasia, eosinophilia Promotes IgE synthesis in asthma Induced goblet cell metaplasia and mucin production Eosinophil growth and maturation Increased in asthma BAL Responsible for lung eosinophilia in asthma

IL-6

macrophage

Promotes IL-4-mediated IgE synthesis Promotes TH2 differentiation

IL-8

airway epithelial cells, neutrophils

Increased in asthma BAL

IL-9

TH2, mast cells

IL-10

TH1(human)/TH2, Treg, monocyte, DC

Sanchez-Guerrero 1997; Bjorck 1998 Rincon 1997 Yousefi 1995; Chanez 1996; Nocker 1996; Ordonez 2000; Jatakanon 1999 Xiu 1995; Sampson 2000 Houssiau 1993 Dugas 1993; Petit-Frere 1993 Hultner 1990; Eklund 1993 McLane 1998; Temann 1998; Temann 2002; Longphre 1999; Louahed 2000 Trincheri 1997; Fiorentino 1991; Moore 1993; De Smedt 1997; Akbari 2001 Borish 1996, Robinson 1996

IL-11

airway structural cells, eosinophils

IL-12

DC, monocytes, B cells

Chemoattractant for neutrophils Promotes activated T cell proliferation Promotes IL-4-mediated IgE synthesis Mast cell growth and differentiation Induces asthma phenotype, stimulates mucus production Inhibits T cell differentiation via APC effects Increased or reduced expression in asthmatic airways

IL-13

TH1/TH2

Increased in severe asthma Airway fibrosis Induces AHR Promotes TH1/inhibits TH2 differentiation Reduced expression in asthmatic airways Inhibits IL-4-induced IgE synthesis Promotes IgE synthesis Increased in asthma airways Promotes airway hyperresponsiveness Airway remodeling

Reviewed in Morel 1998 Robinson 1992 Gavett 1997 Finkelman 1988; Brusselle 1995 Dabbagh 1999 Lopez 1988; Clutterbuck 1989; Yamaguchi 1988 Robinson 1992; Ohnishi 1993; Ying 1993 Dent 1990; Nakajima 1992; Venge1996; Foster 1996

Minshall 2000; Reviewed in Zheng 2001 Zhu 2001 Einarsson 1996 Hsieh 1993; Manetti 1994; Magram 1996; Macatonia 1995; Wu 1997;Kalinski 1999 Naseer 1997 Kiniwa 1992 Punnonen 1993; Punnonen 1995 Kotsimbos 1996; Naseer 1997; Humbert 1997 Grunig 1998; Wills-Karp 1998; Zhu 1999 Zhu 2001, Zhu 2002

TABLE 58.1 Continued Cytokine IL-16

Produced by CD8 T cells, mast cells, eosinophils

IL-17

CD4 memory T cells, eosinophils T cells, B cells, macrophages, DCs TH2

IL-18 IL-25 TNF-a

DC, macrophage, T cells, mast cells

GM-CSF

macrophage, eosinophil, T cells eosinophils, fibroblasts, endothelium airway smooth muscle, epithelium

IFN-c

IL-1RA TGF-b

EGF

TH1, CD8 T cells, NK cells

monocytes, alveolar macrophages Treg, eosinophils, epithelial cells

Role(s) In asthma Induces T cell growth and expression of IL-2R, MHC class II Increased in asthma BAL Promotes IgE synthesis and AHR Stimulates pro-inflammatory cytokine production Chemoattractant for lymphocytes into BAL Increased in asthma BAL Stimulates pro-inflammatory cytokine production Recruitment of neutrophils into airways Inhibition of eosinophilia, mucus production, and AHR Increase of TH2 cytokines, TH2-associated Ab isotypes, and induction of asthma-like lung pathology Increased in asthma BAL Chemoattractant for eosinophils and neutrophils Increased airway smooth muscle contraction Increases cellular adhesion molecules

References Cruikshank 1987 Laberge 2000; Taha 2001 Hessel 1998 Mathy 2000 Mashikian 1998 Molet 2001 Molet 2001 Laan 1999, Hoshino 1999, Linden 2000 Walter 2001, Maecker 2001 Fort 2001

Increased in asthma

Reviewed in Thomas 2001 Ming 1987 Hakonarson 2001; Kips 1992; Thomas 1995 Gamble 1985; Pober 1986 Gauvreau 1998; Sousa 1993

Chemoattractant for eosinophils and macrophages

Xing 1996

Promotes eosinophil survival Possible role in airway remodeling/fibrosis Promotes leukotriene production Inhibition of TH2 Reduced in asthma:correlated with disease activity Increased in pediatric asthma:not correlated with disease activity Inhibits airway eosinophilia

Park 1998 Xing 1996 Howell 1989; Soloperto 1991 Reviewed in Morel 1998 Leonard 1997; Koning 1997 Marguet 2000 Iwamoto 1993, Lack 1996, Coyle 1996; Cohn 2001

Inhibits TH2

Inhibits AHR Inhibits eosinphil migration into airways

Redington 1997 Raghu 1989; Minshall 1997; Redington 2000; Elias 1999 Hansen 2000 Haneda 1999; Hansen 2000

Increased in asthma Increases smooth muscle proliferation Possible role in airway remodeling

Vignola 1997 Cerutis 1997 Holgate 2000

Increased in asthma BAL Induction of fibrosis

IL - 4

et al., 1993; Brusselle et al., 1994), IL-4Ra mice (Barner et al., 1998; Grunewald et al., 1998; Grunig et al., 1998; Cohn et al., 1999) or STAT6 mice (Kaplan et al., 1996; Shimoda et al., 1996; Takeda et al., 1996; Kuperman et al., 1998) are severely impaired in TH2 responses and thus IL-4Ra is critical to induction of TH2 differentiation. The cytokine(s) responsible for residual TH2 responses in IL-4-deficient mice are not known. It is interesting to note, however, that studies with IL-13 mice have revealed an impairment of IL-4 production in the absence of IL-13 from the linked IL-4 gene (Guo et al., 2001). The mechanism by which IL-13 influences IL-4 production is currently unknown. Cord blood lymphocytes of newborns with atopic parents have been shown to generate T cell clones producing higher levels of IL-4 and IL-5 than those derived from newborns born of non-atopic parents (Piccinni et al., 1996). Thus, aberrant production of IL-4 or responsiveness to IL-4 due to polymorphisms in the IL-4R or downstream signaling molecules are likely factors that contribute to the predisposition to TH2 responses in asthmatics. These subsets of asthMonocyte

1319 matics are therefore most likely to benefit from therapeutic approaches that are directed against IL-4, such as the soluble IL-4 receptor, as discussed in the last section of this review. Once TH2 cells are fully differentiated, they make IL-4 in response to activation through the TCR, which along with other cytokines such as IL-5 and IL-9, clearly delineates TH2 from TH1. IL-4 acts as a growth factor for fully differentiated TH2 cells both alone and in synergy with other cytokines, such as IL-2. An important function of IL-4 in this regard is inhibition of T cell apoptosis (Vella et al., 1997). Among the cytokines IL-2, IL-4, IL-7 and IL-15 that have all been shown to have this effect, IL-4 and IL-15 are clearly the most effective (Vella et al., 1997). Importantly in the case of allergic asthma, IL-4 drives isotype switching in B cells towards the generation of IgE. While as the principal TH2-differentiating cytokine IL-4 is central to asthma pathogenesis, its role in chronic asthma may be less relevant. In the chronic phase of the disease, stimulation of allergen-specific memory TH2 cells resulting in the release of other TH2 cytokines such as IL-5, IL-9 and IL-13 will perpetuate

IL-10 ()

IFN-a

IL-10 IL-12 ()

Th1

IL-2

T-bet

IFN-c

() IL-10 TGF-b

DC

T reg

Th p ()

IL-4

Eosinophils

Th2 Mast Cells

IL-4

GATA-3

IL-5 IL-9 IL-10 (mouse)

NKT Cells

FIGURE 58.2 Cytokine effects on dendritic cell-initiated T cell differentiation. CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

IL-13

1320

CYTOKINES AND ASTHMA

the disease process through the actions of these cytokines.

IL-5 Airway eosinophilia is a hallmark of asthma. Although eosinophils have been associated with asthma since the early 1900s, the precise role of eosinophils in the pathophysiology of the disease, especially in the development of AHR, is still unclear. Nonetheless, the close correspondence between eosinophil numbers in blood and airways of asthmatics and the degree of asthma severity and the cytotoxic effects of eosinophil granular proteins, warrant a careful analysis of the role of these cells in asthma pathology (Dahl et al., 1978; Frigas et al., 1980; Gleich et al., 1988; Frick et al., 1989; Walker et al., 1991). The cytokine that is central to eosinophil biology is IL-5 (Bousquet et al., 1990; Sanderson, 1992). The function of IL-5 appears to be specifically focused on the development, differentiation, recruitment, activation and survival of eosinophils. Allergen inhalation results in increased IL-5 levels in the serum, BAL fluid and sputum of patients together with increased eosinophil numbers in these fluids. Eosinophils are recruited in the late phase of the asthmatic response, typically around 24 h after allergen provocation and persist up to 1 week post allergen exposure (Gauvreau et al., 1999). Studies using IL-4 mice have elegantly demonstrated that eosinophils migrate to different compartments in the lung via distinct mechanisms (Cohn et al., 2001). Antigen challenge of IL-4 mice led to eosinophilic inflammation in the lung, but in contrast to wild-type mice, which showed marked influx into the airways as well, IL-4 mice showed reduced eosinophil numbers in the airway lumen (Cohn et al., 2001). Interestingly, this was not due to a deficiency in IL-5 production in IL-4 mice as shown by adoptive transfer of IL-4-deficient in vitro generated TH2 cells into wild-type mice, but was due to increased IFN-c production in the absence of IL-4 as demonstrated using IFNcR mice. The biologic significance of eosinophil compartmentalization is however unknown at the present time. It is becoming increasingly clear that cross-regulation between TH1 and TH2 cytokines, mediated to a large extent by IFN-c and IL-4/IL-10, respectively, is an

important aspect of disease onset and progression in asthma and other diseases which is further discussed in the context of asthma in the next section of this chapter. The development of IL-5 transgenic mice (Clutterbuck et al., 1989; Lee et al., 1997) and IL-5 mice (Kopf et al., 1996) firmly established the important role of IL-5 in the induction of airway eosinophilia in murine models of allergic inflammation. The relationship between airway eosinophilia and AHR has, however, been elusive despite the efforts of multiple investigators in both murine and human studies. Early studies in mice revealed a complex relationship between IL-5, the genetic background of mice and the development of AHR. Use of IL-5 mice on two different genetic backgrounds revealed an IL-5-dependent mechanism for AHR in C57BL/6 mice (Foster et al., 1996), but not in BALB/c mice (Hogan et al., 1998). The studies in BALB/c mice showed a dependence of AHR on CD4 T cells, but not on IL-4 or IL-5 (Hogan et al., 1998). However, conflicting data were obtained with anti-IL-5 antibody. Administration of anti-IL-5 antibody in BALB/c mice resulted in attenuation of both eosinophilia and AHR (Hamelmann et al., 1997). Additional studies in BALB/c mice have shown development of AHR independent of both IL-4 and IL-5 and the cytokine that was implicated in the development of AHR in these mice was IL-13 (Grunig et al., 1998; Wills-Karp et al., 1998). Neutralization of IL-13 showed little effect on eosinophil numbers, but markedly attenuated allergen-induced AHR. Efforts to establish IL-5 as a useful target in asthma therapy in human trials have also not yielded conclusive data (Leckie et al., 2000) and more careful studies are needed before one can completely rule out the role of IL-5 and eosinophils in the development of AHR.

IL-9 IL-9 is another TH2-derived cytokine thought to be involved in asthma pathogenesis. The initial interest in IL-9 in asthma pathogenesis developed from the co-localization of the IL-9 gene with the TH2 cytokine gene cluster on the chromosomal region 5q31–33 (Marsh et al., 1994; Postma et al., 1995). Genetic studies in mice further supported a role for IL-9 in asthma

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

IL - 10

pathogenesis in which AHR was mapped to a region in chromosome 13 harboring the IL-9 gene in mice (Nicolaides et al., 1997). Additionally, AHR in different inbred strains of mice correlated with IL-9 mRNA levels in the lung (Nicolaides et al., 1997). Interestingly, C57BL/6 mice, which show reduced AHR compared with BALB/c mice, are unable to express the IL-9 gene due to a genetic defect in the IL-9 locus (Nicolaides et al., 1997). Although studies in humans and mice have revealed a pleiotropic role for IL-9, an important function of IL-9 is its ability to influence mast cell growth and function (Hultner et al., 1990). IL-9 is a mast cell growth factor, inducer of mast cell proteases and the high-affinity IgE receptor (FceRIa) and thus primes mast cells to respond to allergens (Hultner et al., 1990; Louahed et al., 1995). The effects of IL-9 on asthma pathology include promotion of lung eosinophilia and mastocytosis, mucus hypersecretion and IgE production (Louahed et al., 1995; Dong et al., 1999; Longphre et al., 1999; Gounni et al., 2000; Townsend et al., 2000). This was evident in studies of IL-9 transgenic mice which show increased TH2 cytokine expression accompanied by lung eosinophila, mast cell hyperplasia, mucus hypersecretion and development of AHR (McLane et al., 1998; Temann et al., 1998, 2002). However, these effects of IL-9 were shown to be mediated by the TH2 cytokines IL-4, IL-5 and IL-13 rather than due to direct effects of IL-9 (Temann et al., 2002). In support of this, IL-9 deficiency does not impair development of pulmonary eosinophilia, mucus secretion or AHR in mice (McMillan et al., 2002).

IL-10

1321 have similar functions in down-regulating expression of MHC class II and the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells, such as macrophages and DCs. However, although IL-10 may be considered to be inhibitory for T helper cells in general by virtue of its effects on MHC and costimulatory molecule expression, it is likely that it favors TH2 development due to its strong inhibitory effect on the expression of the TH1-promoting cytokine IL-12 (Moore et al., 1993; De Smedt et al., 1997; Trinchieri, 1997) (Figure 58.3). The actual examination of IL-10 expression in asthmatic lungs has produced data that are inconclusive. Whereas IL-10 expression was reported to be decreased in the lungs of asthmatics, other studies suggest that IL-10-expressing macrophages and T cells are increased in numbers in asthmatics (Borish et al., 1996; Robinson et al., 1996). The first information on regulation of allergen-induced immune responses by IL-10 was provided in a study where IL-10 inhibited the survival and cytokine production by LPSstimulated eosinophils (Takanaski et al., 1994). Similarly, inhibitory actions on mast cell cytokine production (TNF and IL-6) by IL-10 was also reported (Arock et al., 1996; Marshall et al., 1996). In studies in mice, IL-10 has been shown to inhibit the allergic phenotype (Zuany-Amorim et al., 1995; Stampfli et al., 1999; Quinn et al., 2000). In some other studies in Potential roles if IL-10 in Asthma

Prevention

Th2

One of the most intriguing cytokines in the context of allergic airway disease is undoubtedly IL-10. IL-10 has historically been considered to be an antiinflammatory cytokine. This perception is largely based upon the function of an IL-10 homologue, viral IL-10 (vIL-10, also known as BCRF1), which is expressed by the B-lymphotropic virus Epstein–Barr virus (EBV) (Hsu et al., 1990). The main function of vIL-10 associated with inhibition of immune responses is down-regulation of MHC class II expression on cells, which is thought to be a mechanism for viral immune evasion. Both murine and human IL-10

Mast cells

human Th1 Monocytes

Treg

Exacerbation

DCs Th2

IL-10

MHC Class II

DC

IL-12

FIGURE 58.3 Multiple roles of IL-10 in asthma could result in prevention or exacerbation of disease.

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

1322

CYTOKINES AND ASTHMA

mice, however, while IL-10 was shown to inhibit pulmonary eosinophilic inflammation, it was shown to be required for the development of AHR (Makela et al., 2000; van Scott et al., 2000). It is unknown whether these discrepancies are due to strain differences and/or amount and timing of IL-10 expression. The role of IL-10 in TH differentiation or in asthma pathogenesis is difficult to assess due to its highly pleiotropic nature. An additional function of IL-10 is its ability to promote the generation of regulatory T cells (Groux et al., 1997). It has been reported that DCs producing IL-10 are associated with inhibition of airway inflammation in mice (Akbari et al., 2001). Interestingly, peripheral T cell anergy after allergen (bee-venom)-specific immunotherapy was associated with increased IL-10 production, first by CD4CD25 regulatory T cells and subsequently by B cells and monocytes (Akdis et al., 1998). Complicating all of these issues is that in mice, IL-10 is a cytokine produced by TH2, but not TH1 cells, but in humans it is produced by both TH cell subtypes and that in addition to T helper cells, IL-10 can be produced by other cell types including CD8 T cells, monocytes, and mast cells.

IL-13 Several studies have recently established IL-13 as a significant player in asthma. Increase in IL-13 levels has been noted in the lungs of asthmatics (Huang et al., 1995). Although the T cell is an important source of IL-13, other cell types, including mast cells/ basophils, natural killer cells and potentially human alveolar macrophages may also contribute to the IL-13 production in allergic inflammation. As noted above, the IL-13 receptor comprises the heteromeric complex of IL-13Ra1 and IL-4Ra. Another IL-13-binding protein, IL-13Ra2, that binds IL-13 with high affinity, has also been identified but it does not trigger signaling upon ligand binding. The soluble form of this receptor that has been detected could function as a decoy receptor. IL-13 signaling also triggers STAT6 activation in target cells. While IL-13Ra1 is not expressed on murine T cells, human T cells express this receptor subunit. Also, IL-13 elicits IgE production from human, but not murine B cells. Due to the sharing of IL-4Ra between IL-4 and

IL-13, the cytokines have overlapping functions. It is signaling through this receptor subunit that appears to be most critical for the development of the asthma phenotype. Overall, IL-4 may have a more important function in TH2 development and in inhibition of TH1 responses. IL-13, on the other hand, appears to be more important in effector functions, particularly in regards to AHR and mucus production as evident in multiple studies carried out in mice (Grunig et al., 1998; Wills-Karp et al., 1998; Zhu et al., 1999). IL-13 instilled into the airways of BALB/c and A/J mice was sufficient to induce AHR and neutralization of IL-13 before antigen challenge inhibited AHR (Grunig et al., 1998; Wills-Karp et al., 1998). In other studies, neutralization of IL-13 in sensitized mice by the administration of soluble IL-13Ra2 which binds IL-13 but not IL-4, reversed AHR and mucus cell hyperplasia. In contrast, neutralization of IL-4 before challenge did not prevent the development of AHR (Corry et al., 1996). IL-13 transgenic mice develop all the features of asthma, including subepithelial fibrosis (Zhu et al., 1999). IL-4 transgenic mice do not develop AHR or subepithelial fibrosis (Rankin et al., 1996). Neither IL-4 nor IL-13 is critical for eosinophil differentiation and their mobilization from the bone marrow, but by stimulating the induction of cell adhesion molecules such as VCAM-1 on endothelial cells and by stimulating chemokine production (eotaxin), these cytokines mediate recruitment of eosinophils into the lung from the vasculature.

IFN-c The signature cytokine of TH1 cells is IFN-c. Although asthma is a TH2-dominated disease, IFN-c is detectable in the airways and blood of asthmatics. In a recent report, histamine was shown to promote TH1 responses (IFN-c secretion) by stimulating H1 receptor (H1R) expression on TH1 cells and mice lacking H1R displayed enhanced TH2 responses due to diminished IFN-c production (Jutel et al., 2001a). Thus, IFN-a production in allergic responses may be due to a built-in negative feedback mechanism that attempts to temper TH2 responses. Another factor contributing to IFN-c production in asthmatics may be corticosteroids which have been shown inhibit TH2 cytokine gene expression, but increase IFN-c gene

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

IL - 18

expression (Robinson et al., 1993). Since TH1 and TH2 responses cross-regulate each other (discussed in more detail in the following section), attempts have been made to substantiate the notion that TH1 responses would inhibit allergic inflammation. Both human and animal studies have been reported that suggest a protective role for IFN-c in allergic responses when administered in the lung prior to or early in the generation of a TH2 response. In a study in humans, nebulized recombinant IFN-c given to asthmatics improved clinical symptoms and caused a decrease in airway eosinophils (Boguniewicz et al., 1995). Also, treatment with rIFN-c did not cause any apparent adverse effects and did not increase TH1-mediated inflammation. In mice, IFN-c has inhibitory effects on TH2-induced airway eosinophilia and AHR. When administered prior to inhaled antigen challenge, IFN-c reduced the number of CD4 T cells in the respiratory tract (Iwamoto et al., 1993; Nakajima et al., 1993; Li et al., 1996) or reduced TH2 cytokine secretion. These effects may result from the inhibitory effect of IFN-c on TH2 cell recruitment. Once TH2 cells are present in the respiratory tract, IFN-c enhances the resolution of airway eosinophilia and suppresses TH2 cytokine production (Coyle et al., 1996). IFN-c can also dampen the effects of ongoing TH2 cell responses by inhibiting TH2 cell cytokine production and TH2 cell proliferation in vitro (FernandezBotran et al., 1988; Gajewski et al., 1989). In a different study, when both TH1 and TH2 cells were activated together in the respiratory tract, TH1 cells, through the production of IFN-c caused a marked inhibition of airway eosinophilia and mucus production changes by blocking the effects of TH2 cytokines on target tissues (Cohn et al., 1999). Similar to the observations in the human study, TH1 cells did not increase overall inflammation in the lung. Thus, TH1 cells play multiple roles in down-regulating TH2 responses. Shifting the generation of a CD4 T cell population away from TH2 and towards TH1 reduces TH2 cytokines. Over the short term, IFN-c may inhibit TH2 cell function and block the downstream effects of TH2 cytokines on target tissues. In more chronic systems, repeated stimulation with TH1 inducing cytokines, such as IL-12, may help to shift the balance of a TH2 population towards TH1. All of these effects may help to reduce pathological effects induced by TH2 cytokines. While many studies

1323 show that TH1 cells, through IFN-c production, can inhibit TH2 responses, some studies show that TH1 cells can actually enhance inflammation and do not ameliorate disease. It is unknown whether the same mechanisms are also operative during exacerbations of asthma by certain viral infections which tend to induce TH1 responses. Collectively, it appears that the timing of TH1 relative to TH2 cell activation and the ratio of the cytokines produced by these cells may influence the onset and progress of disease.

IL-18 IL-18, originally termed interferon-inducing factor (IGIF), has been shown to promote both TH1 and TH2 responses (Okamura et al., 1995). It is produced by activated macrophages and Kupffer cells and in combination with IL-12 enhances IFN-c production by NK cells, TH1 cells and CD8 T cells (Dinarello, 1999). Increased IL-18 levels have been reported in both TH1 and TH2-dominated diseases. Overall, the effects of IL-18 appear to depend on the presence of other cytokines. Co-administration of IL-18 and IL-12 was shown to inhibit IgE production in helminth-infected mice in an IFN-c-dependent fashion (Yoshimoto et al., 1997). Also, vaccination with IL-18-allergen fusion DNA was shown to protect from and reverse AHR in mice (Maecker et al., 2001). However, TH2-promoting effects of IL-18 have also been noted in other studies. In different animal studies, IL-18 caused increased production of IgE and TH2 cytokines (Kumano et al., 1999; Yoshimoto et al., 1999, 2000; Wild et al., 2000). In combination with IL-2, IL-18 increased IL-13 gene expression in NK and T cells (Hoshino et al., 1999). In combination with IL-3, IL-18 caused increased IL-4 and IL-13 secretion from IL-18Ra-expressing mast cells and basophils both in vitro and in vivo (Yoshimoto et al., 1999). IL-18-induced IgE production was found to be STAT6- and IL-4-dependent (Yoshimoto et al., 2000). In this study, IL-18-induced cytokine (IL-4 and IL-13) and IgE production did not need overt immunization of the animals; IL-18 alone in relatively high doses induced IgE production in vivo. This raises the possibility that increased IL-18 levels in the context of cytokines, such as IL-2 and IL-3 may predispose to an atopic state. TH2-promoting

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

1324

CYTOKINES AND ASTHMA

effects of IL-18 have been also noted in human studies. Histamine was shown to augment IL-18 production from human PBMCs (Kohka et al., 2000). In other studies, increased serum IL-18 levels or increased production from stimulated PBMCs was observed in subjects with asthma (Tanaka et al., 2001). IL-18 levels were found to inversely correlate with peak expiratory flow and in acute asthma, IL-18 levels correlated with serum soluble IL-2 receptor levels (Tanaka et al., 2001). It has been proposed that IL-18 levels may serve to indicate disease activity in mild-to-moderate asthma. Further studies in the future are needed to determine whether IL-18 is a marker of the diseased state or has an important effector function in acute and/or chronic disease.

TH1/TH2 BALANCE IN ASTHMA As discussed above, the immune response in asthmatic lungs is not exclusively TH2 in character, as TH1-associated cytokines are also present in the airways and blood of asthmatics. Therefore, asthma may most correctly be characterized as being a ‘TH2skewed disease’ suggesting an alteration in the balance between TH1 and TH2 in the lungs and the lung-draining lymph nodes. It is to be expected that a great deal of immunologic activity would be taking place in this area since the airways are obviously a major portal of entry for airborne allergens and pathogens. The control of these pathogens is essential, but proper immune system control must also be in place if one is to avoid deleterious reactions. Several mechanisms exist to maintain equilibrium between TH1 and TH2, and these mechanisms operate at both the level of T cell differentiation and at the level of control of mature, fully differentiated T-effector cells. What follows is a discussion about how TH1 and TH2 are regulated developmentally and functionally, and how bias towards TH2 could arise resulting in the characteristic pathology seen in asthma. The two T helper cell subtypes, TH1 and TH2, arise from naive T cell precursors upon their encounter with specific antigenic peptides presented by antigen presenting cells (APCs) in the context of MHC class II. The most important APC in the lung is the dendritic cell

(DC). DCs are the quintessential APCs because in their immature form they are able to take up and process antigens in a very efficient fashion, and in their mature form they express high levels of peptide-loaded MHC class II along with costimulatory molecules such as CD40, CD80 and CD86 which are necessary to fully activate T cells and drive differentiation. Cytokines clearly have a role in T helper cell differentiation. TH1 cells arise from naive precursor T cells following antigen stimulation by DCs under the influence of IL-12 and IFN-c. IL-12, which can induce IFN-c expression in a variety of cells, is primarily responsible for TH1 differentiation in vitro. In vivo, IL-12 also likely plays a major role since it has been shown that DCs can make large amounts of IL-12. The transcription factor T-bet has been shown to direct the development of TH1 cells and the gene for IFN-c is specifically up-regulated by T-bet (Szabo et al., 2000, 2002). IFN-c itself completes a positive feed-back loop by enhancing production of IL-12. Interestingly, IL-12/STAT4 signaling has been shown not to be critical for T-bet expression, but to stabilize the commitment to the TH1 lineage by acting as a growth signal and sustaining IFN-c expression through interactions of STAT4 with the co-activator CREB-binding protein (Mullen et al., 2001). The critical importance of T-bet in IFN-c gene expression has recently been demonstrated using T-bet mice. Mice which fail to express T-bet have a TH2-predominant phenotype which causes lung pathology resembling asthma, and T cells from such mice can adoptively transfer this disease-associated phenotype to SCID mice (Finotto et al., 2002). An analogous transcriptional factor, GATA-3, was previously described which regulates TH2 differentiation (Zhang et al., 1997; Zheng and Flavell, 1997). GATA-3 expression results in the up-regulation of IL-4 and IL-5 production in committed TH2 cells (Siegel et al., 1995; Lee et al., 1998; Ranganath et al., 1998; Zhang et al., 1998). Its predominant role in TH2 development is evident in the fact that GATA-3 expression in developing and even fully differentiated TH1 cells can change the cellular phenotype from TH1 to TH2 (Ferber et al., 1999; Lee et al., 2000). As might be expected, given the predominance of TH2 cells in asthma, GATA-3 expression has been found to be increased in asthma airways (Nakamura et al., 1999, 2000). Its importance in the pathogenesis of asthma

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

T H 1 / T H 2 BALANCE IN ASTHMA

has been directly demonstrated through the use of a dominant negative mutant of GATA-3 in a transgenic mouse, which fails to produce high levels of TH2 cytokines and develop pathologic features of asthma such as airway eosinophilia, mucus production and IgE synthesis (Zhang et al., 1999). Interestingly, although TH2-inducing conditions are generally unable to stimulate TH2-type gene expression in committed TH1 cells, ectopic expression of GATA-3 in fully differentiated TH1 cells was shown to induce TH2 cytokine production (Lee et al., 2000). GATA-3 appears to function in a feedback positive autoregulatory loop in an IL-4/STAT6-independent fashion to up-regulate its own expression, thus stabilizing the TH2 phenotype (Ouyang et al., 2000). Whether IL-4induced STAT6 activation is a key ‘instructive’ step necessary to up-regulate GATA-3 and cause chromatin restructuring is still an open question. An alternate role for IL-4 as a growth factor that ‘selects’ the already committed (by the action of GATA-3) TH2differentiating cell is also being entertained (Mullen et al., 2001). Although the molecular basis of TH2 differentiation appears to be clearly explained by GATA-3 expression, a conceptual problem exists with regard to the cytokines involved in driving the TH2 process. In vitro, it has become a common procedure to drive TH2 differentiation using exogenous IL-4. The problem in vivo is that whereas DC production of IL-12 conveniently explains TH1 differentiation, there are no descriptions of IL-4 production by DCs. Whereas DC-produced IL-12 is thought to drive TH1 differentiation, DC subtypes have been described that direct TH2 differentiation without expression of IL-4 by the DCs themselves (Pulendran et al., 1999). In the mouse spleen a DC subset with greatly reduced expression of IL-12, induces differentiation of TH2 cells (MaldonadoLopez and Moser, 2001). In contrast, another DC subset expressing CD8a makes IL-12 and promotes development of TH1 cells (Maldonado-Lopez and Moser, 2001). A third DC subset expressing B cell and granulocyte markers (B220 and Gr-1, respectively), and a fourth expressing CD4 have also been described but have not yet been associated with an ability to direct TH1 or TH2 responses (Vremec et al., 2000; Nakano et al., 2001). Similar TH1- and TH2 -skewing DC populations have been reported in humans, although they are phenotypically distinct from the subtypes in

1325

mice (Rissoan et al., 1999; Cella et al., 2000). In contrast to mice, human DCs expressing myeloid lineage markers appear to skew towards TH1 while those expressing the lymphoid lineage marker CD4, skew towards TH2 (Rissoan et al., 1999). There are several possible mechanisms by which DCs that do not express IL-4 can nevertheless induce TH2 differentiation. One possibility relates to expression of a different set of genes in TH1-versus TH2skewing DCs. Several publications have recently demonstrated that different maturation signals given to DCs cause vastly different gene expression, irrespective of common cell-surface molecules such as CD80, CD86 and MHC class II (Hashimoto et al., 2000; Granucci et al., 2001). Some of these maturation signals give rise to DCs that promote TH1 and others result in TH2-inducing DCs (Morelli et al., 2001). Another possibility relates to the TH1-skewing cytokine IL-12. Clearly, high IL-12 skews towards TH1, but it has been proposed that complete abrogation of IL-12 expression may not be necessary such that simply reducing the level of IL-12 may be sufficient to allow TH2 differentiation to occur (Kalinski et al., 1999). This idea will be discussed in greater detail below. The preceding paragraphs have shown that a considerable body of literature has emerged on how TH1 and TH2 cells differentiate from naive precursors in vitro and in vivo. The important question for asthma, as for any immunologic disease, is how the balance between TH1 and TH2 is upset, such that inappropriate immune responses occur. TH1/ TH2 balance is established, maintained and perturbed at many levels. There can be alterations in APC function, specifically with regard to DCs, there can be cytokine-mediated modulation of TH differentiation, and finally an entire cytokine network exists that regulates fully differentiated TH1 and TH2 effector cells. As discussed above, unique DC subtypes are able to skew TH development towards TH1 or TH2. What has now become apparent is that these DC subtypes are not terminally programmed to skew TH responses in only one direction. In contrast, DCs appear to possess substantial plasticity in terms of TH1/ TH2 differentiation and this plasticity apparently revolves in large part around the expression of IL-12. Experimentally it has been reported that CD8a DCs (‘TH1 skewing DCs’) from IL-12 knock-out mice induce

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differentiation of TH2 cells (Maldonado-Lopez et al., 2001). More natural modulation of DC expression of IL-12 can occur in response to certain maturation signals such as LPS, TNF-a, or CD40 ligation, qualitative differences in antigens, and also in response to various cytokines and other soluble factors as discussed below. Not all stimuli that drive the maturation of DCs from an antigen-uptake to an antigen-presenting phenotype result in functionally equivalent DCs. Granucci et al., used DNA microarray to show that LPS versus TNF-a maturation result in a large number of differentially expressed genes including membranebound proteins, cell cycle proteins, chemokines and cytokines (Granucci et al., 2001). The level of expression of the p40 subunit of IL-12 was approximately 800-fold higher DCs matured with LPS versus TNF-a, suggesting that these cells could be relatively better at stimulating TH1 differentiation. Alterations in the relative expression of IL-12 by DCs can also be accomplished based upon qualitative differences in antigens. For example, intracellular pathogens, such as viruses, tend to give rise to TH1 responses while extracellular pathogens such as helminths tend to induce TH2. However, even among antigens that would initially appear to be very similar, significantly different effects on DCs can be observed. Pulendran et al., have reported that LPS from Escherichia coli induces high IL-12 expression in murine CD8a DCs, whereas LPS from another bacterium, Porphyromonas gingivalis, fails to induce IL-12 production resulting in TH cells with a TH2skewed phenotype (Pulendran et al., 2001). In spite of the failure of P. gingivalis LPS to induce IL-12 production, presumably because it signals through a different Toll-like receptor, it induces DC maturation (assessed by MHC class II, CD40, CD80 and CD86 expression) to a comparable level as E. coli LPS. Cytokines also seem to be able to alter the THskewing capacity of DCs. The effects of IL-10 on APCs, including DCs, were discussed above and it was suggested that IL-10 might favor TH2 development. In support of this idea, it was recently reported that incubation of DCs with IL-10 in vitro resulted in ‘TH2skewing’ DCs with an enhanced ability to stimulate TH2 responses (Maldonado-Lopez et al., 2001). A novel TH2-derived cytokine, IL-25, has been implicated in the induction of TH2 cytokine expression via its

effects on an MHC class II-expressing cell which may be a DC (Fort et al., 2001). Interestingly, mice treated with IL-25 display a TH2-dominated phenotype, including increased serum IgE and eosinophilia, as well as asthma-associated lung pathology such as eosinophilia, enhanced mucus production and epithelial cell hyperplasia. Non-cytokine soluble factors apparently can act similarly. For example, histamine has been reported to act on DCs to reduce IL-12 expression, while increasing IL-10 expression, thus resulting in cells that can skew towards TH2 (Mazzoni et al., 2001). These authors hypothesized that mast cell-derived histamine in the lung could therefore promote TH2 responses associated with asthma. This is in contrast to the report that histamine inhibits TH2 cytokine production, while stimulating TH1 cytokine secretion (Jutel et al., 2001a 2001b). Thus, these seemingly conflicting studies re-emphasize the inherent mechanisms balancing the TH1/TH2 system and therefore, the complexity in understanding the basic immunology of errant responses, such as those occurring in asthma. Finally, cytokines regulate the function of fully differentiated T effector cells (reviewed in Morel and Oriss 1998). IFN-c directly inhibits proliferation of activated TH2 cells. IL-10 inhibits TH1 proliferation, but this inhibition is mediated indirectly through downregulation of APC function. So in parallel with regulation of T cell differentiation, expansion of TH1 and TH2 effector cells is balanced by both inhibitory, as well as stimulatory components. Among different novel therapeutic approaches that are being attempted to tip the TH cell balance away from TH2 without the complete abrogation of immunity in the lung, some cytokine-directed/based therapies show promise and are discussed below.

CYTOKINE-DIRECTED THERAPIES FOR ASTHMA Great strides have been made in the last 20 years in asthma therapy. Asthma can be controlled in most patients through the use of inhaled corticosteroids combined with bronchodilators such as b2-agonists and theophylline. However, in very severe cases these drugs may require oral administration in high doses over a long period of time which can cause side-

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CYTOKINE - DIRECTED THERAPIES FOR ASTHMA

effects. Therefore, the morbidity and costs associated with these severe cases are grounds for pursuing novel therapies which could more efficiently and safely treat asthma. In fact, as immunological aspects of the disease are being elucidated, a great deal of effort is being expended to exploit various pathways for the development of novel treatments. They are beyond the scope of this chapter, but new treatments are being developed or envisioned to block: inflammatory mediators such as IgE, leukotrenes, histamine and nitric oxide; proteases such as tryptase, elastase, and metalloelastase; adhesion molecules such as VLA-4, ICAM-1 and VCAM-1; and cell signaling molecules/transcription factors such as NF-jB, GATA-3, MAP kinase, and STAT6. These therapeutic strategies have recently been reviewed (Ray and Ray, 2001). Here, we will concentrate on therapies based upon blocking or promoting the function of cytokines.

Soluble IL-4 receptor Soluble IL-4 receptor (sIL-4R) binds and sequesters secreted IL-4 making it unavailable for the IL-4 receptor on the cell surface. In a phase I study to test sIL-4R for safety, moderate atopic asthmatics received a single inhaled dose of recombinant human sIL-4R (rhuIL-4R; 0.5–1.5 mg). Over a 15-day period, symptoms were modestly reduced and FEV1 was improved compared with placebo, particularly at the higher dose (Borish et al., 1999). Similarly, a murine study also showed effectiveness of sIL-4R in inhibiting airway inflammation in antigen-challenged mice (Henderson et al., 2000). In a subsequent phase I/II double-blind placebo-controlled study, 3.0 mg of rhuIL-4R caused a significant decline in FEV1 compared with placebo in moderate asthmatics and caused no adverse effects (Borish et al., 2000). All of these patients were maintained without corticosteroids during the study. Further studies are ongoing to test the long-term efficacy in asthma management using this new treatment modality.

Soluble IL-13 receptor Neutralizing IL-13 alone may be effective in treating asthma. In animal studies, selective neutralization of IL-13 using an IL-13 receptor-human Fc fusion pro-

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tein reduced airway eosinophilia, mucus production and AHR (Grunig et al., 1998; Wills-Karp et al., 1998). Although IL-13 plays a less important role in inducing TH2 cell differentiation, IFN-c suppression and IgE production, it is a more potent stimulus for mucus production and AHR. Therefore, an IL-13 inhibitor has the potential benefit of allowing essential TH2 responses to develop in the respiratory tract, while blocking the pathologic changes more specific to asthma.

Anti-IL-5 antibody In an animal study, inhibition of IL-5 using an antiIL-5 monoclonal antibody (TRFK-5) reversed established eosinophilia without inhibiting established AHR (Mathur et al., 1999). More recently, a humanized anti-IL-5 monoclonal antibody (SB-240563) was used in mild asthmatics to determine the effects on blood and airway eosinophilia and AHR. In a double-blind, placebo-controlled, parallel group randomized investigation, allergic subjects were treated the antibody (2.5 or 10 mg kg1 given as a single intravenous infusion) or with placebo (Leckie et al., 2000). Effects on blood, sputum and airway eosinophilia and on allergen-induced AHR at 1 week and 4 weeks after the treatment were recorded. While, the treatment caused a significant decline in eosinophilia in all compartments, no effect on allergen- or histamine-induced AHR before or after challenge was observed. Although the authors concluded ineffectiveness of this form of therapy in controlling late phase responses, limitations of their study were noted by other investigators (O’Byrne et al., 2001). The principal limitations in the study appear to have been inappropriate choice of the patient population and lack of sufficient power in the study. The subjects, who were mild asthmatics, did not develop allergen-induced AHR on one of the two occasions and histamine-induced responses on two of three occasions when measurements were made. Second, the study did not include the number of patients that was necessary to have the study adequately powered to demonstrate that the treatment had a negative outcome. Therefore, while this study prompted many to conclude that anti-IL-5 therapy would be ineffective in the treatment of asthma, additional studies are needed before this form of treatment is rejected for good.

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CONCLUSION In conclusion, it is obvious that a great deal of information is available regarding the involvement of cytokines in asthma induction, progression and control. The myriad of cytokines involved, along with their many and varied functions, makes asthma a very complex situation to understand. However, the generally favorable results of early attempts to generate novel therapies targeting biologic modifiers (cytokines) suggest that further expanding the understanding of the immunologic basis of asthma will be well worth the effort.

ACKNOWLEDGMENTS The authors wish to thank Prabir Ray for suggestions and critical reviewing of the manuscript. This work was supported by grants from the National Institutes of Health (to A.R.).

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or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect ofIL-4. J. Exp. Med. 186, 325–330. Venge, J., Lampinen, M., Hakansson, L. et al. (1996). Identification of IL-5 and RANTES as the major eosinophil chemoattractants in the asthmatic lung. J. Allergy Clin. Immunol. 97, 1110–1115. Vignola, A.M., Chanez, P., Chiappara, G. et al. (1997). Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis. Am. J. Respir. Crit. Care Med. 156, 591–599. Vremec, D., Pooley, J., Hochrein, H., Wu, L. and Shortman, K. (2000). CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164, 2978–2986. Walker, C., Kaegi, M.K., Braun, P. and Blaser, K. (1991). Activated T cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlated with disease severity. J. Allergy Clin. Immunol. 88, 935–942. Walker, C., Bode, E., Boer, L. et al. (1992). Allergic and nonallergic asthmatics have distinct patterns of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. Respir. Dis. 146, 109–115. Walter, D.M., Wong, C.P., DeKruyff, R.H. et al. (2001). Il-18 gene transfer by adenovirus prevents the development of and reverses established allergen-induced airway hyperreactivity. J. Immunol. 166, 6392–6398. Wild, J.S., Sigounas, A., Sur, N. et al. (2000). IFN-gammainducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma. J. Immunol. 164, 2701–2710. Wills-Karp, M., Luyimbazi, J., Xu, X. et al. (1998). Interleukin13: central mediator of allergic asthma (see comments). Science 282, 2258–2261. Wu, C., Ferrante, J., Gately, M.K. and Magram, J. (1997). Characterization of IL-12 receptor beta1 chain (IL12Rbeta1)-deficient mice: IL-12Rbeta1 is an essential component of the functional mouse IL- 12 receptor. J. Immunol. 159, 1658–1665. Xing, Z., Braciak, T., Ohkawara, Y. et al. (1996). Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation. J. Leukoc. Biol. 59, 481–488. Xiu, Q., Fujimura, M., Nomura, M. et al. (1995). Bronchial hyperresponsiveness and airway neutrophil accumulation induced by interleukin-8 and the effect of the thromboxane A2 antagonist S-1452 in guinea-pigs. Clin. Exp. Allergy 25, 51–59. Yamaguchi, Y., Suda, T., Suda, J. et al. (1988). Purified interleukin 5 supports the terminal differentiation and proliferation of murine eosinophilic precursors. J. Exp. Med. 167, 43–56. Ying, S., Durham, S.R., Barkans, J. et al. (1993). T cells are the

principal source of interleukin-5 mRNA in allergeninduced rhinitis. Am. J. Respir. Cell Mol. Biol. 9, 356–360. Yoshimoto, T., Bendelac, A., Watson, C. et al. (1995). Role of NK1.1 T cells in a TH2 response and in immunoglobulin E production. Science 270, 1845–1847. Yoshimoto, T., Okamura, H., Tagawa, Y.I. et al. (1997). Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-gamma production from activated B cells. Proc. Natl Acad. Sci. USA 94, 3948–3953. Yoshimoto, T., Tsutsui, H., Tominaga, K. et al. (1999). IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc. Natl Acad. Sci. USA 96, 13962–13966. Yoshimoto, T., Mizutani, H., Tsutsui, H. et al. (2000). IL-18 induction of IgE: dependence on CD4 T cells, IL-4 and STAT6. Nat. Immunol. 1, 132–137. Yousefi, S., Hemmann, S., Weber, M. et al. (1995). IL-8 is expressed by human peripheral blood eosinophils. Evidence for increased secretion in asthma. J. Immunol. 154, 5481–5490. Zhang, D.H., Cohn, L., Ray, P. et al. (1997). Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272, 21597–21603. Zhang, D.H., Yang, L. and Ray, A. (1998). Differential responsiveness of the interleukin-5 (IL-5) and IL-4 genes to transcription factor GATA-3. J. Immunol. 161, 3817–3821. Zhang, D.H., Yang, L., Cohn, L. et al. (1999). Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11, 473–482. Zheng, T., Zhu, Z., Wang, J. et al. (2001). IL-11: insights in asthma from overexpression transgenic modeling. J. Allergy Clin. Immunol. 108, 489–496. Zheng, W. and Flavell, R.A. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596. Zhu, Z., Homer, R.J., Wang, Z. et al. (1999). Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103, 779–788. Zhu, Z., Lee, C.G., Zheng, T. et al. (2001). Airway inflammation and remodeling in asthma. Lessons from interleukin 11 and interleukin 13 transgenic mice. Am. J. Respir. Crit. Care Med. 164, S67–S70. Zhu, Z., Ma, B., Zheng, T. et al. (2002). IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling. J. Immunol. 168, 2953–2962. Zuany-Amorim, C., Haile, S., Leduc, D. et al. (1995). Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice. J. Clin. Invest. 95, 2644–2651

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59 Cytokine gene transfer Paul D. Robbins, Walter J. Storkus and Andrea Gambotto University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Life is what we make it, always has been, always will be. Grandma Moses

INTRODUCTION Over the last quarter of a century, more than 20 interleukins as well as multiple interferons, growth factors and other cytokines have been identified and characterized. A significant number of the identified cytokines have been implicated in either the progression or treatment of disease. More recently, the complete sequencing of the human genome has led to the identification of additional cytokine homologues that presumably play important roles in regulating the immune system. Many of these cytokines can be produced as recombinant proteins that can be administered systemically for regulation of immune response. Indeed, the activities of the identified cytokines have been elucidated mostly through the use of recombinant proteins. However, the delivery and subsequent expression of cytokines by gene transfer allows for

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

both analysis of cytokine function, as well as for potential therapeutic applications. Transfer of a cytokine gene allows for synthesis of the cytokine in eukaryotic cells where it is appropriately modified, bypassing the need to produce high levels of biologically active protein. Moreover, it is possible to target production of the cytokine to specific sites in vivo, eliminating possible toxicity of systemic administration. It is important to note that gene transfer can be used for applications other than developing therapeutic strategies. As outlined below, gene transfer can be used as a tool to identify the biological activities of a cytokine with unknown function. Moreover, gene transfer can be used to examine the role of a cytokine in the pathogenesis of disease. This chapter will focus on gene transfer approaches that can be used to identify and characterize the biological activities of cytokines, as well as treat human disease.

Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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APPROACHES TO GENE THERAPY There are two general approaches for gene therapy, ex vivo and in vivo gene delivery (Figure 59.1). The ex vivo, indirect approach involves modifying cells in culture by gene transfer that are then transplanted into the recipient. The advantage to this approach is that the target cell for gene modification can be carefully controlled and characterized. Moreover, the levels of expression of a specific therapeutic cytokine can be determined before transplantation. The in vivo, direct approach involves the direct delivery of the gene into the recipient, resulting in the genetic modification of cells at the site of injection. The advantage of this approach is that it does not require the culturing of primary cells that then need to be tested prior to transplantation. However, it is difficult to control exactly which cells are being modified in vivo. In addition, there can be an immune response to the injected virus. Thus depending upon the application, either ex vivo or in vivo gene transfer methods can be utilized. It is important to note that both ex vivo and in vivo gene transfer can be used to express cytokines either locally or systemically (Figure 59.2). Even though cytokines, for the most part, are secreted proteins, it is possible to target expression to a specific site where the biological activities of a specific cytokine can be enhanced, for instance, direct injection of a vector carrying a cytokine gene into a tumor results in elevated intra-articular levels of the factor

IN VIVO

with only low systemic levels. Alternatively, the modification of a hematopoietic stem cell to express a cytokine most likely will result in high systemic levels following transplantation of the genetically modified cell.

Gene transfer vectors The rate-limiting step for successful gene therapy is the efficiency of gene transfer, especially for in vivo applications. Given that for most cell types the efficiency of uptake of naked, plasmid DNA is extremely low, vectors that are able to transfer DNA more efficiently have been developed. There are two general types of vectors that have been developed for gene transfer, viral and non-viral. Viruses have evolved to be highly efficient at delivery of nucleic acid, either DNA or RNA, to specific cell types while avoiding the host immune response. If the pathogenicity of a virus can be eliminated, while still retaining the efficiency of gene transfer and expression, then it is well suited for gene therapy/gene transfer applications. The basic strategy utilized for generating non-pathogenic viruses is to deleted the genes that are required for viral replication, rendering the virus replication defective. The defective virus can be grown in a complementing cell line that expresses the viral proteins necessary for viral replication. In contrast to vector derived from viruses, non-viral vectors such as liposomes or DNA conjugates have the advantage of being non-pathogenic, but also are less efficient in the

EX VIVO Local

Systemic

• Intracellular protein • Non-trafficking cell • Low level secreted protein

• High level secreted protein • Hematopoletic stem cell

Direct gene transfer

Gene transfer

FIGURE 59.1 Gene therapy approaches. The approaches for in vivo, direct and ex vivo, indirect gene transfer are shown.

FIGURE 59.2 Local and systemic gene transfer. Cytokines can be delivered by ex vivo or in vivo gene transfer, resulting in either localized or systemic expression levels.

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transfer of nucleic acid to cells. Each of these vector systems, retrovirus, lentivirus, adenovirus, herpes simplex virus, adeno-associated virus and non-viral DNA formulations, has its strengths, as well as weaknesses that require further modification to make them suited for general gene therapy applications.

LTR

LTR

w

w

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TG

Pol

Env

LTR

LTR

Retroviral vectors Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate, integrating into the host DNA where they then are able to express a viral RNA for the life of the infected cell (Guild et al., 1988; Miller, 1992; Miller et al., 1993; Byun et al., 1996; Kim et al., 1997). Thus retroviruses and vectors based on retroviruses have the advantage of permanently modifying cells. MLV is a simple RNA virus in that is genome encodes for three polyproteins, gag, pol and env. These three proteins are further proteolyzed into smaller polypeptides that are required in trans for viral replication and packaging. All that is required for viral replication in cis are the 5 and 3 long terminal repeats (LTRs) that contain promoter, polyadenylation and integration sequences, a packaging site termed w, and a tRNA binding site, as well as several additional sequences involved in reverse transcription. In order to generate a MLV-based vectors, the genes encoding the three viral proteins are removed and heterologous genes and transcriptional regulatory sequences inserted.To produce replicationdefective MLV vectors, packaging lines that express the three viral genes, Gag, Pol and Env are utilized. The vector, in the proviral form of double-stranded DNA, is introduced into the packaging cells either

Gag, Pol, Env

LTR

Packaging cell

TG

LTR

Target cell

FIGURE 59.3 Production of replication defective retroviral vectors. A retroviral vector is generated by inserted the therapeutic gene (TG) in place of the viral proteins, gag, pol and env. The vector is then transfected into a packaging cell, stably expressing the viral proteins necessary for viral packaging. The resulting virus is able to infect target cells where it can integrate into the genome of the target cell, but is unable to replicate further due to the absence of the viral proteins. transiently or stably by transfection, resulting in the production of infectious virus (Figure 59.3). The virus produced from the transfected packaging cells is replication defective in that following infection of a target cell and integration of the vector into the host DNA, the virus is unable to replicate due to the absence of the viral trans-acting proteins. As described above, MLV-based retroviral vectors have the advantage of stably integrating into the host DNA of the infected cell where it can express a therapeutic gene for extended periods of time. The provirus

TABLE 59.1 Vectors Vector

Integration

Stable/Transient

Cell division

Retrovirus Lentivirus Adenovirus Adeno-associated Herpes simplex Naked DNA Lipsomes Electroporation Conjugates

Yes Yes No Wt virus No No No No No

Stable Stable Transient Stable(?) Transient Transient Transient Transient Transient

Yes No No No No No No No No

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contained in the host DNA is stable during cell division, so stem cells genetically modified by retroviral infection give rise to differentiated progeny that all contain the same viral integrant. The main disadvantage to the MLV-based retroviral vectors is that they require cell division, in particular, mitosis for integration. Thus the current retroviral vectors are better suited for ex vivo gene therapy where isolated cells can be propagated in culture, genetically modified by retroviral infection, and then transplanted into the recipient. It is important to note that retroviral vectors with titers as high as 1010 have been produced that can be used for infection of dividing cells in vivo, albeit at a low efficiency. The use of a class of retroviruses (see below) that is able to infect non-dividing cells improves in vivo gene transfer efficiency.

Lentiviruses Lentiviruses are a class of retroviruses that include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). Although the life cycle of lentiviruses is similar to MLV and other retroviruses, lentiviruses have the advantage of being able to integrate into the host DNA of non-dividing cells. Thus they are significantly more useful for in vivo applications than conventional retroviral vectors. However, lentiviruses are more complex than MLV-based retroviruses since they encode for accessory proteins in addition to Gag, Pol and Env encoded by multiply spliced viral RNAs. Recently, methods for production of vectors based on HIV, SIV, FIV and EIAV have been developed that allow for infection of slowly or non-dividing cell types, like b-cells from pancreatic islets, hepatocyes, muscle, tumor cells and neurons (Naldini et al., 1996a, 1996b). However, there is still a concern regarding safety of lentiviral vectors for clinical applications, in particular HIV-based vectors. The generation of replication-competent lentiviruses may have significant associated pathologies following in vivo gene transfer. Thus methods for determining whether clinical preparations of HIV-based vectors are contaminated with replication-competent virus still need to be developed and validated prior to the initiation of clinical trials.

Adenoviruses Adenoviruses are linear, double-stranded DNA viruses that cause respiratory and eye infections in a number of mammalian species, including humans. Although there are many serotypes of adenovirus, it is adenovirus serotype 5 that is the best characterized and which is used predominantly as a vector for gene transfer (Krougliak and Graham, 1995). However, the lack of antibodies against other serotypes such as sertoype 35 may allow for their more widespread clinical applications. Unlike retroviral vectors, adenoviruses can infect a wide variety of non-dividing cell types, resulting in transient gene expression. Moreover, the virus can be grown to titers that are significantly higher than retroviral and lentiviral vectors, reaching 1014 viral particles ml-1. Adenoviruses carry four early genes, expressed in a temporal manner that encode for multiple polypeptides important for the virus life cycle. The proteins encoded by the E1 region of the virus are essential to activate transcription of the E2, E3 and E4 genes that encode for additional regulatory proteins, as well as polypeptides important for viral replication. Late in infection, the major late promoter (MLP) is activated, resulting in expression of the late proteins important for packaging of the viral DNA. In order to generate a replication-defective virus from Ad-5 (Graham and Prevec, 1995; Krougliak and Graham, 1995), a strategy similar to the generation of retroviral vectors is used. The E1 gene is important for temporal expression of all the other adenoviral genes and thus is required for viral replication. In the first generation vectors, the E1 gene, encoding for multiple E1A and E1B polypeptides, is deleted from the viral genome and an expression cassette carrying the gene of interested inserted, resulting in a virus that is replication defective unless the E1 polypeptides are provided in trans. The cell line 293, derived from a human embryonic kidney cell line, stably expresses E1A and E1B, allowing for replication and propagation of a DE1 virus (Figure 59.4). However, the virus is replication defective in that after infection of a target cell, the virus is unable to replicate due to the absence of the E1 proteins. The transgene carried by the vector potentially can be expressed for as long as the viral DNA is maintained as an episome. The advantages of adenoviral vectors is that they can be grown to a high titer, greater than 1014 particles ml-1,

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I

HCMV

MCS

PA

E1

w5 adenovirus genomic DNA

E3

Cre 8 293 cells

Transgene Co-transfect

I

HCMV

MCS

PA

E1A, E1B

PAdlox recombination plasmid

FIGURE 59.4 Production of replication defective adenoviral vectors. A first generation DE1 adenoviral vector is generated using a shuttle plasmid containing sequences flanking the E1gene. The therapeutic gene (TG) is inserted in place of E1 in the shuttle vector under the regulation of a heterologous promoter. The shuttle vector is then cotransfected with the adenovirus vector into 293 cells, which stably express adenovirus E1 proteins. The shuttle plasmid and the vector are then able to recombine, resulting in production of a replication-defective adenoviral vector.

and are able to infect a wide variety of dividing and non-dividing cell types. However, the viral DNA that is unable to replicate in the infected cell is eventually degraded, resulting in loss of gene expression. In addition, the ‘first generation’ DE1 vectors still express a low level of viral proteins, resulting in a stimulation of an immune response to the virally infected cell in vivo. The first generation adenoviral vectors may be well suited for cancer gene therapy where a stimulation of a local immune response to the tumor is needed, but for other applications the vector needs to be further developed. One approach to reduce the immunogenicity of the virus after infection in vivo is to remove some or all of the open reading frames (ORFs) of the E4 gene that encode trans-acting regulatory proteins (Kochanek et al., 1996; Wang and Finer, 1996). The ‘second generation’ vectors are grown on 293 cells modified to express the polypeptides encoded by the E4 gene (Wang and Finer, 1996). These vectors have a reduced

immunogenicity in vivo, but there also appears to have a corresponding decrease in transgene expression since it appears as if E4 polypeptides play a role in enhancing gene expression from the viral genome. An alternative approach for making a more defective, non-immunogenic virus is to completely remove all the viral genes, leaving only the inverted terminal repeats (ITRs) and the packaging site (Kochanek et al., 1996). This ‘gutted’ or ‘gutless’ virus is grown using a first or second generation virus as a helper virus in 293 cells. The only difficulty with this approach is how to enrich for the gutted vector in relation to the helper virus. The insertion of sequences into the helper virus that hinder its growth specifically in modified 293 cells has resulted in 1000-fold better production of the gutted vectors compared with the helper virus. However, it is still unclear whether the virus is able to express high levels of the transgene for longer periods of time in vivo.

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Herpes simplex virus vectors Herpes simplex viruses (HSV) are large linear DNA viruses of approximately 150 kb that are able to infect cells lytically, as well as infect certain cell types latently such as neurons. Similar to adenovirus, HSV can infect a wide variety of cell types including muscle, tumor cells, liver and pancreatic islets. The normal life cycle of HSV following uptake of the virus into cells involves expression of a set of immediate–early genes (e.g. ICP0, ICP4, ICP22, ICP27 and ICP47). ICP4 and ICP27 are essential genes for expression of early and late gene products important for virus morphogenesis and infectious particle production. In order to generate a vector based on HSV, the immediate–early genes important for subsequently activating the early and late genes are deleted or inactivated (Fink and Glorioso, 1997a, 1997b; Fink et al., 1995; Glorioso et al., 1997). The resulting virus can be grown on complementing cell lines expressing the appropriate immediate–early genes. The first HSV vector had a deletion in the ICP4 gene (Wu et al., 1996), but was still able to express the other immediate–early genes products that are toxic to certain cell types. Recently, further deletion of ICP0, ICP22 and ICP27 has resulted in a virus that is non-toxic and can express a transgene for extended periods in culture and in vivo (Marconi et al., 1996). These HSV vectors are able to incorporate exogenous DNA of up to 35 kb, allowing for insertion of large fragments of DNA. Alternatively, it is possible to package plasmid vectors, termed amplicons, using defective HSV helper virus or plasmids encoding helper functions (Geller, 1997). To generate helper free amplicons vectors, a plasmid carrying the HSV origin of replication and packaging signals is co-transfected with a series of plasmids that contain all of the HSV genes, but are missing the packaging sequence. The vector plasmid is packaged into infectious HSV particles and used for gene transfer.

virus contains two genes, Rep and Cap, that encode polypeptides important for viral replication and encapsidation. Similar to retroviruses, Rep and Cap can be supplied in trans with only the inverted terminal repeat (ITR) sequences required in cis for the virus life cycle. Thus therapeutic genes can be inserted between the ITRs and infectious virus generated by co-transfection of the 293 cells line with the vector and a Rep and Cap expression plasmid followed by infection with a first generation adenoviral vector (Samulski, 1993; Rolling and Samulski, 1995). The resulting virus can be purified away from the contaminating adenovirus by centrifugation. However, to avoid possible adenoviral contamination, infectious recombinant AAV can also be generated by co-transfection of a plasmid expressing the complementing adenoviral genes. Wild-type AAV can be grown to high titers and infect a wide variety of cell types. Following infection, the virus forms a double-stranded DNA intermediate that can be integrated at a certain frequency into chromosome 19 through a Rep-dependent mechanism. However, it appears as if recombinant AAV vectors are unable to integrate specifically and may actually persist as episomal DNA in non-dividing cells. One of the rate-limiting steps in the virus life cycle is the rate of second-strand synthesis, necessary for expression of the transgene (Ferrari et al., 1996). Normally, adenoviral gene products are able to stimulate the rate of second-strand synthesis, but in the absence of adenoviral helper functions, AAV requires cellular factors. These unknown factors are either high in certain cell types or can be induced by DNA damaging agents (Russell et al., 1995). In particular AAV has been shown to stably infect muscle and liver following delivery of recombinant AAV produced using adenoviral complementing plasmids (Fisher et al., 1997; Snyder et al., 1999).

Non-viral vectors Adeno-associated virus Adeno-associated virus (AAV) is a member of the parvovirus family of single-stranded small DNA viruses that require a helper virus, such as adenovirus or herpes simplex virus for replication. Although the majority of the population is seropositive for AAV, the virus is not associated with any pathology in humans. The

Although non-viral gene transfer is less efficient than viral mediated DNA delivery, it has several advantages. First, it is considerably cheaper to produce large amounts of DNA than viruses for clinical application. Second, there is no possibility of recombination giving rise to replication-competent virus that can result in significant pathology. Third, it may be possible to

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repeat the dose with DNA formulations that are non- or weakly immunogenic. For these reasons, many laboratories and biotechnology companies have focused on improving non-viral gene delivery methods. Although certain cells such as myoblasts are able to take up naked DNA at some efficiency, complexing of the DNA to different chemical formulations increases DNA uptake and subsequent gene expression (Gao and Huang, 1995; Liu et al., 1996). In particular, cationic and anionic liposomes are able to increase the uptake of DNA in tissue culture cells with cationic liposomes improving the efficiency in vivo. It is also possible to couple the DNA to polypeptides to target the DNA to specific cell surface proteins. Condensation of the DNA using different polycations is able to improve DNA uptake and prevent degradation.

THERAPEUTIC APPLICATIONS OF GENE TRANSFER Cytokine gene transfer can be used to treat a wide variety of diseases, including cancer and autoimmune diseases. Moreover, cytokine gene transfer can be used to enhance the immune response to viral and bacterial vaccines, as well as to specific tumor antigen. Given the large number of cytokines that have been identified to date, it is impossible to provide a complete description of the different cytokines used for gene therapy. Instead, the focus of this chapter will be on the general approaches used that could be applied to a wide variety of cytokines.

Cancer Cytokine gene therapy of cancer has been evaluated extensively over the past 10 years, and an evaluation of novel cytokines for its anti-tumor effects has become a first-line screening procedure. Cytokine gene therapy has been applied in many formats, with most early evaluations implementing cytokine cDNA transfected tumors in murine models (Lotze et al., 1996; Morris and Scher, 2000; O’Malley and Crouch, 2000; Tahara et al., 2002). More recent studies have applied cytokine gene therapy via intratumoral delivery of transfected fibroblasts or dendritic cells, or via

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direct gene delivery into the tumor or dermis/muscle using either plasmid or recombinant virus (Lotze et al., 1996; Tueting et al., 1999a). As noted elsewhere in this chapter, the paracrine delivery of cytokines into the tumor microenvironment optimizes their biologic efficacy, while limiting toxicities typically associated with systemic applications of these potent factors. An early comprehensive comparison of cytokine gene therapy in the B16 melanoma model in B6 mice demonstrated a clear benefit of granulocyte– macrophage colony-stimulating factor (GM-CSF)based gene therapy in promoting protective immunity (Dranoff et al., 1993). This resulted in one of the first clinical trials for GM-CSF gene therapy for renal cell carcinoma (RCC) (Berns et al., 1995). These preclinical and clinical applications supported the critical involvement of vaccine (autologous GM-CSF cDNA-engineered RCC) infiltrating DCs in promoting enhanced CD4 and CD8 T cell responses and objective clinical responses (Dranoff et al., 1993; Berns et al., 1995). Since these early studies, many other cytokines applied either as single agents or in combinational gene therapies, have provided prophylactic and/or therapeutic benefit in animal models (Forni et al., 1994; Hock et al., 1994; Blankenstein and Qin, 1996; McBride et al., 1996; Tueting et al., 1997; Ferrantini and Belardelli, 2000; Lotze et al., 2000). Depending on the cytokine(s) applied, diverse leukocytic subpopulations served as the ‘rate-limiting’ effector cells in a given model system, for example CD4 T cells, CD8 T cells, NK cells, macrophages, PMNs and eosinophils. Perhaps the reason for the ability of such a diverse array of cytokines with often times antagonistic immunobiologies (i.e. IL-4 or IL-10 and IL-12), is that the induction and recruitment of therapeutic T cells into the tumor microenvironment requires two distinct and temporally separate operations. The first requires the activation and generation of tumor-specific effector cells, which based on our current understanding in the cancer setting, occurs in the tumor-draining lymph node(s) via a process termed ‘cross-priming’ (Huang et al., 1994; Chiodoni et al., 1999; Ranieri et al., 2000; Hoffman et al., 2000; Berard et al., 2000; Gao et al., 2000). Theoretically, cytokine gene therapy provided intratumorally (i.t.) may also activate or recruit antigen-presenting cells (APCs, including dendritic cells (DCs)) that can

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acquire tumor antigens in situ and serve as an effective ‘vaccine’ that can activate T cells in the draining lymphatic tissues. Elegant studies by Levitsky et al. (1996) and recent clinical trial data from our own group and others suggests that DC-mediated ‘crosspriming’ may prove critical to disease regression. ‘Cross-priming’ results in the temporal evolution of a broad repertoire of tumor-reactive CD4 and CD8 T cells. Once DC-primed T cells have expanded and have begun to circulate systemically, a second guidance cue must then be provided so that these anti-tumor T cells and other tumoricidal effector cells (i.e. NK cells, PMNs, eosinophils, basophils) can be recruited into residual tumor sites to promote destruction of tumor-apoptosis and tumorassociated vasculature. This damage again provides a rich source of tumor antigen, that once acquired by tumor-associated and/or recruited DCs, may result in reiterated rounds of ‘cross-priming’ and ‘epitope spreading’ within the host T cell repertoire. Such an evolving cascade of multi-specific CD4 and CD8 anti-tumor T cell reactivity would be anticipated to result in a more effective systemic clearance of existing disease and result in durable protective immunity. Hence combinational cytokine gene therapy, particularly those approaches that consider the temporal staging of delivery to accommodate the mechanism by which cellular anti-tumor is promoted, may ultimately prove most effective in the clinic. Based on the observation that TH1-type immunity appears most effective in promoting tumor regression in situ, TH1-biasing cytokines such IFN-alpha, IL-12 and IL-18 all represent very attractive candidate cytokines for use in gene therapy approaches for cancer (Sinigaglia et al., 1999a; Adorini, 1999; Dinarello 2000; O’Garra and Arai, 2000). Of note, IFN-alpha is produced principally by human and murine ‘plasmacytoid’ DC subsets in response to viral and bacterial infections (Pulendran et al., 2000; Hochrein et al., 2001), while IL-12 and IL-18 are produced principally by human ‘myeloid’ and murine ‘lymphoid’ DCs after cognate interaction with CD4 T cells (Wilson et al., 1997; Stoll et al., 1998; Rissoan et al., 1999; Mantovani et al., 2000). Interestingly, each of these cytokines synergizes with the others in promoting increased expression of each other’s receptors on treated T cells, and in particular TH1 clones appear to be predominantly impacted in this respect. Hence, IFN-alpha

and IL-18 each increase IL-12 receptor expression on TH1 CD4 T cells (Sinigaglia et al., 1999b; Wu et al., 2000), IFN-alpha and IL-12 increase IL-18 receptor expression (Dinarello et al., 1998; Sareneva et al., 2000). This mechanism, in part, may serve to explain the noted synergy in driving TH1-type immunity that we have observed in preliminary studies for these cytokines when applied in combinations (Osaki et al., 1999; Tueting et al., 1999b; Tanaka et al., 2000). Remarkably, recent evidence from our group also suggests that even TH2-biased immunity that may prevent effective immunotherapy in cancer bearing patients (Goto et al., 1999; Filella et al., 2000; Yoshino et al., 2000) can be converted to mixed TH1/TH2 (i.e. TH0) immunity in the presence of IL-12 and potentially IFN-alpha and IFN-alpha and IL-18 (Kalinski et al., 2000). This provides increased enthusiasm that such immune deviation may be corrected in patients, allowing for enhanced therapeutic benefit. Intratumoral delivery of IFN-alpha, IL-12 and IL-18 can all lead to local inflammatory reactions, to the activation of the tumor-associated endothelium, and to the production of chemokines that recruit and maintain TH1 CD4 T cells, CD8 CTL, activated NK cells and DCs into/within the treated tumor microenvironment (Yu et al., 1997; Tannenbaum et al., 1998; Yao et al., 1999; Okada et al., 2000).

Autoimmune diseases In contrast to cancer therapies, the treatment of autoimmune diseases by gene transfer focuses on the inhibition, not the stimulation, of the immune response. Gene transfer has been reported to be therapeutic for the treatment of an animal models of arthritis, type I diabetes, lupus, multiple sclerosis, as well as models of inflammation (Chernjovsky et al., 1995; Bertera et al., 1999; Bandara et al., 1993; Giannonkakis et al., 1999a, 1999b; Croxford et al., 2000). In general, the focus of the use of cytokine gene transfer to inhibit autoimmune diseases has focused on the delivery of anti-inflammatory cytokines, such as IL-1Ra, to down-regulate expression of the proinflammatory cytokines IL-1 and TNF. Alternatively, gene transfer of immunosuppressive cytokines such as IL-10 that are able to block activation of DC, rendering the DC tolerogenic, appears able to confer inhibition of antigen-specific immune response

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(Lechman et al., 1999; Whalen et al., 1999). Gene transfer of immunosuppressive or anti-inflammatory cytokines can be injected directly at the site of the immune response. For example, for the treatment of arthritis, the therapeutic cytokines can be delivered intra-articularly where they can inhibit many of the pathologies associated with disease (Bandara et al., 1993; Evans and Robbins, 1997; Ghivizzani et al., 1998; Kim et al., 2000). Alternatively, antigen-specific T cells can be genetically modified in culture to express the immunosuppressive cytokines that are then able to home to sites of disease following systemic administration (Chernajovsky et al., 1995; Mageed et al., 1998; Nakajima et al., 2001; Shaw et al., 1997). Modification of T cells to express IL-4, IL-10 or a soluble TNF receptor has been shown to be therapeutic for the treatment of murine EAE and arthritis. The modified T cells are able to home to either brain lesions or joints, respectively, where expression of the cytokine is able to reduce disease pathology. Similarly, dendritic cells can be modified in culture to express antiinflammatory or immunosuppressive genes that can then modify the immune response in the lymph nodes or spleen following injection (Whalen et al., 2001). Systemic injection of DC, genetically modified by adenoviral infection to express IL-4, has been shown to result in the complete suppression of collagen-induced arthritis in the rabbit (Kim et al., 2000). The advantage to using genetically modified DC is that a single treatment has been shown to confer long-lasting suppression of immune disease in animal models of arthritis and diabetes. In studies designed to treat arthritis by local, intraarticular gene transfer, it was observed that local adenovirus-mediated gene transfer of viral IL-10 to rabbit knee joints with antigen-induced arthritis ameliorated disease, not only within the treated joints, but also significantly reduced all associated pathologies in the contralateral joints as well. In addition, peri-articular administration of adenovirus expressing vIL-10 into one paw of mice immunized with collagen type II prevented the development of collagen-induced arthritis not only in the injected paw, but also in non-treated ipsilateral and contralateral paws (Whalen et al., 1999). Thus the observation that local treatment of one joint results in a therapeutic effect in the contralateral joint was termed the ‘contralateral effect’. We have proposed a model

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where local intra-articular gene delivery is able to functionally alter the activity of a subset of immunoregulatory cells, APCs, that in turn can downmodulate the immune response at distant sites in an antigen-specific manner. In support of this model, we have recently demonstrated that local injection of Ad.vIL-10 into inflamed mouse paws could generate APC that could inhibit local and distal DTH reactions in recipient mice following adoptive transfer (Whalen et al., 2001). The anti-inflammatory effect of the adoptively transferred APC was only mediated when the donor mice had been sensitized to the antigen used to incite the DTH response in the recipient mice. Moreover, bone marrow-derived DC cultured in the presence of recombinant IL-10 or infected ex vivo with Ad.vIL-10 were shown to suppress DTH reactions by adoptive transfer, but only when pulsed with the inciting antigen (Whalen et al., 2001). Together, these data support the role of antigen-primed APC in the down-regulation of both local and distal joint inflammation. These results also suggest that local treatment of autoimmune or inflammatory diseases by gene transfer is able to confer a systemic therapeutic effect that could be useful for the treatment of arthritis, diabetes and multiple sclerosis.

Transplantation Similar to the treatment of autoimmune disease, cytokine gene transfer can be used to block the immune response to transplanted tissues. Given that transplantation requires the isolation of tissue and cells from a donor followed by transplantation into a recipient, ex vivo gene transfer to the isolated tissue can be readily performed. Thus the allografted cells or tissue can be modified to express immunosuppressive or anti-inflammatory cytokines, resulting in local expression following transplantation. The genetic modification of tissues to express IL-1Ra or IL-10 has been shown to result in longer survival of the transplanted organ. Alternatively, administration of donorderived DC, treated with recombinant cytokines in culture to render them more tolerogenic through the down-regulation of costimulatory molecules, has improved graft survival. Moreover, the donor-derived DC can be genetically modified to express immunosuppressive agents, such as IL-10 or IL-4 that are able to inhibit the CTL response to the allografted tissues.

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Vaccines The importance of cellular immune responses in the control of several infectious diseases has been recognized (Rowland-Jones et al., 1998; Ogg et al., 1998). Such observations provide justification for the development of DNA-based vaccines, through viral vectors or naked plasmid DNA, which induce potent cellular immune responses. Co-administration of cytokines with DNA vaccines has the potential to direct such a response. As an example interleukin-12 (IL-12) and interleukin-18 (IL-18), like IFNc, promote the development of cellular immune responses and the two cytokines may synergize to enhance production of IFNc and other cytokines, so amplifying NK cell and T-lymphocyte responses (Micallef et al., 1996; Okamoto et al., 1999). The use of IL-12 and IL-18 in combination as adjuvants may therefore prove more effective than IFNc alone. Hence a large number of reports demonstrate the ability of these cytokines to enhance immune responses to pathogens including HIV-1, FIV (Gherardi et al., 1999; Billaut-Mulot et al., 2000; Hanlon et al., 2001; Dunham et al., 2002), herpes simplex virus (Sin et al., 1999), Mycobacteria (Lindblad et al., 1997) and Leishmania (Afonso et al., 1994). Other cytokines such granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2) and interleukin-15 (IL-15) have been extensively tested as adjuvants of DNA vaccination. GMCSF have shown in the context of HIV, HCV and malaria mouse vaccine model to elicit CTL responses (Ou-Yang et al., 2002; Moore et al., 2002), remarkably potent CD4() T cell responses (Barouch et al., 2002; Baruch and Letvin, 2000) and increase in antigenspecific antibodies (Hoffman and Doolan, 2000; Ou-Yang et al., 2002) further suggesting that optimal TH cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF. Several reports have also demonstrated IL-2 and IL-15 to augment DNA vaccine-mediated antibody and CTL responses against HIV, SIV and HSV in various animal models (Xin et al., 1999; Inoue et al., 2001).

Examining the role of cytokines in disease by gene transfer In addition to using gene transfer as therapeutic strategies for treating disease, gene transfer can also

be used to dissect the role of specific cytokines in disease. For example, there are several animal models of arthritis where a large number of cytokines have been implicated in disease progression. In the rabbit and murine models of arthritis, the disease is usually induced by immunization to an antigen followed by induction of disease through subsequent injection of the antigen. The role of specific cytokines is determined by blocking the activity of the cytokines through the use of antibodies, small molecules or soluble receptors. However, this approach does not necessarily determine the role of a specific cytokine in disease. Thus, we and others have used gene transfer of specific disease-associated cytokines to examine the specific pathologies induced. For example, we have used gene transfer to examine the role of specific cytokines in the intra-articular pathologies of arthritis. Intra-articular injection of cells, expressing high levels of IL-1b, resulted in many of the pathologies associated with rheumatoid arthritis. Elevated intraarticular IL-1b resulted in an increase in the white blood cell infiltrate, as well as synovial proliferation. In addition, germinal centers were observed in the synovial pannus 2 weeks following injection of the IL-1b expressing cells. There was also an increase in cartilage degradation and bone erosion, similar to human disease. Moreover, intra-articular IL-1b expression resulted in systemic effects, including an increased erythrocyte sedimentation rate (ESR), a decrease in body weight and an increase in body temperature. Thus increased intra-articular expression of a single cytokine was able to confer significant intraarticular and systemic pathologies that are similar to those observed during the course of human disease. Given that the IL-1b rabbit knee model of arthritis is more reproducible and faster than antigen-induced arthritis, but with the same pathologies, it represents an attractive model for testing anti-arthritic therapies.

Identification of cytokine function by gene transfer The recent completion of the human genome sequence is now allowing for the identification of additional cytokine family members. For instance, there are now eight members of the IL-1 family of cytokines, IL-1b, IL-1alpha, IL-1Ra, IL-18, IL-1H1, IL-1H2, IL-1H3 and IL-1H4. In addition, there are

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REFERENCES

multiple IL-10 family members including IL-19, IL-20, IL-22 and IL-24. Previously, identification and characterization of cytokines involved first identifying an activity followed by purification of the factor, protein sequencing and eventually cloning of the gene. However, there are now many cytokine family members where the gene has been cloned, but the activity of the putative cytokine is unknown. Recombinant protein can be generated, but without an assay to characterize the cytokine it is difficult to demonstrate that the purified recombinant protein is functional. Gene transfer is one approach that can facilitate the identification of biological activities of new putative cytokines, as well as demonstrate a possible therapeutic application. For example, we have been trying to determine the possible activities of the IL-1b family members. Recombinant adenoviral vectors have been generated expressing each of the new family members that were then tested in different animal models of disease. Interestingly, treatment of an established MCA205 mouse fibrosarcoma by single intratumoral injection of Ad-hIL-1H4 resulted in significant growth suppression (Gao et al., 2002). Furthermore, complete inhibition of tumor growth was observed following multiple injections of Ad-hIL-1H4. The anti-tumor activity of human IL-1H4 was abrogated in nude and SCID mice and in IL-12, IFNc or Fas ligand-deficient mice. In contrast, IL-1H4 was able to confer an antitumor effect in NKT-deficient mice. These results suggest that IL-1H4 could play an important role in the link between innate and adaptive immunity and may be useful for tumor immunotherapy.

SUMMARY Gene transfer has played a significant role in understanding the biological activity of cytokines, the role of cytokines in disease and allowed for the development of clinical approaches for treating a wide variety of diseases. Gene transfer of cytokines to cells in culture or in vivo can be accomplished by a variety of gene transfer vectors, including both viral- and nonviral-based vectors. For specific gene transfer applications, certain vectors have their own advantages and disadvantages. The majority of phase I clinical trials using cytokine gene transfer have not shown significant efficacy. However, it is likely that as we learn

more about the biology of cytokines, especially how multiple cytokines work synergistically to regulate the immune response, it is likely that cytokine gene therapy will play a significant role in the treatment of many human diseases.

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60 Antisense therapy Stanley T. Crooke Isis Pharmaceuticals, Inc., Carlsbad, CA, USA

Be careful about reading health books. You may die of a misprint. Mark Twain

INTRODUCTION During the past decade, antisense technology has matured. Today, antisense technology is generally accepted as a broadly useful method for gene functionalization and target validation. Further, with the approval by regulatory agencies around the world of Vitravene™, the first antisense drug to be commercialized, the emerging data showing activity of a number of antisense drugs in clinical trials and the overwhelming evidence from studies in animals, the potential of antisense as a therapeutic technology is now better appreciated. Antisense technology exploits oligonucleotide analogs (typically 15–20 nucleotides) to bind to cognate RNA sequences through Watson–Crick hybridization resulting in the destruction or disablement of the target RNA. Thus antisense technology represents a ‘new pharmacology’. The receptor, mRNA, has never before been considered in the context of drug receptor interactions. Prior to the advent The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

of antisense technology, no medicinal chemistry had been practised on the putative ‘drugs’, oligonucleotides. The basis of the drug receptor interaction, Watson–Crick hybridization, had never been considered as a potential binding event for drugs and put into a pharmacological context. Finally, postbinding events, such as recruitment of nucleases to degrade the receptor RNA, had never been considered from a pharmacological perspective. A key to understanding antisense technology is to consider it in pharmacological context. It is essential to understand the structure, function and metabolism of the receptors for these drugs. As with any of the class of drugs, it is essential to consider the effects of antisense oligonucleotides in the context of dose– response curves. It is essential to consider the future in the context of advances in antisense biology, and medicinal chemistry that result in improved pharmacological behaviors. Numerous factors may influence the interpretation of experimental results with regard to mechanism of Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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action. Many, for example oligonucleotide purity and unpredicted structures in oligonucleotides, of these factors that caused problems early in the development of the technology we now understand well enough to avoid. Others, such as the influence of RNA structure, or interactions with proteins are dramatically better understood, but information is still insufficient to support the creation of algorithms that provide clear explanations and design direction. Thus they require continued research and careful experimental interpretation (for review see Crooke 2001a). Consequently, it remains as essential with antisense drugs as another other class of drugs to characterize the mechanism of action before drawing conclusions about biological effects observed. Thus, in our laboratories we demand a positive demonstration that an antisense mechanism is the most likely mechanism to explain the biological effects observed. For RNase H activating oligonucleotides, Northern blot, RT-PCR, RNase protection assays or transcriptional array analyses showing selective loss of the target RNA are the ideal choices and many laboratories are publishing reports of such activities in vitro and in vivo (Chiang et al., 1991; Dean et al., 1994; Hijiya et al., 1994; Skorski et al., 1994). Ideally, a demonstration that closely related isotypes are unaffected should be included. In brief, then, for proof of mechanism, the following steps are recommended. 1.

2.

3. 4. 5.

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Perform careful dose response curves in vitro using several cell lines and methods of in vitro delivery. Correlate the rank order potency in vivo with that observed in vitro after thorough dose response curves are generated in vivo. Perform careful ‘gene walks’ for all RNA species and oligonucleotide chemical classes. Perform careful time courses before drawing conclusions about potency. Directly demonstrate proposed mechanism of action by measuring the target RNA and/or protein. Evaluate specificity and therapeutic indices via studies on closely related isotypes and with appropriate toxicological studies. Use RNase H protection assays and transcriptional arrays to provide broader analyses of specificity where the assays have been validated.

8.

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Perform sufficient pharmacokinetics to define rational dosing schedules for pharmacological studies. When control oligonucleotides display surprising activities, determine the mechanisms involved.

MOLECULAR MECHANISMS OF ANTISENSE DRUGS Today, a wide variety of mechanisms of action by which antisense drugs may work have been identified, including inhibition of key steps in RNA processing, such as 5 capping or 3 polyadenylation, inhibition of translation, cleavage of the target RNA by double-strand RNases and others (for review see Crooke, 1998b, 2001a). However, two mechanisms of action have been exploited most extensively for the development of antisense and inflammatory agents, activation of RNase H and inhibition of splicing, so these will be discussed.

Activation of RNase H RNase H is a ubiquitous enzyme that degrades the RNA strand of an RNA–DNA duplex. It has been identified in organisms as diverse as viruses and human cells (Crouch and Dirksen, 1985). At least two classes of RNase H have been identified in eukaryotic cells. Multiple enzymes with RNase H activity have been observed in prokaryotes (Crouch and Dirksen, 1985). Although RNase H is involved in DNA replication, it may play other roles in the cell and is found in the cytoplasm, as well as the nucleus (Crum et al., 1988). However, the concentration of the enzyme in the nucleus is thought to be greater and some of the enzyme found in cytoplasmic preparations may be due to nuclear leakage. The precise recognition elements for RNase H are not known. However, it has been shown that oligonucleotides with DNA-like properties, as short as tetramers can activate RNase H (Donis-Keller, 1979). Changes in the sugar influence RNase H activation as sugar modifications that result in RNA-like oligonucleotides, e.g. 2-fluoro or 2-methoxy do not appear to serve as substrates for RNase H (Sproat et al., 1989; Kawasaki et al., 1993). Alterations in the orientation of

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the sugar to the base can also affect RNase H activation as a-oligonucleotides are unable to induce RNase H or may require parallel annealing (Gagnor et al., 1989; Morvan et al., 1991). Additionally, backbone modifications influence the ability of oligonucleotides to activate RNase H. Methylphosphonates do not activate RNase H (Maher et al., 1989; Miller, 1989). In contrast, phosphorothioates are excellent substrates (Cazenave et al., 1989; Mirabelli et al., 1991; Stein and Cheng, 1993). In addition, chimeric molecules have been studied as oligonucleotides that bind to RNA and activate RNase H (Furdon et al., 1989; Quartin et al., 1989). For example, oligonucleotides comprised of wings of 2-methoxy phosphonates and a five-base gap of deoxyoligonucleotides bind to their target RNA and activate RNase H (Furdon et al., 1989; Quartin et al., 1989). Furthermore, a single ribonucleotide in a sequence of deoxyribonucleotides was shown to be sufficient to serve as a substrate for RNase H when bound to its complementary deoxyoligonucleotide (Eder and Walder, 1991). That it is possible to take advantage of chimeric oligonucleotides designed to activate RNase H and have greater affinity for their RNA receptors and to enhance specificity has also been demonstrated (Giles and Tidd, 1992; Monia et al., 1993). In one study, RNase H-mediated cleavage of target transcript was much more selective when deoxyoligonucleotides comprised of methylphosphonate deoxyoligonucleotide wings and phosphodiester gaps were compared with full phosphodiester oligonucleotides (Giles and Tidd, 1992). Despite the information about RNase H and the demonstration that many oligonucleotides may activate RNase H in lysate and purified enzyme assays, relatively little is yet known about the role of structural features in RNA targets in activating RNase H (Minshull and Hunt, 1986; Gagnor et al., 1987; Walder and Walder, 1988). In fact, direct proof that RNase H activation is, in fact, the mechanism of action of oligonucleotides in cells has, until very recently, been lacking. Recent studies in our laboratories provide additional, albeit indirect, insights into these questions. ISIS 1939 is a 20 mer phosphorothioate complementary to a sequence in the 3-untranslated region of ICAM-1 RNA (Chiang et al., 1991). It inhibits ICAM production in human umbilical vein endothelial cells

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and Northern blots demonstrate that ICAM-1 mRNA is rapidly degraded. A 2-methoxy analog of ISIS 1939 displays higher affinity for the RNA than the phosphorothioate, is stable in cells, but inhibits ICAM-1 protein production much less potently than ISIS 1939. It is likely that ISIS 1939 destabilizes the RNA and activates RNase H. In contrast, ISIS 1570, an 18-mer phosphorothioate that is complementary to the translation initiation codon of the ICAM-1 message inhibited production of the protein, but caused no degradation of the RNA. Thus, two oligonucleotides that are capable of activating RNase H had different effects depending on the site in the mRNA at which they bound (Chiang et al., 1991). A more direct demonstration that RNase H is likely a key factor in the activity of many antisense oligonucleotide was provided by studies in which reverse-ligation PCR was used to identify cleavage products from bcrabl mRNA in cells treated with phosphorothioate oligonucleotides (Crooke et al., 1995). Given the emerging role of chimeric oligonucleotides with modifications in the 3- and 5-wings designed to enhance affinity for the target RNA and nuclease stability and a DNA-type gap to serve as a substrate for RNase H, studies focused on understanding the effects of various modifications on the efficiency of the enzyme(s) are also of considerable importance. In one such study on E. coli RNase H, we have reported that the enzyme displays minimal sequence specificity and is processive. When a chimeric oligonucleotide with 2-modified sugars in the wings was hybridized to the RNA, the initial site of cleavage were the nucleotide adjacent to the methoxy–deoxy junction closest to the 3-end of the RNA substrate. The initial rate of cleavage increased as the size of the DNA gap increased and the efficiency of the enzyme was considerably less against an RNA target duplexed with a chimeric antisense oligonucleotide than a full DNA-type oligonucleotide (Crooke et al., 1995). In subsequent studies, we have evaluated the interactions of antisense oligonucleotides with structured and unstructured targets, and the impacts of these interactions on RNase H in more detail (Lima and Crooke, 1997). Using a series of non-cleavable substrates and Michaelis-Menten analyses, we were able to evaluate both binding and cleavage. We showed that, in fact, E. coli RNase H1 is a double-strand RNA

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binding protein. The Kd for the RNA duplex was 1.6 lM; the Kd for a DNA duplex was 176 lM; and the Kd for single-strand DNA was 942 lM. In contrast, the enzyme could only cleave RNA in an RNA–DNA duplex. Any 2-modification in the antisense drug at the cleavage site inhibited cleavage, but significant charge reduction and 2-modifications were tolerated at the binding site. Finally, placing a positive charge (e.g. 2-propoxyamine) in the antisense drug reduced affinity and cleavage. We have also examined the effects of antisense oligonucleotideinduced RNA structures on the activity of E. coli RNase H1 (Lima and Crooke, 1997). Any structure in the duplex substrate was found to have a significant negative effect on the cleavage rate. Further, cleavage of selected sites was inhibited entirely, and this was explained by steric hindrance imposed by the RNA loop traversing either the minor or major grooves or the heteroduplex. Recently we have cloned and expressed human RNase H1. The protein is homologous to E. coli RNase H1, but has properties similar to those described for human RNase H type 2 (Frank et al., 1994; Wu et al., 1998). The enzyme is stimulated by low concentrations of Mg2, inhibited by higher concentrations, and is inhibited by Mn2 in the presence of Mg2. It is 33 kDa in molecular weight. It is double-strand RNA binding protein and exhibits unique positional and sequence preferences for cleavage (Wu et al., 1999). Additionally, human RNase H2 has been cloned, but to date the expressed protein has not been shown to be active (Frank et al., 1998). Very recently, we have reported on the effects of several mutations introduced into human RNase H1 on the activity of the enzyme (Wu et al., 2000). Thus, we now have the necessary tools to begin to explore the roles of human RNase H in biological and pharmacological processes and to begin to develop drugs designed to interact with them more effectively. Finally, at least with regard to RNase H-induced degradation of targeted RNA, we have recently demonstrated that in the range of 1–150 copies of RNA per cell, the level of target RNA has no effect on the potency of antisense inhibitors (Miraglia, 2000). This is due to the fact that the number of molecules of antisense drug per cell exceeds the number of copies of RNA by several orders of magnitude. Thus, other factors must be rate limiting.

Inhibition of splicing A key step in the intermediary metabolism of most mRNA molecules is the excision of introns. These ‘splicing’ reactions are sequence-specific and require the concerted action of spliceosomes. Consequently, oligonucleotides that bind to sequences required for splicing may prevent binding of the necessary factors or physically prevent the required cleavage reactions. This then would result in inhibition of the production of the mature mRNA. Activities have been reported for anti-c-myc and antiviral oligonucleotides with phosphodiester, methylphosphonate and phosphorothioate backbones (Smith et al., 1986; Zamecnik et al., 1986; Kulka et al., 1989; McManaway et al., 1990). Kole and colleagues (Dominski and Kole, 1993, 1994; Sierakowska et al., 1996) were the first to use modified oligonucleotides to inhibit splicing. They showed that 2-MOE phosphorothioate oligonucleotides could correct aberrant beta-globin splicing in a cell-free system. Similar observations were in a cellular system (Sierakowska et al., 1996). In our laboratory, we have attempted to characterize the factors that determine whether splicing inhibition is effected by an antisense drug (Hodges and Crooke, 1995). To this end, a number of luciferasereporter plasmids containing various introns were constructed and transfected into HeLa cells. Then the effects of antisense drugs designed to bind to various sites were characterized. The effects of RNase Hcompetent oligonucleotides were compared with those of oligonucleotides that do not serve as RNase H substrates. The major conclusions from this study were: first, that most of the earlier studies in which splicing inhibition was reported were probably due to non-specific effects. Second, less effectively spliced introns are better targets than those with strong consensus splicing signals. Third, the 3-splice site and branchpoint are usually the best sites to which to target to the oligonucleotide to inhibit splicing. Several studies have now demonstrated antisense-mediated redirection of slicing of an endogenous cellular mRNA. In one study, 2-O-methoxyethyl oligonucleotides redirected splicing of IL-5 receptor pre-mRNA (Karras et al., 2000). Similar results were reported for antisense agents designed to bind to Bclx pre-mRNA (Taylor, 1999; Mercatante et al., 2001). Thus, antisensemediated alternative splicing is a potentially powerful

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CHARACTERISTICS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES

tool with which to investigate this important source of biological diversity, and to create focused therapies for diseases caused by disorders in splicing.

THE MEDICINAL CHEMISTRY OF OLIGONUCLEOTIDES Because it was apparent almost immediately that native phosphodiester oligodeoxy- or ribo-nucleotides are unsatisfactory as drugs because of rapid degradation (Wickstrom, 1986), a variety of modifications were rapidly tested. As previously mentioned, perhaps the most interesting of the initial modifications were the phosphate analogs, the phosphorothioates, (De Clercq et al., 1969) and the methylphosphonates (Ts’o et al., 1983). Both fully modified oligonucleotides and oligonucleotides ‘capped’ at the 3- and/or 5termini with phosphorothioate or methylphosphonate moieties were tested (Tidd and Warenius, 1989). However, studies from many laboratories demonstrated that capped oligonucleotides were relatively rapidly degraded in cells (Dagle et al., 1990, 1991; Crooke, 1991). Nor were point modifications with intercalators that enhanced binding to RNA (Asseline et al., 1984; Cazenave et al., 1989), cholesterol (Letsinger et al., 1989), or poly-L-lysine (Lemaitre et al., 1987; Leonetti et al., 1988) sufficiently active or selective to warrant broad-based exploration. Since the initial approaches to modifications of oligonucleotides, an enormous range of modifications, including novel bases, sugars, backbones, conjugates and chimeric oligonucleotides have been tested (for review see Crooke, 1998a). Many of these modifications have proven to be quite useful and are progressing in testing leading to clinical trials. 2-O(2-methoxyethyl) chimeric antisense inhibitors are, in fact, now in clinical trials.

CHARACTERISTICS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES Of the first-generation oligonucleotide analogs, the class that has resulted in the broadcast range of activities and about which most is known, is the phosphorothioate class. Phosphorothioate oligonucleotides

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were first synthesized in 1969 when a poly (rlrC) phosphorothioate was synthesized. This modification clearly achieves the objective of increased nuclease stability. In this class of oligonucleotides, one of the oxygen atoms in the phosphate group is replaced with a sulfur. The resulting compound is negatively charged, is chiral at each phosphorothioate phosphodiester, and is much more resistant to nucleases than the parent phosphorothioate (Cohen, 1993). These oligonucleotides hybridize to RNA with an affinity slightly lower per nucleotide unit than DNA. Affinity is measured by thermal melting of hybrids and is reported as the temperature at which 50% of a population of duplexes is disrupted (tm). Phosphorothioates are approximately 0.5°C per nucleotide less stable than corresponding oligodeoxynucleotides or about 10°C per molecule as most antisense drugs are 20 nucleotides long (for review see Crooke, 2001a).

Interactions with proteins Phosphorothioate oligonucleotides bind to proteins. The interactions with proteins can be divided into non-specific, sequence-specific and structure-specific binding events, each of which may have different characteristics and effects. Non-specific binding to a wide variety of proteins has been demonstrated. Exemplary of this type of binding is the interaction of phosphorothioate oligonucleotides with serum albumin. The affinity of such interactions is low. The Kd for albumin is approximately 200 lM, thus, in a similar range with aspirin or penicillin (Joos and Hall, 1969; Crooke et al., 1996b). Furthermore, in this study, no competition between phosphorothioate oligonucleotides and several drugs that bind to bovine serum albumin was observed. These drugs interact with many other proteins with low affinity and a few with higher affinity. Among the more important protein interactions are interactions with nucleases and polymerases (for review see Crooke, 2001a). These interactions may influence the pharmacokinetic, pharmacologic and toxicologic properties of these molecules. They may also complicate studies on the mechanism of action of these drugs, and may, in fact, obscure an antisense activity. For example, phosphorothioate oligonucleotides were reported to enhance lipopolysacchoride stimulated synthesis or tumor

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necrosis factor (Hartmann et al., 1996). This would obviously obscure antisense effects on this target.

In vitro cellular uptake Phosphorothioate oligonucleotides are taken up by a wide range of cells in vitro (Crooke, 1991, 1993b, 1995; Gao et al., 1992; Neckers, 1993). In fact, uptake of phosphorothioate oligonucleotides into a prokaryote, Vibrio parahaemoyticus, has been reported, as has uptake into Schistosoma mansoni (Chrisey et al., 1993; Tao et al., 1995). Uptake is time- and temperaturedependent. It is also influenced by cell type, cellculture conditions, media and sequence, and length of the oligonucleotide (Crooke, 1995). No obvious correlation between the lineage of cells, whether the cells are transformed or whether the cells are virally infected, and uptake has been identified (Crooke, 1995). Nor are the factors that result in differences in uptake of different sequences of oligonucleotide understood. Although several studies have suggested that receptor-mediated endocytosis may be a significant mechanism of cellular uptake, the data are not yet compelling enough to conclude that receptormediated endocytosis accounts for a significant proportion of the uptake in most cells (Loke et al., 1989). Numerous studies have shown that phosphorothioate oligonucleotides distribute broadly in most cells once taken up (Crooke, 1993a, 1995). Again, however, significant differences in subcellular distribution between various types of cells have been noted. Cationic lipids and other approaches have been used to enhance uptake of phosphorothioate oligonucleotides in cells that take up little oligonucleotide in vitro (Bennett et al., 1992, 1993; Quattrone et al., 1994). Again, however, there are substantial variations from cell type to cell type. Other approaches to enhanced intracellular uptake in vitro have included streptolysin D treatment of cells and the use of dextran sulfate and other liposome formulations, as well as physical means such as microinjections (Crooke, 1995; Giles et al., 1995; Wang et al., 1995).

In vivo pharmacokinetics Phosphorothioate oligonucleotides bind to serum albumin and a-2 macroglobulin. The apparent affinity

for albumin is quite low (200–400 lM) and comparable to the low affinity binding observed for a number of drugs, e.g. aspirin and penicillin (Joos and Hall, 1969; Srinivasan et al., 1995; Crooke et al., 1996a). Serum protein binding, therefore, provides a repository for these drugs and prevents rapid renal excretion. As serum protein binding is saturable, at higher doses, intact oligomer may be found in urine (Agrawal et al., 1991; Iversen, 1991). Studies in our laboratory suggest that in rats, oligonucleotides administered intravenously at doses of 15–20 mg kg1 saturate the serum protein binding capacity (Yu et al., 2000). Phosphorothioate oligonucleotides are rapidly and extensively absorbed after parenteral administration. For example, in rats, after an intradermal dose 3.6 mg kg1 of 14C-ISIS 2105, a 20-mer phosphorothioate, approximately 70% of the dose was absorbed within 4 h and total systemic bioavailability was in excess of 90% (Cossum et al., 1994). After intradermal injection in man, absorption of ISIS 2105 was similar to that observed in rats (Crooke, et al., 1994). Subcutaneous administration to rats and monkeys results in somewhat lower bioavailability and greater distribution to lymph nodes, as would be expected (Leeds et al., 2000). Distribution of phosphorothioate oligonucleotides from blood after absorption or intravenous administration is extremely rapid. We have reported distribution half-lives of less than 1 hour, and similar data have been reported by others (Agrawal et al., 1991; Iversen, 1991; Cossum et al., 1993, 1994). Blood and plasma clearance is multi-exponential with a terminal elimination half-life from 40 to 60 h in all species except man. In man, the terminal elimination half-life may be somewhat longer (Crooke et al., 1994). Phosphorothioates distribute broadly to all peripheral tissues. Liver, kidney, bone marrow, skeletal muscle and skin accumulate the highest percentage of a dose, but other tissues display small quantities of drug (Cossum et al., 1993, 1994). No evidence of significant penetration of the blood–brain barrier has been reported. The rates of incorporation and clearance from tissues vary as a function of the organ studied, with liver accumulating drug most rapidly (20% of a dose within 1–2 hour) and other tissues accumulating drug more slowly. Similarly, elimination of drug relatively rapid from liver compared with many other tissues, e.g. terminal half-life from liver: 62 hour; from

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renal medulla: 156 hour. The distribution into the kidney has been studied more extensively and drugs are shown to be present in Bowman’s capsule, the proximal convoluted tubule, the bush border membrane and within renal tubular epithelial cells (Rappaport et al., 1995). The data suggested that the oligonucleotides are filtered by the glomerulus, then reabsorbed by the proximal convoluted tubule epithelial cells. Moreover, the authors suggested that reabsorption might be mediated by interactions with specific proteins in the bush border membranes. In addition, oligonucleotide is accumulated in a non-filtering kidney, suggesting that there is uptake from the basal side also. Clearance of phosphorothioate oligonucleotides is due primarily to metabolism (Iverson, 1992; Cossum et al., 1993, 1994). Metabolism is mediated by exoand endonucleases that result in shorter oligonucleotides and, ultimately, nucleosides that are degraded by normal metabolic pathways. Although no direct evidence of base excision or modification has been reported, these are theoretical possibilities that may occur. In one study, a larger molecular weight radioactive material was observed in urine, but not fully characterized (Agrawal et al., 1991). Clearly, the potential for conjugation reactions and extension of oligonucleotides via these drugs serving as primers for polymerases must be explored in more detail. In a very thorough study, 20 nucleotide phosphodiester and phosphorothioate oligonucleotides were administered intravenously at a dose of 6 mg kg1 to mice. The oligonucleotides were internally labeled with 3H-CH3 by methylation of an internal deoxycytidine residue using HhaI methylase and S-(3H)-adenosyl methionine (Sands et al., 1994). The pharmacokinetic properties observed were consistent with our results as described above. Additionally, in this paper, autoradiographic analyses showed drug in renal cortical cells (Sands et al., 1994). In a recent study in humans in which the level of intact drug was carefully evaluated using capillary gel electrophoresis, the pharmacokinetics of ISIS 2302, a 20-mer phosphorothioate oligodeoxynucleotide, after a 2 hour infusion, were determined. Doses from 0.06 mg kg1 to 2.0 mg kg1 were studied and the peak plasma concentrations were shown to increase linearly with dose with the 2 mg kg1 dose resulting in peak plasma concentrations of intact drug of approx-

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imately 9.5 lg ml1. Clearance from plasma, however, was dose-dependent with the 2 mg kg1 dose having a clearance of 1.28 ml min1 kg1, while that of 0.5 mg kg1 was 2.07 ml min1 kg1. Essentially, no intact drug was found in urine. In addition to the pharmacological effects that have been observed after phosphorothioate oligonucleotides have been administered to animals (and humans), a number of other lines of evidence show that these drugs enter cells in organs. Autoradiographic, fluorescent and immunohistochemical approaches have shown that these drugs are localized in endopromal convoluted tubular cells, various bone marrow cells, cells in the skin and liver (Rappaport et al., 1995; Takakura et al., 1996; Butler et al., 1997) (for review see Levin et al., 2001).

Aerosol administration Phosphorothioate oligodeoxynucleotides have been shown to be attractive for inhalation delivery to the lung and upper airway (Nyce, 1997, 2001; Templin et al., 2000). Target reduction in the lung has been demonstrated (Nyce and Metzger, 1997) and these drugs have been shown to distribute broadly to all cell types in the lung after aerosol administration. Further, these drugs were shown to be well tolerated at doses up to 12 mg kg1 (Templin et al., 2000).

Topical administration Phosphorothioate oligonucleotides formulated in very simple cream formulation have been shown to penetrate normal mouse, pig and human skin and to penetrate and accumulate in human psoriatic skin grown on nude mice. Further, in a phase IIa study in patients with plaque psoriasis, ISIS 2302, in ICAM-1 inhibitor, was shown to accumulate throughout the dermis and epidermis after topical administration and to result in a positive trend in the primary endpoint, induration (Kruger, unpublished observations). Thus, studies have demonstrated substantial accumulation of drug throughout the dermis and epidermis and reduction of targets, such as ICAM-1 and B71 and B72 (for review see Hardee et al., 2001).

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Toxicological properties Complement activation Rapid infusion of phosphorothioate oligodeoxynucleotides to nonhuman primates can result in cardiovascular collapse (Cornish et al., 1993; Galbraith et al., 1994; Henry et al., 1997b). These hemodynamic effects are associated with complement activation, but complement activation is necessary but not sufficient to produce the observed cardiovascular effects (for review see Levin et al., 2001). The other factors contributing to cardivascular collapse are not yet fully elucidated. However, it has been suggested that dosing monkeys that are restrained may result in exacerbating cardiovascular events. Activation of the complement cascade is due to activation of the alternate pathway is relatively insensitive to sequence, but is absolutely related to peak plasma concentration (Henry et al., 1997c). The threshold concentration for activation of complement in the monkey is 40–50 lg ml1 phosphorothioate oligodeoxynucleotide and once the threshold is reached, variable but potentially dangerous levels of complement activation are observed. Studies in man have avoided peak plasma concentrations that would induce complement. However, it appears that monkeys are substantially more sensitive to complement activation than man. A comparison of the effects on complement activation in human versus monkey serum demonstrates a dramatic difference. In monkey serum, phosphorothioate oligodeoxynucleotides activate complement in a concentration-dependent fashion. In human serum, complement activation is actually inhibited at higher concentrations of oligonucleotide (Levin et al., 2001). This is thought to be due to the sensitivity of human serum to inhibition of complement activators. The mechanism of complement activation is currently believed to be due to an interaction with factor H (Levin et al., 2001). These effects can be reduced by chemical modifications and formulations that reduce plasma protein binding (Levin et al., 2001).

Inhibition of clotting In all species studies, phosphorothioate oligodeoxynucleotides induce a transient, apparently self-

limited, peak plasma concentration-related inhibition of clotting manifested as an increase in activated partial thromboplastin time (aPTT) (Griffin et al., 1993; Wallace et al., 1996; Henry et al., 1997d; Nicklin et al., 1997). Increases in aPTT are minimally affected by sequence, but the effects are directly proportional to the length of the phosphorothioate oligodeoxynucleotide (Levin et al., 2001). The mechanisms of aPTT increase appears to an interaction with the intrinsic tenase complex (Henry et al., 1994; Sheehan and Lan, 1998). This interaction is complex and involves effects on multiple clotting factors including factors VIIIa, IXa and X. The effects on clotting are transient, appear to be self-limited and have not resulted in bleeding diatheses in animals or man. Effects on clotting can be ameliorated by chemical modifications such as 2-O-methoxyethyl substitution (Levin et al., 2001).

Immune stimulation In the rodent, the most prominent toxicity is immune stimulation. This effect is also frequently a confounding variable that must be evaluated with regard to the mechanism of action of pharmacological effects (for review see Levin et al., 1998). Subchronic administration of doses as low as 10 mg kg1 day1 in rodents results in splenomegaly, lymphoid hyperplasia and diffuse multiorgan mixed mononuclear cell infiltrates (Sarmiento et al., 1994; Henry et al., 1997a, 1997c; Monteith et al., 1998). These effects are reminiscent of stimulator effects observed on isolated splemocytes (Pisetsky and Reich, 1994; Liang et al., 1996; Lemonidis-Farrar et al., 2000). In fact these in vitro studies are reasonably predictive of the relative potencies of phosphorothioates in moving in vivo immune stimulation. Immune stimulation in the rodent is a property common to all phosphorothioate oligodeoxynucleotides, but potency varies substantially as a function of sequence. Rodent immune stimulatory motifs include palindromic sequences and CG motifs (Davis et al., 1998; Krieg, 1998). In primates, immune stimulation is far less prominent than in the rodent. In fact, in the monkey, doses necessary to produce immune stimulation have not been identified despite evaluation of numerous oligonucleotides at a variety of doses and schedules (Levin et al., 2001). At least one factor contributing to

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CHARACTERISTICS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES

these species differences is that the optimal rodent immune stimulator sequence is simple and immune stimulation is induced by all sequences. In contrast in the primate, the optimal sequences are different and more complex (Harmann and Krieg, 2000), and the response is much less promiscuous with regard to sequence. Modifications of the base, sugar and the backbone can reduce immune stimulation in the rodent. For example, 5-methycytosine and 2-Omethoxyethyl containing oligonucleotides display substantially reduced potency for immune stimulation in the rodent (Levin et al., 2001).

Other toxicities Occasionally, in the rodent but not the monkey, single cell hepatolyte neurons are observed and this has been related to immunostimulation (Levin et al., 1998). Also, on occasion, in the monkey transient thrombocytopenia is observed, perhaps associated with complement activation (for review see Levin et al., 2001). Other toxicities noted in animals are mild and occur infrequently at therapeutic doses. For example, occasional increases in liver function enzymes are noted, but these occur at high doses and are not associated with histopathological changes (Levin et al., 2001).

Human safety At Isis, we have had the opportunity to study approximately 10 phosphorothioate oligonucleotides in humans. We have studied antisense drugs administered intravitreally, intradermally, subcutaneously, intravenously and topically. Vitravene, an intravitreally administered drug is commercially available around the world. After systemic administration, in clinical trials, we have studied more than 2000 patients treated with more than 70 000 doses. I.V. doses have ranged from 0.5 mg kg1 every other day to as much as 30 mg kg1 day1 at a continuous infusion. Most patients have been treated with multiple doses and many have been treated for prolonged periods, i.e. multiple months.

Complement activation With 2-hour infusions at a dose of 2 mg kg1, no increases in complement split products were

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observed in more than 300 patients with inflammatory diseases treated with ISIS 2302, a 20 nucleotide phosphorothioate antisense inhibitor of ICAM-1. Similarly, ISIS 5132, an inhibitor of C-raf kinase was dosed from 0.5 to 6.0 mg kg1 and no meaningful increases in complement split products were observed. ISIS 3521, a PKCa inhibitor gave equivalent data and both of these drugs were studied in patients with various malignant diseases. Similar data were observed with all the phosphorothioate oligodeoxynucleotides administered on this schedule in a variety of patients (for review see Nemunaitis et al., 1999b; Dorr et al., 2001). Three phosphorothioate oligodeoxynucleotides, ISIS 3521, a PKCa inhibitor, ISIS 5132, a C-raf kinase inhibitor, and ISIS 2503, an H-ras inhibitor have been thoroughly characterized in patients with malignant diseases and dosed with long-term infusions. When given as 21-day antitumor infusions at doses as high as 10 mg kg1 day1, no increases in complement split products were observed (Holmlund et al., 1998; Sikic et al., 1998; Dorr et al., 1999). In contrast, when these drugs were given as 24 hour infusions, significant increases in complement split products were observed at doses of 18 mg kg1 day1 and greater (Dorr et al., 2001). Despite these increases in complement split products, only a small number of patients experienced mild fevers and myalgias at these very high doses.

Cytokines At very high doses, i.e. 24 mg kg1 day1 as a continuous i.v. infusion, significant increases in IL-6, IL-IRa and TNFa were observed and these often correlated with flu-like syndromes (Dorr et al., 2001). At present, the precise roles of each of the cytokines and complement activation in the clinical signs and symptoms (myalgia and fever) at high doses are not defined.

Coagulation All phosphorothioate oligodeoxynucleotides studied in normal volunteers and in patients have resulted in transient, self-limited increases in aPTT. The effects are dose- and peak plasma concentration-dependent (Dorr et al., 2001). These effects appear to be more prominent after the first dose. In no patients treated have we observed any evidence of bleeding, so

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the effects on aPTT have not proven to be a problem in the clinic and humans appear to behave similarly to other animals with regard to this side effect.

Platelet effects When phosphorothioate oligodeoxynucleotides have been administered by continuous i.v. infusion in patients with malignant diseases, transient thrombocytoplastin has been observed in a few patients (Dorr et al., 2001). These effects were more frequent during the first course of therapy, were not obviously doserelated and not associated with bone marrow effects (Nemunaitis et al., 1999a; Yuen et al., 1999). Most of the time, platelet counts returned to normal, while dosing was continued and no bleeding was observed. The mechanisms for these effects are not clear, but probably involve margination.

Other toxicities At no dose studied have significant effects on liver, kidney or other organ performance been observed (Dorr et al., 2001).

Clinical activities Vitravene, a phosphorothioate oligonucleotide, designed to inhibit cytomegalovirus-induced retinitis has been shown to be safe and effective in the treatment of this disease after intravitreal injection and has been approved for sale by regulatory agency in the USA, Europe and South America (for review see Grillone, 2001).

ISIS 2302 ISIS 2302 is a phosphorothioate oligonucleotide inhibitor of human ICAM-1. It has been demonstrated to reduce ICAM-1 levels in which a number of organs in a variety of animal models and to result in potent anti-inflammatory effects (for review see Bennett, 2001). ISIS 2302 has been evaluated in a number of clinical trials in patients with inflammatory diseases (for review, see Shanahan, 2001). The drug was evaluated in a randomized placebo-controlled phase 2a trial in 43 patients with moderate to severe rheumatoid arthritis (Maksymowych, 1999). In this trial the

effects of 0.5, 1.0 and 2.0 mg kg1 i.v. three times weekly for 1 month were compared with placebo. The 0.5 mg kg1 and 2.0 mg kg1 doses resulted in prolonged improvement in rheumatoid arthritis activity, compared with placebo. All doses were extremely well tolerated. ISIS 2302 was evaluated both by i.v. and topical routes as a potential therapeutic for patients with psoriasis, the dosing for the i.v. study was equivalent to that used for the rheumatoid arthritis study, but only 17 patients were evaluated. Again, all doses were well tolerated. At the end of the treatment period, an approximately 30% reduction on psoriasis activity index score (a measure of psoriasis activity) was reported (Fredriksson and Pettersson, 1978). Additionally, several concentrations of ISIS 2302 were evaluated in patients with moderate plaque psoriasis. In this study, the drug was applied topically in a simple cream formulation once a day for 3 months. The patients were followed for a total of 6 months. In approximately half the patients, skin biopsies were taken. The study showed that ISIS 2302 achieved very high dermal and epidermal concentrations that increased as the concentration in the cream was increased with 2% and 4% creams given dermal concentrations similar to those at which pharmacological effects were observed in animals. Further, there was a trend (P  0.053) in favor of increasing ISIS 2302 concentrations with regard to the primary endpoint, induration. Again the drug was very well tolerated. Additional studies with topical ISIS 2302 are in progress. ISIS 2302 has been evaluated most extensively in Crohn’s disease. In the initial placebo controlled study in steroid-dependent patients, ISIS 2302 resulted in a substantial reduction in symptoms and a dramatic reduction in steroid use and reduction of ICAM-1 levels in the small intestines of patients treated and evaluated with serial colonoscopies. The duration of effect after a month of dosing was in excess of 6 months. Further, serial colonoscopic biopsies demonstrated reduction of ICAM-1 protein in the small bowels of treated patients (Yacyshyn et al., 1998). In a subsequent 300 patient trial in which patients were treated with placebo or 2 mg kg1 every other day for 2 or 4 weeks, again the drug was shown to be extremely well tolerated. A population pharmacokinetic study demonstrated that heavier patients and women achieved greater exposures to the drug and that in

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the patients in the upper two quartiles of drug exposure, the drug produced a statistically significant increase in complete remissions compared with placebo (Yacyshyn et al., 1997). Additional phase III trials at higher doses are in progress.

ISIS 3521 ISIS 3521 is a phosphorothioate oligodeoxynucleotide inhibitor of protein kinase C a (for review see Holmlund, 2001). This drug has been evaluated as a single agent in a variety of solid tumors, using a variety of dose schedules at doses as high as 30 mg kg1 day1 and the drug was well tolerated. As a single agent, ISIS 3521 has shown modest activity in patients with a variety of tumors (Holmlund, 2001). ISIS 3521 has also been evaluated in combination in a number of chemotherapeutic agents in a number of solid tumors. The most advanced experience is a phase I/II study in 53 patients with non-small cell carcinoma of the lung in combination with carboplatin and taxol. In this study, with no increase in toxicity, ISIS 3521 was associated with a substantial improvement in survival (16 months) compared with historical controls (Yuen, 2001). Based on these data, a large phase III program is in progress.

G3139 G3139 is a phosphorothioate oligodeoxynucleotide designed to inhibit BCL2. It has demonstrated single agent activity in non-Hodgkin’s lymphoma. In the non-Hodgkin’s lymphoma trial, BCL2 levels in peripheral blood cells were reduced by G3139 (Waters et al., 2000). G3139 is also being studied in combination with chemotherapeutic agents. In a phase I study with decarbazine in patients with malignant melanoma, a 21% response rate in 14 patients was reported (Jansen et al., 2000). This drug is being evaluated in a number of other malignant diseases and in a number of other combinations.

ISIS 2503 ISIS 2503 is a phosphorothioate oligodeoxynucleotide designed to inhibit Ha-ras. It is currently completing phase II single agent and combination studies in patients with a variety of solid malignancies (for

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review see Holmlund, 2001). It has been reported to be well tolerated and preliminary suggestions of activity have been reported (see Table 60.1).

ISIS 5132 ISIS 5231 is a phosphorothioate oligodeoxynucleotide designed to inhibit C-raf kinase. In phase I studies, this drug was shown to reduce C-raf kinase levels in peripheral blood cells and displayed activity in patients with ovarian cancer (O’Dwyer et al., 1999). It too is completing phase II evaluation (for review see Holmlund, 2001).

CHARACTERISTICS OF 2O-(2 METHOXYETHYL) CHIMERAS In the past 5 years, enormous advances in the medicinal chemistry of oligonucleotides have been reported. Modifications at nearly every position in oligonucleotides have been attempted and numerous potentially interesting analogs have been identified. Although it is far too early to determine which of the modifications may be most useful for particular purposes, it is clear that a wealth of new chemicals is available for systematic evaluation and that these studies should provide important insights into the SAR of oligonucleotide analogs. The 2-O-(2-methoxyethyl) (Figure 60.1) substitution represents a significant advance in antisense therapeutics and the culmination of more than a decade of progress in antisense technology. Although oligonucleotides in which every nucleotide contains a 2-O(2-methoxyethyl) modification have proven of great value when used for occupancy only mediated mechanisms, e.g. induction of alternative splicing (for review see Kole and Mercatante, 2001). They have been used most broadly in chimeric structures designed to serve as substrates for RNase H. Numerous chimeric 2-O(2-methoxyethyl) oligonucleotides have not been studied extensively. Although such oligonucleotides typically display affinity for target RNA that is several orders of magnitude greater than for phosphorothioate oligodeoxynucleotides, they are less attractive substrates for RNase H (for review see Crooke, 2001b). Consequently, they typically display only five- to

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TABLE 60.1 In vivo studies using antisense oligonucleotides: inflammatory targets Therapeutic area

Molecular target

Animal model

Reference

Colitis

NFjB p65 NFjB p65

Mouse TNBS colitis; IL10/ mouse colitis E. histolytica infection in human intestinal xenografts in SCID mice Mouse DSS colitis Rabbit dust mite-induced asthma

Neurath et al., 1996 Seydel et al., 1998

Rat ovalbumin-induced asthma Mouse ovalbumin-induced asthma Mouse LPS-induced lung inflammation Mouse LPS-induced lung inflammation Rat traumatic brain injury Mouse experimental autoimmune encephalomyelitis Rat carrageenan-induced inflammation Rat experimental autoimmune encephalomyelitis Mouse contact hypersensitivity Mouse IL-1-induced skin inflammation Mouse cytotoxic dermatitis

Nyce and Metzger, 1997 Karras et al., 2000 Kumasaka et al., 1996; Qin et al., 2000 Ghirnikar et al., 1998 Ding et al., 1998

Lung inflammation

Central nervous system inflammation

ICAM-1 Adenosine A1 receptor IL-4, IL-5 IL-5 ICAM-1 TNFa MCP-1 iNOS c-fos IP-10

Skin inflammation

ICAM-1 IL-1 receptor ICAM-1

Shock Ocular disease Lupus

iNOS VEGF NFjB p50

Transplantation rejection

Reperfusion injury

ICAM-1

Rat LPS-induced hypotensive shock Mouse model of retinopathy of prematurity BXSB mouse model of autoantibody production Mouse heart allograft

ICAM-1 ICAM-1 ICAM-1 Cdc2 kinase Cdk2 kinase Granzyme C ICAM-1 ICAM-1

Mouse islet cell allograft Rat heart, kidney allograft Rat heart allograft Mouse heart allograft (arteriopathy) Mouse heart allograft (neointimal formation) Mouse interstitial nephritis Rat kidney isograft reperfusion injury Rat heart allograft reperfusion injury

15-fold increases in potency both in vitro and in vivo. For example, a direct comparison of two antisense inhibitors of C-raf kinase with the same sequence, one being a phosphorothioate oligodeoxynucleotide the other a 2-O-(2-methoxyethyl) chimera, showed that in vitro the 2-O-(2-methoxyethyl) chimera was approximately five-fold more potent. Similarly, after i.v. administration in a rat heart allograph model, the modified oligonucleotide was at least five-fold more potent (Stepkowski et al., 2000). Similar data were reported from a direct comparison of antisense inhibitors of survivin in an in vitro and in vivo human tumor xenograft model (Zellweger et al., 2001). Phar-

Bennett et al., 1997 Nyce and Metzger, 1997

Gillardon et al., 1997; Wojcik et al., 1996) Klimuk et al., 2000 Burch and Mahan, 1991 Christofidou-Solomidou et al., 1997 Hoque et al., 1998 Robinson et al., 1996 Khaled et al., 1997 Stepkowski et al., 1994 Stepkowski, 1998 Stepkowski et al., 1997 Mann et al., 1999 Suzuki et al., 1999 Suzuki et al., 1997 Bailey and Kelley, 1997 Dragun et al., 1998 Feeley et al., 1999

macological studies of antisense oligomers modified with 2-O-(2-methoxyethyl) chemistry have recently been reviewed (Dean, 2001). Perhaps more importantly, 2-O-(2-methoxyethyl) chimeras are substantially more stable than phosphorothioate oligonucleotides. In fact, in mice, rats and monkeys, the elimination half-life is nearly 30 days in plasma and in several tissues (Crooke and Graham, 2001; Geary et al., 2001a, 2001b; Manoharan, 2001). Furthermore, in the liver, elegant correlations between pharmacokinetic and pharmacodynamic effects have been reported showing a duration of pharmacological action of nearly 20 days in the

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FIGURE 60.1 2-O-MOE:2-O-(2-methoxyethyl)RNA.

mouse (Zhang et al., 2000). Studies in animals have recently been extended to man. ISIS 104838, a TNFa antisense inhibitor, a 2-O-(2-methoxyethyl) chimera, was shown to reduce TNFa secretion at doses at least 10-fold lower than would be expected in man by first generation antisense drugs (Dorr, unpublished observations) and to have an elimination half-life in plasma of approximately 30 days (Geary, unpublished observations). These studies demonstrate the feasibility of once a month dosing. Although significant progress in achieving acceptable oral bioavailability of antisense inhibitors has been reported, much remains to be accomplished and key clinical studies in progress will help determine how feasible oral delivery is (for review see Hardee et al., 2001). Based on the progress, two key barriers to oral bioavailability have been identified and partially overcome: presystemic metabolism and penetration across the G.I. mucosa. Because the gut has a very high level of nucleases contributed by both the host and bacteria resident in the G.I. tract, metabolism of phosphorothioate oligodeoxynucleotides in the gut occurs much too rapidly to support adequate oral bioavailabity (Nicklin et al., 1998). Chimeric modifications of the oligonucleotides have been shown to enhance stability and oral bioavailability (Zhang et al., 1995, 1995b). With the development of 2-O-MOE phosphorothioates, oral bioavailability is potentially feasible (Hardee et al., 2001).

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In rodents, dogs and monkeys, the permeability of the G.I. tract to antisense inhibitors has been significantly enhanced by using formulations containing penetration enhancers, such as bile acid salts and fatty acids. After intrajejunal administration of several 2-O-MOE-modified oligonucleotides in the presence of penetration enhancer containing formulations, systemic bioavailability in excess of 20% was observed in all three species (Hardee et al., 2001). Initial studies with solid dose forms containing penetration enhancers resulted in significantly less systemic bioavailability (5.5%). Clearly, therefore, more progress is required before solid dose forms with adequate (10–20%) oral bioavailability are available (Geary et al., 2001, 2001b; Hardee et al., 2001). Initial clinical trials in which a variety of solid dose forms containing ISIS 104838, a 2-O-MOE chimeric antisense inhibitor of TNFa, and various penetration enhancers are in progress, so within the next years we should have some sense of the near term potential for oral delivery of antisense inhibitors in man. Finally, 2-O-(2-methoxyethyl) chimeras have been shown to be less proinflammatory (Henry et al., 2000; Levin et al., 2001). After repeated subcutaneous dosing in man, ISIS 104838 produced dramatically lower local inflammation than first generation antisense drugs. Thus, more convenient and better locally tolerated subcutaneous administration may be feasible. In conclusion, although many more questions about antisense remain to be asked than are answered, progress has continued to be gratifying. Clearly, as more is learned, we will be in the position to perform progressively more sophisticated studies and to understand more of the factors that determine whether an oligonucleotide actually works via an antisense mechanisms. We should also have the opportunity to learn a great deal more about this class of drugs as additional studies are completed in humans.

ANTISENSE INHIBITORS FOR INFLAMMATORY TARGETS Table 60.1 summarizes the activities reported for antisense inhibitors with various inflammatory targets in vivo. Most of the data derive from studies using the first generation antisense drugs, the phosphorothioates.

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More recent studies in our laboratories and with our collaborators have employed 2-methoxyethyl chimeras, the second generation antisense drugs currently being evaluated in clinical trials. A wide range of dose administration schedules and animal models have been studied.

EFFECTS ON INTERFERON FAMILY MEMBERS Interferon c A few studies have used antisense oligonucleotides to down-regulate IFN-c. In a recent report, Kuo and coworkers utilized an anti-IFN-c oligonucleotide to demonstrate the autocrine role of the cytokine in expression of isoform of nitric oxide synthase (iNOS) in IL-1b-treated rat hepatocytes (Schroeder et al., 1998). They first demonstrated that anti-IFN-c oligonucleotide treatment effectively reduced IL-1binduced expression of the target cytokine to basal levels, whereas the sense control oligonucleotide had no effect on expression. Further analysis showed that antisense knockdown of IFN-c expression also inhibited expression of the inducible iNOS and subsequent production of nitrite in a sequence-specific manner. In vivo effects of an antisense oligonucleotide to IFN-c have yet to be reported.

Interleukin 10 Several research groups have utilized antisense oligonucleotides to reduce expression of IL-10 in cell culture. In addition to work by the Hauber group and Salazar-Onfray and colleagues discussed previously, which showed pharmacological effects by IL-10 antisense on MHC expression, Tsuchiya and coworkers identified two antisense oligonucleotides that efficiently reduced IL-10 mRNA and protein levels in phorbol ester-activated U937 cells, a transformed monocytic cell line. These oligonucleotides were found to reduce IL-10 levels in a dose- and sequencespecific manner (Arima et al., 1999). The compounds, which targeted sites in the coding region and the 3 untranslated region, were both more active than those targeted to the AUG region.

THE TNF FAMILY TNFa To date, a variety of antisense oligonucleotides has been reported that reduce human, mouse, or rat TNFa expression in cell culture. The range of activity observed has been broad, however, ranging from 36% to 90% reduction in expression levels. This variation appears to be based upon the use of different cell types and transfection methods, the number of sites screened for activity, and the type and degree of oligonucleotide modifications (Taylor et al., 1996, 1997, 1998; Rojanasakul, 1997; Tu, 1998; Mayne, 1999). Recently it has been reported that administration of an anti-TNFa morpholino oligomer into mice, by intranasal insufflation, inhibited TNFa protein expression resulting from exposure to aerosolized endotoxin by approximately 30%, as measured from the bronchoaveolar lavage fluid (Qin et al., 2000). Reduction of TNFa protein levels occurred in a doseresponsive manner and was sequence-specific, as shown by comparison to a scramble morpholino control. Unlike oligodeoxynucleotides, morpholino oligomers are not substrates for RNase H or other endogenous enzymes, and reduce target protein expression via an occupancy-only mechanism (Baker and Monia, 1999). Incomplete inhibition was attributed to heterogeneous delivery of the oligomers to the cells throughout the lungs by the intranasal insufflation procedure. The effect of anti-TNFa oligomer treatment on LPS-induced pulmonary inflammation, as measured by lung lavage neutrophil counts, was dependent on the time between treatment with the oligomer and exposure to LPS. The sequence specificity of the effect of morpholino treatment on pulmonary inflammation was not determined. In an in vitro study, bacterial lipopolysaccharide (LPS)-induced TNFa production from lung alveolar epithelial cells was dose-dependently inhibited in a sequence-specific manner by an 18mer TNFa antisense oligonucleotide (first described by Rojanasakul et al., as noted above) targeted to the translation initiation site (Xavier et al., 1999). Lipofectin-mediated transfection was also found to block macrophage inflammatory protein (MIP)-2 production by these cells, similar to coincubation with TNFa polyclonal or

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THE TNF FAMILY

monoclonal antibodies. However, the magnitude of inhibition of either TNFa or MIP-2 protein was no better than 50% in this study. These observations suggest that MIP-2 expression is controlled in part in an autoregulatory fashion by TNFa produced from the epithelial cells following LPS stimulation, as might occur during an acute inflammatory reaction. TNFa antisense has also been shown to protect against LPS-induced, Kupffer cell-mediated hepatocyte injury in vitro (Kurose et al., 1996). In this hepatocyte–Kupffer cell co-culture model, TNFa released from Kupffer cells after endotoxin exposure was implicated in compromised hepatocyte mitochondrial energy synthesis, as measured by decreased oxidative phosphorylation. The mitochondrial dysfunction was followed by positive propidium iodide staining, indicating loss of membrane barrier function and lethality. In these experiments, 18mer antisense and sense oligonucleotides were utilized that were free of CpG motifs and incubated in cultures in the absence of transfection enhancers. However, the authors did not show a dose–response effect. Antisense treatment (at 20 lM) of Kupffer cells, but not hepatocytes resulted in reduction of TNFa protein levels, as would be expected since the Kupffer cells are the major source of TNFa in this system. TNFa antisense treatment of Kupffer cells was also found to attenuate the decrease in oxidative phosphorylation, pointing to a role for TNFa in endotoxin-induced liver injury. In our laboratories we have created 2methoxyethyl chimeric antisense inhibitors of TNFa and evaluated them in a number of animals (unpublished data, B. Baker, Isis Pharmaceuticals, Inc.). For example, a mouse TNFa antisense inhibitor, ISIS 2302 was shown to reduce TNFa expression and improve the disease activity index in a mouse colitis model in a dosedependent and sequence-specific manner (0.25, 2.5 and 12 mg kg1 subcutaneous every other day) (Baker et al., 2000). The antisense inhibitor to TNFa of the same chemistry, ISIS 104838 is currently in phase II trials both i.v. and s.c. In phase I trials, pharmacokinetics showed an elimination of half-life of 30 days, so once a month dosing may be satisfactory. In phase I trials, no adverse events were noted (Crooke, 2001a).

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Tumor necrosis factor receptor 1 (TNF-R1 OR P55) In two recent reports, Ojwang and coworkers evaluated the effects of anti-TNF-R1 oligonucleotides in a transformed human lung embryonic fibroblast cell line, and in human newborn foreskin fibroblasts (Ojwang et al., 1997a, 1997b). In these studies, the investigators evaluated a number of differentially modified analogs of an oligodeoxynucleotide that targeted the poly(A) signal site of the TNF-R1 mRNA. These modifications included C5-propynyl or -hexynyl derivatives of 2-deoxyuridine, the C5propynyl derivative of 2-deoxycytidine, and the 7-deaza-7-propynyl derivative of 2-deoxy guanosine. Although antisense knockdown of target mRNA and protein was 50% or less, reduction of TNFa-induced IL-6 and IL-8 expression by treatment with these antiTNF-R1 oligonucleotides ranged from 40 to 80%. Antisense treatment was selective for TNF-R1, as shown by selective decrease of TNF-R1 protein in comparison to TNF-R2 and lack of effect on IL-1induced IL-6 production. The modest activity of these oligonucleotides against TNF-R1 may be a reflection of the metabolism or turnover rate of the protein. In this respect, only a minor subpopulation of TNF-R1 has been shown to be expressed in the plasma membrane of endothelial cells and monocytic U937 cells. The majority of the protein is associated with the Golgi apparatus (Jones et al., 1999). The significance of this distribution pattern is unknown, but it may have an impact on antisense efficacy.

Tumor necrosis factor receptor 2 (TNF-R2 OR P75) Less is known about the functional role of TNF-R2 in comparison to TNF-R1. Antisense knock out of TNFR2 in human neuronal-like cells increased the degree of cell death induced by its ligand, TNFa, and cell injury induced by hypoxic conditions or exposure to the b-amyloid peptide (Shen et al., 1997). Treatment was selective for TNF-R2 in comparison to TNF-R1, as shown by radioimmunoprecipitation and Western blot, respectively. These results indicate that TNF-R2 may protect human neurons from cell death.

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FAS In a series of studies in our laboratory (Zhang et al., 2000), antisense inhibitors a 2-O-(2-methoxyethyl) chimeras to mouse Fas were investigated. These antisense inhibitors reduced Fas RNA and protein levels in vitro and in mouse livers after i.v. administration in a highly sequence-specific and dose-dependent fashion. The duration and extent of the effects correlate with the concentration of drug in liver. The drug ablated the effects (death) of Fas antibody in treated animals.

EFFECTS ON THE INTERLEUKIN FAMILY Interleukin 1a (IL-1a) Kawaguchi and colleagues hypothesized that IL-1a expression in these fibroblasts acted in an autocrine manner to promote production of collagen and thus sclerosis of the skin. To test this hypothesis, they treated these fibroblast cells with a phosphorothioate-modified antisense oligonucleotide complementary to the AUG region of the IL-1a transcript, and then examined the effects of treatment on expression of two factors that promote synthesis of collagen, IL-6 and PDGF-a (Kawaguchi et al., 1999). IL-1a antisense oligonucleotide treatment reduced target IL-1a mRNA and protein levels in a dose- and sequencedependent manner. Further analysis showed that antisense reduction of IL-1a levels reduced expression of IL-6 at both the RNA and protein level, and PDGF-a at the RNA level.

Interleukin 4 (IL-4) and 5 (IL-5) Several attempts have been made to use antisense oligonucleotides to modulate the TH2 response in asthma models by targeting either IL-4 or IL-5. In a rat model of asthma, investigators treated CD4 cells ex vivo with antisense oligonucleotides specific to either IL-4 or IL-5, and then injected the cells back into rats prior to challenge with an ovalbumin aerosol. They found a significant reduction in airway resistance, airway eosinophilia and both IL-4 and IL-5 expression, when the rats received CD4 cells treated with IL-4 but

not IL-5 antisense, or a control oligonucleotide (Molet et al., 1999). IL-5 expression in lung lavage fluid was only slightly decreased by the transfer of IL-5 antisense-treated T cells, suggesting that the early production of IL-4, but not IL-5, may be critical in the late phase response. Results may differ, however, when animals are treated systemically with antisense oligonucleotide. Our own group has found that a systemically administered methoxy-ethyl-modified phosphorothioate oligonucleotide to IL-5 was effective in reducing both lung eosinophilia and the late phase response in a mouse model of asthma (Karras et al., 2000). These results may differ from those of Molet et al., because systemically administered IL-5 antisense is likely to have a different site of action than antisense oligonucleotide administered ex vivo to isolated T cells. Likely target cells of systemically administered IL-5 antisense could include eosinophils and/or mast cells, both of which can secrete IL-5, or precursor cells in bone marrow.

Interleukin 6 (IL-6) Two reports describe the use of antisense oligonucleotides to reduce expression and function of human IL-6 in cell culture. In the most recent report, the investigators used a phosphorothioate-modified 15base oligonucleotide, complementary to a sequence in the coding region of the transcript, to reduce IL-6 production in B cells stimulated with CD40 antibodies and IL-4 (Bjorck et al., 1998). Although treatment of the B45 B cell hybridoma with the oligonucleotide resulted in reduction of IL-6 production, this activity may have been due to the non-antisense effects of a string of guanine residues nested in this particular oligonucleotide sequence (Benimetskaya et al., 1997; Wang et al., 1998). The control oligonucleotide did not produce this kind of activity. Treatment of activated tonsillar B cells with the anti-IL-6 oligonucleotide was shown to result in a reduction in IgE production. Reduction of proteoglycan synthesis by chondrocytes is believed to play a role in the loss of cartilage during rheumatoid arthritis. Exposure of chondrocytes to IL-1 effectively inhibits proteoglycan synthesis, and also activates expression of many inflammatory genes, including IL-6. With this in mind, Nietfeld and coworkers evaluated the effects of an IL-6 antisense oligonucleotide on synthesis of

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EFFECTS ON THE INTERLEUKIN FAMILY

proteoglycans in cartilage explant cultures induced with IL-1 (Nietfeld et al., 1994). Pretreatment of the explants with the IL-6 phosphorothioate-modified antisense oligonucleotide inhibited IL-1-induced production of IL-6 protein in a dose- and sequencespecific manner. Further analysis showed that antisense reduction of IL-6 expression interfered with the inhibition of proteoglycan synthesis that occurred upon IL-1 induction. Antisense efficacy was notably good in these experiments considering the highly electronegative extracellular matrix that surrounds the chondrocytes in cartilage, given the overall negative charge of oligonucleotides. The accessibility of the cells to the oligonucleotide may be an attribute of the explants however, since chondrocyte uptake is not observed in vivo, as determined by IHC staining of fixed tissue (unpublished results, M. Butler, ISIS Pharmaceuticals).

Interleukin 10 (IL-10) Several research groups have utilized antisense oligonucleotides to reduce expression of IL-10 in cell culture. In addition to work by the Hauber group and Salazar-Onfray and colleagues discussed previously which showed pharmacological effects by IL-10 antisense on MHC expression, Tsuchiya and coworkers identified two antisense oligonucleotides that efficiently reduced IL-10 mRNA and protein levels in phorbol ester-activated U937 cells, a transformed monocytic cell line. These oligonucleotides were found to reduce IL-10 levels in a dose- and sequence-specific manner (Arima et al., 1999). The compounds, which targeted sites in the coding region and the 3 untranslated region, were both more active than those targeted to the AUG region.

Interleukin receptor I, type (IL-1RI) There are two IL-1 receptor proteins, type I and type II. Type I transduces signals across the cell membrane, whereas type II acts as a decoy or ligand sink. One of the earliest in vivo demonstrations of antisense efficacy involved the type I interleukin-1 receptor (Burch and Mahan, 1991). These experiments involved subcutaneous injection of an 18-base phosphorothioate oligonucleotide which targeted the AUG region of the

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type 1 IL-1R. IL-1-induced neutrophil migration into skin was reduced by 37%. Although the authors did not investigate whether antisense treatment affected IL-1R expression in skin, a human-specific IL-1R oligo which contained five mismatched bases to the murine sequence failed to modulate neutrophil influx, suggesting an antisense mechanism in this system. A more recent report (Miraglia et al., 1996) has described the evaluation of 36 phosphorothioatemodified oligonucleotides for activity towards IL-1RI in A549 cells and normal human dermal fibroblasts, where target sites were located in the 5 UTR, coding region, splice junctions, and 3 UTR of the transcript. In these experiments, antisense activity, as measured by reduction of RNA levels, was only observed at sites in the 3 UTR. The oligonucleotide targeting the AUG region, as previously described, was inactive. Activity of the lead compound, ISIS 8807, was dose- and sequence-dependent, and treatment of A549 cells with this compound reduced IL-1RI protein and mRNA by 60 and 90%, respectively. In addition, this oligonucleotide reduced IL1a-induced expression of ICAM-1 mRNA in A549 cells by 66%, in comparison to saline or control oligonucleotide-treated cells.

Interleukin 4 receptor, alpha chain (IL-4Ra) To date, one research group has utilized antisense oligonucleotides to down-regulate IL-4Ra expression in B cells (Ikizawa et al., 1995). In this study, three phosphorothioate-modified antisense oligonucleotides were tested for activity in Daudi cells, which constitutively express the receptor. The oligonucleotide that targeted the AUG region was the most effective at reduction of receptor surface expression. This oligonucleotide was evaluated for its ability to inhibit production of IgE by normal B cells costimulated with IL-4 and CD40 monoclonal antibody. A dose-dependent decrease in IgE protein production was observed in the antisense-treated group, with no effect on IgG production. Reduction of total Ce mRNA and mature Ce mRNA levels was observed, but germline Ce was unaffected. These investigators, however, did not determine if inhibition of IgE production was sequence-dependent.

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EFFECTS ON CHEMOKINES AND CHEMOKINE RECEPTORS Monocyte chemotactic protein (MCP)-1 The application of antisense technology to chemokinemediated processes has focused chiefly on monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), which were two of the earliest described members of the family. MCP-1, a CC chemokine, has been shown to regulate chemotaxis of monocytes, T lymphocytes and basophils (Baggiolini et al., 1997). In an in vivo model of traumatic brain injury in rats, MCP-1 expression has been linked to macrophage infiltration and inflammation of the CNS. Administration of MCP-1 antisense, but not sense oligodeoxynucleotides, for 3 days via an implanted Alzet miniosmotic pump prior to traumatic brain injury inhibited the induction of gliosis, as measured by glial fibrillary acidic protein immunostaining, and reduced the number of macrophages that were able to cross the damaged blood–brain barrier (Ghirnikar et al., 1998). In addition, a role for MCP-1 in recruitment of proinflammatory monocytes to atherosclerotic plaques was suggested by studies showing that MCP-1 antisense oligonucleotides or neutralizing antibodies inhibited monocyte chemotactic activity produced by cultured rat aortic vascular smooth muscle cells treated with platelet-derived growth factor (PDGF), using a Boyden chamber assay (Poon et al., 1996). These investigators showed that two separate 18mer antisense sequences devoid of CpG motifs produced similar effects and that antisense treatment resulted in decreased levels of PDGF-induced MCP-1 mRNA. Published data also claim that MCP-1 can directly modulate lymphocyte activation, as MCP-1 antisense oligonucleotide treatment of either lung fibroblasts or splenic macrophages both selectively inhibits MCP-1 synthesis by these cells and enhances CD4 T cell proliferation (Hogaboam et al., 1998). However, in this study, the antisense 29mer sequence contains a near-canonical CpG motif, whereas the sense oligonucleotide does not.

Macrophage inflammatory protein (MIP)-1a, b The related CC chemokines, MIP-1a and b, are spontaneously expressed by adult CD4 T cell leukemia cells (ATL). Antisense oligonucleotides directed at MIP-1a and b have been shown to reduce ATL adhesion to endothelial cells and inhibit expression of the spontaneously activated form of LFA-1, events that are hypothesized to control pathologic ATL extravasation into tissues (Tanaka et al., 1998). Inhibition of either MIP-1a or MIP-1b with these antisense 15mers was found to result in decreased ICAM-1-dependent adhesion to endothelial cells. The investigators directly measured transendothelial migration using microchemotaxis chambers precoated with activated endothelial cells and found that either MIP-1a or MIP-1b antisense pretreatment of ATL cells blocked their spontaneous transmigration across the endothelial layer. Thus, these activities appear to be mediated by endogenous production of these chemokines by the leukemic cells. However, in this study, it is not clear whether the observed effects are due to an antisense mechanism, since target mRNA or protein reduction following antisense treatment was not shown and the antisense, but not the sense, sequences did contain non-canonical CpG motifs.

Interleukin 8 (IL-8) Pleiotropic effects of IL-8 on leukocytes have also been delineated in part by the use of antisense approaches. An IL-8 antisense oligonucleotide was found to specifically block the production of monocyte-derived angiogenic activity (Koch et al., 1992). Hypoxia-induced IL-8 production by epithelial cells was blocked by treatment with IL-8 antisense and this resulted in decreased adherence of polymorphonuclear cells (PMNs), suggesting that IL-8 acts as a chemotactic signal for PMNs to aggregate at the basal surface of epithelia (Colgan et al., 1996). Furthermore, an antisense, but not a scrambled, IL-8 antisense oligonucleotide was found to inhibit endometrial stromal cell proliferation in vitro and this effect was reversed by addition of recombinant IL-8 (Arici et al., 1998).

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EFFECTS ON VASCULAR ENDOTHELIAL GROWTH FACTOR ( VEGF )

IFN-c-inducible protein (IP)-10/CRG-2 Antisense oligonucleotides targeting the chemokine crg-2 (more recently named interferon-c-inducible protein or IP-10) were evaluated in a rodent model of experimental autoimmune encephalomyelitis (EAE) (Wojcik et al., 1996). Diester backbone and phosphorothioate backbone oligonucleotides targeting the same region of IP-10 mRNA were compared, and the oligonucleotides were injected into the CNS by osmotic minipump infusion into the lumbar subarachnoid space. The phosphorothioate, but not the diester backbone oligonucleotides proved to be nonspecifically toxic to CNS tissue. Although rats treated with phosphorothioate oligonucleotides developed paralytic symptoms typical of EAE, the paralysis occurred in rats given both IP-10 antisense oligonucleotides and control oligonucleotides. Moreover, when rats were not immunized to develop EAE, but were given the IP-10 phosphorothioate antisense oligonucleotide, they developed paralysis as well. A histological examination of rat spinal cords showed that the phosphorothioate oligonucleotides were causing an atypical necrosis of CNS parenchymal tissue instead of the expected inflammatory infiltration of mononuclear cells, with the necrosis presumably causing the paralytic symptoms. Interestingly, the IP-10 phosphorothioate antisense did cause a specific reduction in target mRNA in spinal cord when compared with a scrambled control sequence. The diester backbone oligonucleotides used by Wojcik et al. did not cause CNS tissue necrosis, and the antisense sequence produced a statistically significant reduction in the disease activity (i.e. a reduction in paralytic symptoms) compared with the control oligonucleotide-dosed group. Although the diester IP-10 antisense failed to reduce IP-10 mRNA levels when sampling was conducted at the height of clinical disease, a significant decrease in target mRNA was achieved when IP-10 levels were measured earlier in the disease course. Although it is somewhat surprising that a diester backbone oligonucleotide would have sufficient potency to produce the effects that it did in these experiments, little is known about nuclease activity in cerebrospinal fluid. This was one of the first studies using antisense oligonucleotides to treat EAE, and the results obtained suggest that some complex interactions may be

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occurring between the locally administered phosphorothioate oligonucleotides and the inflamed CNS tissue. With respect to the toxicity observed with the intrathecally administered phosphorothioates in this model, it is worth noting that other groups have directly injected phosphorothioate oligonucleotides into the CNS without observing any local tissue necrosis (e.g. Ding et al., 1998). Our group, moreover, has had extensive experience using systemically administered phosphorothioate oligonucleotides in mouse models of EAE without any such toxicity. The toxicity seen by Wojcik et al. may therefore be related to interactions between specific factors induced during the EAE model and the intrathecal injection of phosphorothioates. Alternatively, but less likely, is the possibility that heightened IP-10 target reduction with the phosphorothioate oligonucleotides could result in CNS toxicity and tissue destruction. Although somewhat limited in scope, together these studies support the multi-functional nature of the chemokine molecules in experimental model systems, and indicate the potential for therapeutic benefit through pharmacological intervention in this pathway. A challenge for the future development of antisense or other drugs targeted to chemokines or chemokine receptors will be to further understand the functional redundancy of the chemokine system in inflammation.

EFFECTS ON VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) Dysregulated angiogenesis is critical to the development of several different ocular diseases, including macular degeneration, diabetic retinopathy and corneal neovascularization. The role of vascular endothelial growth factor (VEGF) was investigated in a mouse model of retinopathy of prematurity. Antisense oligonucleotides targeting VEGF were injected intravitreally into the eyes of neonatal mice prior to the onset of proliferative retinopathy. The VEGF antisense treatment caused a statistically significant reduction in retinal VEGF levels and in the level of retinal neovascularization which was not seen when control oligonucleotides were injected (Robinson et al., 1996). The inhibition of neovascularization was,

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however, incomplete, implying that other angiogenic factors could be involved in this model.

CONCLUSION Antisense technology continues to advance. Today, answers to most of the basic questions are published. Clinical experience with first generation antisense drugs now exceeds several thousand patients and a number of studies now suggest therapeutic activity. Second generation antisense drugs with dramatically improved properties are now available and in clinical trials. Antisense technology has also been automated such that it is useful as a tool to determine gene function and it is being used to determine the biological roles of various cytokine and related genes. Further, antisense inhibitors of cytokines and related genes are progressing in evaluations for therapeutic potential.

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‘antisense’ oligodeoxynucleotide. Nucelic Acids Res. 25, 2648–2656. Bennett, F. (2001). General pharmacology of phosphorothioate oligodeoxynucleotides. In: S.T. Crooke (ed.). Antisense Therapeutics: Principles, Strategies and Applications. New York: Marcel Dekker, pp. 291–318. Bennett, C.F., Chiang, M.Y., Chan, H. et al. (1992). Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 41, 1023–1033 Bennett, C.F., Chiang, M.Y., Chan, H. and Grimm, S. (1993). Use of cationic lipids to enhance the biological activity of antisense oligonucleotides. J. Liposome Res. 3, 85–102. Bjorck, P., Larsson, S., Andang, M. et al. (1998). IL-6 antisense oligonucleotides inhibit IgE production in IL-4 and antiCD40-stimulated human B-lymphocytes. Immunol. Lett. 61, 1–5. Burch, R.M. and Mahan, L.C. (1991). Oligonucleotides antisense to the interleukin 1 receptor mRNA block the effects of interleukin 1 in cultured murine and human fibroblasts and in mice. J. Clin. Invest. 88, 1190–1196. Butler, M., Stecker, K. and Bennett, C.F. (1997). Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues. Lab. Invest. 77, 379–388. Cazenave, C., Stein, C.A., Loreau, N. et al. (1989). Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. Nucleic Acids Res. 17, 4255–4273. Chiang, M.Y., Chan, H., Zounes, M.A. et al.. (1991). Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. J. Biol. Chem. 266, 18162–18171. Chrisey, L.A., Walz, S.E., Pazirandeh, M. and Campbell, J.R. (1993). Internalization of oligodeoxyribonucleotides by Vibrio parahaemolyticus. Antisense Res. Dev. 3, 367–381. Cohen, J.S. (1993). In: S.T. Crooke and B. Lebleu (eds). Antisense Research and Applications Boca Raton: CRC Press, pp. 205. Colgan, S.P., Dzus, A.L. and Parkos, C.A. (1996). Epithelial exposure to hypoxia modulates neutrophil transepithelial migration. J. Exp. Med. 184, 1003–1015. Cornish, K.G., Iversen, P., Smith, L. et al. (1993). Cardiovascular effects of a phosphorothioate oligonucleotide with sequence antisense to p53 in the conscious rhesus monkey. Pharmacol. Commun. 3, 239–247. Cossum, P.A., Sasmor, H., Dellinger, D. et al. (1993). Disposition of the 14C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats. J. Pharmacol. Exp. Ther. 267, 1181–1190. Cossum, P.A., Truong, L., Owens, S.R. et al. (1994). Pharmacokinetics of a 14C-labeled phosphorothioate oligonucleotide, ISIS 2105, after intradermal administration to rats. J. Pharmacol. Exp. Ther. 269, 89–94. Crooke, R.M. (1991). In vitro toxicology and pharmacokinetics of antisense oligonucleotides. Anti-Cancer Drug Design 6, 609–646. Crooke, R.M. (1993a). Cellular uptake, distribution and metabolism of phosphorothioate, phosphodiester, and methylphosphonate oligonucleotides In: S.T. Crooke and B. Lebleu (eds). Antisense Research and Applications. Boca Raton: CRC Press, pp. 427–339. Crooke, R.M. (1993b). In vitro and in vivo toxicology of first generation analogs In: S.T. Crooke and B. Lebleu (eds).

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61 Assays for cytokines Theresa L. Whiteside University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

The mind of man is capable of anything – because everything is in it, all the past as well as all the future. Joseph Conrad, Heart of Darkness

INTRODUCTION Assays for cytokines are becoming an important component of laboratory evaluations of immune responses in humans. Recent expansion in the knowledge of the cytokine biology and the realization that cytokines play a role in human diseases have created a need for the precise assessment and accurate interpretation of their presence and activity in body fluids, tissues and cells. Ever increasing utilization of cytokines as therapeutic agents is likely to result in the transfer of assays for cytokines from the research domain to clinical laboratories responsible for monitoring of patients in the near future. Cytokines are multifunctional molecules, which mediate interactions between various cells and contribute to the pathogenesis of many diseases. The correct analysis of their involvement in a disease process is a crucial, but by no means simple, task. Current approaches to cytokine analysis emphasize a need for defining and validating the clinical usefulness of individual cytokine assays, as well as The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0–12–689663–1, London

simultaneous evaluations of many cytokines to be able to establish a relevant ‘cytokine profile’. A wide range of cytokine assays available today offers means for evaluating diverse aspects of the cytokine biology. For example, it is possible to investigate the cellular source of a cytokine, measure its intracytoplasmic content, its level in the extracellular space or to evaluate effects elicited by the interaction of this cytokine with its receptor on a target cell. In fact, a single assay is unlikely to confirm the role of a given cytokine in disease. For this reason, the emphasis has shifted from single cytokine assays to the definition of a cytokine profile in the tissue microenvironment. This, in turn, introduces a demand for the availability of relatively simple but specific, sensitive and reliable measurements that can be readily performed in a laboratory. The choice of a right panel of assays for cytokines is clearly not a simple matter, and it requires understanding of the cytokine biology, as well as expertise in assay development. Cytokines are low molecular-weight glycoproteins Copyright © 2003 Elsevier Science Ltd. All rights of reproduction in any form reserved.

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produced by immune as well as non-immune cells, which have been shown to regulate immunologic responses, hematopoietic development, cell-to-cell communication, as well as host responses to infectious agents and inflammatory stimuli (Oppenheim, 1993; Aggarwal and Puri, 1995). Cytokines are transiently expressed and produced after cellular activation, and they regulate events occurring during normal and pathologic conditions (Dinarello et al., 1990; Meager, 1998). A ubiquitous presence of cytokines in most physiologic and pathologic responses suggests that they are important for health, and that the cytokine imbalance, whether an excess or deficiency, might have undesirable consequences. Delivery of an exogenous cytokine can, in some cases, lead to clinical benefits. There are several features of cytokines that may be clinically relevant. All cytokines are pleiotropic, i.e. they have the ability to interact with a variety of cellular targets, usually via the specific receptors expressed on the surface of a target cell. Binding of cytokines to cell surface receptors initiates signaling and new RNA and protein synthesis. Cytokines are redundant in that the same biologic function can be executed by several distinct cytokines. This overlapping of cytokine activities, where a single cell target shows seemingly identical responses to multiple cytokines, has been referred to as cytokine cross talk. They are known to function as networks or cascades of interacting cytokines, which are able to induce each other. They regulate their own production via autocrine, juxtacrine or paracrine pathways in response to microenvironmental stimuli. Cytokines induce biologic effects at minute pharmacologic doses and, once released by the producing cell, have a short half-life. They are powerful physiologic mediators with a potential for inducing tissue damage. Cytokines are local mediators meant to exert biologic effects in their microenvironment. Systemic effects of cytokines are often quite different and more dramatic than those mediated locally (Dinarello and Wolff, 1993). The general properties of cytokines imply that more than one cytokine may be involved in a defined biologic response and that the concept of a ‘cytokine profile’ may be a valid one. In practical terms, it means that not one but several cytokines acting in concert are responsible for a specific functional phenotype. A division of labor among cytokines can be best illus-

trated by considering Th1 and Th2 immune responses: the Th1-type cytokines (e.g. IL-2, IL-12, IFN-c, TNF-a) are thought to be involved in elimination of intracellular infections, while B cell or antibody responses are mediated by Th2-type cytokines, such as IL-3, IL-4 and IL-5. The third group of cytokines, Th3, includes regulatory mediators such as IL-10 or TGF-b, which are responsible for maintaining a balance in the host microenvironment (Mosmann et al., 1997). This function-based profiling of cytokines or cytokine polarization appears to be useful in the classification of certain human diseases into the Th1 or Th2 categories. Thus, IL-2, IL-12 and IFN-c play an important role in eliminating intracellular parasites, notably Leishmania and related organisms, in organ-specific autoimmune diseases, allograft rejection and recurrent abortions (Romagnani, 1997a, 1997b). Other human diseases are associated with the predominance of the Th2-type cytokines. For example, atopic disorders, chronic graft-versus-host disease (GVHD), progression to AIDS, metastatic disease and the hypereosinophilic syndrome have all been linked with the dysregulated production of the Th2-type cytokines (reviewed in Romagnani, 1997). Furthermore, recent evidence indicates that a switch from the Th1 to Th2 cytokine profile might accompany progression of the disease (Grewe et al., 1998). Because cytokine polarization occurs in many diseases, it appears that the definition of the cytokine profile might become clinically useful. Cytokine assays of the near future are likely to be focused on a simultaneous assessment of many cytokines in a single biologic sample. In this chapter, cytokine assays that best lend themselves to multiple-type formats are featured, although traditional biological and immunologic cytokine assays that have been a part of a familiar laboratory repertoire will be covered as well.

PRINCIPLES OF CYTOKINE MEASUREMENTS AND TYPES OF ASSAYS AVAILABLE A variety of assays and assay formats are available for cytokine measurements (Table 61.1). The choice of the most appropriate assay depends on the scope of the experiment at hand, the end points to be established and the available specimens. Thus, document-

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TABLE 61.1 Summary of the methods available for detection of cytokines or cytokine receptors 1. Body fluids or cellular supernatants: (a) Cytokine levels measured by bioassays or immunoassays (b) Cytokine soluble receptors measured by bioassays or immunoassays (c) Definition of cytokine/cytokine receptor profiles 2. Cellular populations: (a) Spontaneous cytokine production (b) Stimulated cytokine production (c) Multiparameter flow cytometry for cytokinepositive cells (d) Magnetic bead-based quantification or isolation of cells secreting cytokines (e) mRNA for cytokines in situ hybridization (ISH), real-time qRT-PCR, Northern blots, RNase protection assays (RPA) 3. Single cell assays for cytokine expression or secretion: (a) Intracytoplasmic cytokine staining (ICC) (b) ELISPOT (c) mRNA-based assays (see above) 4. Detection of cytokines in tissues: (a) Immunocytochemistry (b) Immunofluorescence (c) mRNA-based assays (see above) 5. Cytokine proteomics 6. Cytokine genomics

ing the presence of a cytokine in culture supernatants or body fluids is simpler than documenting its presence at a tissue site. Monitoring of changes in single cytokines or establishing the cytokine profile during disease progression or during therapy is considerably more complex and required special quality control measures not required for single-sample estimates. Cytokine production by cells responding in vitro to a specific stimulus is an excellent measure of cellular activation. At the same time, this assay calls for isolation of cells and their in vitro activation by a stimulant. The obvious concern with this type of an assay is that it utilizes isolated or cultured cells, which are out of the context of their natural microenvironment. Hence, it has been argued that in vivo assays of cytokines, which provide a true measure of their biologic activity, are more useful and relevant. These involve injections of a cytokine into experimental animals or humans and monitoring of its biologic effects. Perhaps the best example of in vivo assays is the pyrogen assay for interleukin-1 (IL-1) (Dinarello, 1991).

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While informative, this and similar types of biologic assays are expensive, utilize large numbers of animals and require special facilities as well as expertise. In humans, the approach is only possible in the context of clinical trials utilizing cytokines for therapy, where the cytokine pharmacokinetics and its toxicity are often measured in lieu of its biologic activities. Although in vivo cytokine assays are essential for defining biologic activities of cytokines, particularly when working with newly discovered cytokines or in preparation for clinical trials, they have been largely replaced by in vitro-based bioassays, which depend on the utilization of cytokine-responsive cell lines for determining of biologic effects of body fluids obtained from animals or humans treated with cytokines (for a comprehensive review see Wadhwa and Thorpe, 1998). Of the two general categories of ex vivo cytokine assays, i.e. bioassays and immunoassays, the latter have gained a much wider acceptance. In either case, however, analysis of the cytokine content in biologic samples has proven to be clinically useful. Under normal circumstances, no or low levels of cytokines are detectable in body fluids or tissues. Therefore, their presence and level of expression point to the activation of cytokine pathways associated with a particular disease or its progression. Certain cytokines may be present early, others late in the disease, providing a way for discerning its stages. Acute disease is associated with a cytokine profile that is distinct from that detectable in chronic conditions. Also, the consistent association of elevated cytokine levels with a disease process is a likely indication that anti-cytokine therapy might be effective. In such cases, it might be important to serially monitor plasma levels of cytokine before, during and after therapy and to attempt to correlate its levels with clinical responses. Although there are many pitfalls associated with bioassays and immunoassays for cytokines, as discussed below, the availability of these assays has rapidly advanced the knowledge of cytokines in health and disease. One principle that has emerged from this collective experience is that bioactivity of a cytokine is always more important than its presence or its level. Immunoassays only measure the latter. It follows that an investigator interested in defining the involvement of a cytokine in a specific biologic process has to

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select the combination of assays that can best inform him of the potential of this cytokine for modulating functions of its target(s). Clearly, this calls for a combination of bioassays and immunoassays performed in parallel on samples of body fluids, supernatants of isolated cells or supernatants of established and cultured cell lines. For practical reasons, immunoassays are generally selected as the assays of choice, although it is clear that they may not be as informative as bioassays in some cases. Cytokine production or expression by various cells can be measured at the protein or mRNA levels in cells separated from body fluids or tissues or directly in tissue biopsies, using immunochemistry, RT-PCR or in situ hybridization (ISH). In this type of analysis, the protein-based assays are perhaps more informative, because mRNAs for cytokines tend to be rapidly processed and may not be detectable even with very sensitive procedures. Assays based on cytokine production are preferable to those relying on expression, simply because function is more convincing than appearance, especially in pathologic conditions, where translational defects could occur, resulting in a cytokine accumulation in the Golgi without its concomitant secretion. Some of these assays can be performed with individual cells, allowing for the frequency analysis of cytokine producing cells in the populations of hematopoietic or tissue cells. Singlecell cytokine assays have gained a wide acceptance of late, largely because they are both quantitative and able to provide a definition of cellular types involved. Cytokine profiling, as opposed to measurements of individual cytokines, introduces an added complexity to the task. Nevertheless, to accomplish this task, rapid and cost-effective assays that utilize small sample volumes have been introduced. The newest methodologies for cytokine measurements employ multiplex formats, allowing for the simultaneous measurement of many different proteins. The advent of large-scale genome sequencing and application of the cytokine genes to microarrays is likely to further revolutionize cytokine measurements. In the near future, it will be possible to simultaneously evaluate changes in expression of many cytokine genes in response to a unique signal. Similarly, using tissue microarrays, it will become feasible to simultaneously observe the presence of one or more cytokines in cells located in different tissue sites. The usefulness of such

cytokine profiling for diagnosis or therapy will have to be determined in the future. However, considering the properties of cytokines, as described above, it seems that the definition of a cytokine cascade or a cytokine profile in response to a specific stimulus is much more likely to correlate with physiologic processes and perhaps therapeutic interventions than the currently available single-cytokine assays.

CYTOKINE MEASUREMENTS IN BODY FLUIDS OR CELLULAR SUPERNATANTS By far the most frequently used cytokine assays measure levels and/or activity of these mediators in body fluids obtained from patients or generated in cultures of various cells stimulated with specific antigens. These assays fall into two general categories: immunoassays and bioassays. The former, as exemplified by ELISA, are more widely utilized because of their specificity, ease of performance, rapid turnaround time and a relatively low cost. Like with all other antibody-based assays, however, the performance of ELISAs for cytokines, and more precisely the determination of their sensitivity and specificity, is entirely dependent on the quality of antibodies selected for the cytokine capture and detection. Importantly, biologically inactive cytokines or their fragments are detectable in immunoassays, if they are recognized by the antibodies employed for cytokine capture. In contrast, bioassays measure only biologically active cytokines, regardless of whether they are available as soluble proteins, cytokine–receptor complexes or cytokine–serum protein aggregates. While highly sensitive (in the picogram range), bioassays are not specific, and neutralization with cytokine-specific antibodies is necessary to confirm the identity of a cytokine being measured. This is the consequence of cytokine redundancy, which means that several different cytokines in the unknown sample could mediate the same biologic activity. In addition, the presence in body fluids of cytokine inhibitors (e.g. soluble cytokine receptors) might interfere with bioassays. Bioassays are also time-consuming as well as cumbersome to perform, because of the requirement for a target cell line, which serves as a read-out of the biologic response. Today, bioassays are mainly

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used for validation purposes rather than routine cytokine assessments. It remains unconfirmed at this time which of the two approaches to cytokine assessments is more clinically useful.

Bioassays for cytokines Bioassays require knowledge of tissue culture techniques, because they depend on the use of a cell line as a cytokine target. Generally, cell line-based assays provide accurate estimates of biologically active cytokines. They require a tissue culture facility or at least an access to a laminar flow hood, as the sterility of cells is essential for meaningful data interpretation. Bacterial growth or mycoplasma infection of the target cells must be avoided. To minimize the potential for contamination, all cell work should be performed under sterile conditions and all solutions prepared and aliquoted in a laminar flow hood. A CO2 incubator is used to grow the cells and to perform the bioassay. All materials that come into contact with the cells (tissue culture flasks, pipettes and pipette tips) must be sterile. These items may be purchased already sterile or may be sterilized, if an autoclave is available. Tissue culture media may also be prepared and then filter sterilized, or they may be purchased in an already sterile condition. Some cells require multiple, special additives for optimal growth. There may be a great variation in the reagent quality, and the best supplier for each reagent must be determined to assure the optimal growth conditions for the selected target cell lines. Some investigators pre-test for the best reagents and then buy a large quantity of the particular lot. The success of bioassays is strictly dependent on the characteristics of target cells, and any deviation from the established optimal conditions might be detrimental to the assay reliability. Bioassays for cytokines are generally performed with carefully selected cell lines, which can be grown indefinitely in vitro. The cell lines are selected for responsiveness to or dependence on a particular cytokine. Such cell lines have an absolute requirement for a particular cytokine (i.e. they stop growing unless the cytokine is present). This forms the basis for an anti-proliferation assay, since the cells die out if the sample does not contain the required cytokine. Other bioassays depend on the ability of cytokines to induce proliferation or death of target cells, as

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described below. For example, tumor necrosis factor (TNF) is measured based on its ability to kill the susceptible target cells, and cell death indicates the presence of the cytokine (Higuchi et al., 1995). Cell lines are advantageous, since they provide a uniform population of cells that are expected to respond to the tested cytokine in a similar manner. Cell lines used as targets may be obtained from the American Type Culture Collection (ATCC; www. atcc.org), a nonprofit organization in Manassas, VA, USA. Similar cell line repositories are available in Europe. The ATCC serves as a central repository for cell lines of various origins, and frozen cells may be purchased for a minimal price. In many instances, however, target cell lines may be donated by other investigators. Although the practice of exchanging cell lines is convenient, each investigator is obliged to assure that these donated cell lines are free of mycoplasma contamination. The presence of mycoplasma infection can alter functional characteristics and growth of cells used as targets in bioassays, and thus it is not acceptable. In general, cell lines used as targets should be screened for mycoplasma at regular intervals, using commercially available DNA hybridization-based assays.

Types of bioassay Bioassays may be divided into four broad categories based on the type of function measured in the target cells incubated in the presence of a cytokine: (1) assays which measure proliferation or growth inhibition; (2) assays of cytotoxicity; (3) assays dependent on the induction of a specific cell function, such as chemotaxis; and (4) assays estimating the quantity of a protein induced in the target cell. In cytokine-driven proliferation or proliferation inhibition assays, the level of a cytokine is determined by its ability to stimulate or inhibit proliferation of selected target cells (Randall et al., 1993). Increasing the concentration of the cytokine increases cellular proliferation or leads to cell death. At the end of the assay, there are increased (or decreased) numbers of cells, which are detected using cell counts or tritiated thymidine incorporation into cellular DNA. Proliferation of B9 cells in response to IL-6 is an excellent example, as described by Remick (2002). These cells will not only increase in number in the presence of

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IL-6, but in the absence of IL-6, they will die out over time. Both of these types of responses are measured by determining the number of cells at the end of the assay. Another well-known bioassay utilizes a CTLL-2 cell line as a target for measuring proliferative activity of IL-2 (Gillis et al., 1978) (Figure 61.1). While incorporation of tritiated thymidine or manual cell counts are classic methods for measuring proliferation, alternative non-radioactive procedures have been introduced and gained popularity. For example, the cells may be pulsed with MTT-tetrazolium (3-[4,5dimethylthiazol-2-yl-]2,5-diphenyltetrazolium bromide) for 4 to 6 h, as described (Mosmann, 1983; Tada et al., 1986). As the proliferating target cells undergo oxidative respiration, the dye is metabolized to form insoluble dark purple formazan crystals. The crystals are dissolved by removing some of the supernatant and adding an appropriate solvent, such as a detergent or acidified isopropanol (Tada et al., 1986). The 96-well plates are then conveniently read in a microELISA reader. If the plate reader is interfaced with a computer, the acquired data may then be easily

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FIGURE 61.1 A bioassay for IL-2 using CTLL-2 cells. Supernatants of tumor cells (PCI-13) transduced with the gene for human IL-2 were collected and tested for the ability to promote growth of CTLL cells. Thymidine incorporation was measured as an indicator of cellular proliferation. As the concentrations of IL-2 increased in the tested supernatant, so did the proliferation of CTLL-2 cells.

analyzed. There are newer, water soluble tetrazolium salts available, such as XTT (Roehm et al., 1991), MTS (Buttke et al., 1993) or WST-1 that produce soluble formazan products and do not require the solubilization step (Goodwin et al., 1995; Marshall et al., 1995; Ishiyama et al., 1996). The results of proliferation assays are expressed in c.p.m. (for tritiated thymidine incorporation) or in arbitrary half-maximal units (for metabolic assays) defined as the dilution which produces a half-maximal response (Remick et al., 1989). Two different approaches may be used for calculations of activity: if a recombinant cytokine is used as a standard, the activity of a test sample may be directly compared with the standard; alternatively, activity of the test sample may be compared only with the maximal response of the target cells, by plotting responses against the dilutions (with both axes in a logarithmic scale) and calculating a half maximal response (Remick et al., 1989). Several computer programs are available which will calculate the equation for a curve generated with the recombinant standard. In cytotoxicity assays, the cytokine exerts a direct toxic effect on target cells growing as an adherent monolayer. Some cytokines, such as TNF-a or TNF-b, are cytotoxic for susceptible cell lines. As the concentration of these cytokines increases, more cells are killed during the assay. The uptake of a dye (e.g. naphthol blue-black or neutral red) by residual viable cells at the end of the assay can be quantitated using an ELISA reader. This response is inversely proportional to cytotoxic activity of the cytokine, and it often occurs in a log-linear fashion, where the toxicity of the cytokine declines logarithmically with its dilution. An example of this response is the WEHI 164 cell line, which is very sensitive to the toxic effects of TNF (Remick, 2002). Increasing concentrations of TNF increases the death rate of the cells. The third type of a bioassay evaluates the induction of a specific cell function other than proliferation. The cytokine is mixed with the target cells and a cellular function is measured. Various cellular functions can be used for building a bioassay. These range from secretion of secondary cytokines (Callard et al., 1987), changes in phosphorylation of signaling molecules (Sadick et al., 1996), alterations in the calcium flux (Yu et al., 2000), up-regulation of the MHC molecules (Gibson and Kramer, 1989), release of cytoplasmic granules (Schroder et al., 1987), induction of respi-

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ratory burst in macrophages or granulocytes (Doyle et al., 1994) or promotion of chemotaxis by the chemokines (Boyden, 1962; Junger et al., 1993; Taub et al., 1995a, 1995b). The selection of the appropriate assay is generally governed by the known or suspected ability of the cytokine being evaluated to mediate the specific function. Although these bioassays are firmly based on functional assessments of cytokines, none has so far been adapted to a routine clinically applicable format. These assays are primarily used in research laboratories. Among them, those that are flow cytometry-based have an excellent potential for developing into rapid, robust and relevant cytokine bioassays pending their validation. The fourth type of bioassay is a two-step procedure. The cytokine will induce a specific cellular function, which will then be measured in the next step. An example of this is the bioassay for IL-18. IL-18 will induce T cells to secrete interferon-c (Konishi et al., 1997; Dinarello et al., 1998). The first step of the assay mixes the T cells with the IL-18 and a co-stimulant. The second step measures the amount of IFN-c secreted into the supernatant. Antiviral assays can also be included in this category (Lewis, 1995a, 1995b). They involve the pre-incubation of susceptible cells with interferons followed by measurements of an increased survival and protection from cytopathic effects of a challenge virus, such as encephalomyocarditis virus or vesicular stomatitis virus. Target cells are human primary fibroblasts or cultured Hep 2C, WISH or A549 cell lines, and the number of viable target cells is determined at the end of the assay. Bioassays for individual cytokines are not described in this chapter. However, the reader is referred to three recent reviews, one of which is available on line, for operating procedures and useful tips on bioassays measuring various cytokines (Mire-Sluis and Thorpe, 1998; Remick and Whiteside, 2000; Remick, 2002).

Advantages and pitfalls of bioassays for cytokines The fact that bioassays are infrequently performed on a routine basis is indicative of their complexity and their cumbersome, labor-demanding nature (Bienvenu et al., 1998). The requirements for tissue culture and for neutralization of biologic activity with cytokinespecific antibodies to confirm assay specificity are

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difficult to implement and expensive. In addition, the analysis of S-shaped dose–response curves generated in the bioassay is not a simple matter, requiring special computer programs (Rossio, 1997) and careful data interpretation. The bioassay generally requires days as opposed to hours to complete, and the overall time of performance, as well as of labor invested, compare unfavorably with the ELISA-type immunoassay. The bioassay does not lend itself to automation or a rapid throughput, and it is not appropriate for a large-scale screening of specimens. For these reasons, it has largely remained in the research domain. It should be remembered, however, that the bioassay is a ‘gold standard’ of cytokine testing, not only because it is more sensitive than the immunoassay, but primarily because it is biologically relevant. Since it measures bioactivity of cytokines and not only their presence or levels, it is the only biologically relevant cytokine assay, aside from in vivo animal-based experiments. For this reason, the bioassay is most often used for confirmation of results obtained in immunoassays or for validation purposes. The major problem associated with bioassays is that they are not specific for a single cytokine. Cells used as targets are generally responsive to a range of cytokines or growth factors, and the presence of inhibitory cytokines can mask effects of the analyses (Randall et al., 1993). Synergistic effects of two or more cytokines present in the tested sample could also result in an overestimated cytokine content, as described previously (Wadhwa et al., 1995, 1996). To confirm the identity of the cytokine responsible for the effect, it is necessary to neutralize it by using cytokine-specific antibodies. While often successful in dissecting the responses of target cell lines to cytokines, neutralization is strictly dependent on the quality of available antibodies, and it considerably adds to the complexity of the assay. Attempts have been made to increase the specificity of bioassays by transfecting cytokine receptors into cell lines (e.g. Hu-Li et al., 1989), but even these transfected target cells respond to other cytokines. Similarly, reporter gene assays have been developed to increase the sensitivity of cytokine detection by transfecting target cells with a construct consisting of the promoter of a gene known to be induced by the tested cytokine fused to a reporter gene system (Abe et al., 1994).

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Immunoassays for cytokines Immunological assays for cytokines are more convenient, easier, quicker and less expensive than bioassays (Dinarello, 1992; Whiteside, 1994a). Today, immunoassays for most known cytokines and chemokines are available in pre-packed kits, which are convenient and easy to use. Immunoassays can discriminate between cytokines with similar biologic activities (e.g. TNF-a and TNF-b or IL-1a and IL-1b) based on their distinct immune reactivity. The methodology relies on the ability of a specific antibody to recognize the cytokine being tested. Both radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) exist. With RIAs, the radioactivity (usually 125 I) is detected in a gamma counter, and the c.p.m. are then used to construct a standard curve to allow for establishing of a correlation between the amount of cytokine and the c.p.m. Conventional competitive binding RIAs are rapid and easily quantifiable, but not sufficiently sensitive to measure physiologic or even pathologic levels of cytokines. They frequently suffer from unspecified ‘matrix’ effects when complex biological samples are tested, which means that high and unpredictable background values interfere with the assay interpretation. To avoid this, semi-purification or extraction of the sample is required (Cannon et al., 1988). Furthermore, RIA users have the disadvantage of working with radioactivity, especially the gamma emitter, 125 I. The most widely utilized immunoassays are based on a ‘two-site’ principle, where one antibody coated on to a plate is used to capture the antigen, and another antibody is used to detect the captured antigen (Plate 61.2) (see Plate section). The detection antibody is frequently monoclonal and can be radioactively labeled for immunometric assays (IRMA) or conjugated to an enzyme for ELISA. In either case, the assay can be used to measure cytokines in complex biologic fluids without pretreatment of samples (Thorpe et al., 1988). ELISA is routinely performed in 96-well plates and, at the end of the assay, a color reaction is developed. The intensity of the color in each well is quantitated spectrophotometrically, using an ELISA reader, and the degree of activity is directly related to a standard curve run on every plate. ELISA is usually easy to perform, and several of the steps may be automated. ELISA may be performed as either a direct or indirect

assay. In the direct assay, the cytokine to be tested is directly bound, or captured, to the plastic of a 96-well plate. After the cytokine (antigen) is firmly adhered to the plate, unbound substances are washed away, and an antibody recognizing the cytokine is added to each well. This antibody is conjugated to an enzyme. After washing away unbound antibody, a substrate solution is added. The enzyme conjugated to the antibody will reduce/oxidize the substrate to yield a colored reaction. The intensity of the color reaction is directly proportional to the amount of antibody on the plate, which in turn is related to the amount of cytokine. This method is convenient, rapid and very specific, provided the antibody directed against the cytokine has specificity. However, it relies on the ability of the cytokine to bind to the plate before it is detected. This may be difficult, either because the cytokine does not bind well, or because the cytokine is present in a complex protein mixture, such as plasma or serum, where several other proteins will compete for binding. Also, immobilization of capture antibody or capture antigen sometimes results in steric changes that compromise binding of subsequent assay components. It has been suggested that allowing for the antigen– antibody reaction to occur in the liquid phase, followed by capture of antigen–antibody complexes by anti-immunoglobulin antibody bound to the plate can successfully resolve the problem (Wadhwa et al., 1990). In an indirect or sandwich ELISA shown in Plate 60.2, several of these difficulties are eliminated. In the first step, an antibody directed against the cytokine is bound to the ELISA plate. Antibodies, which are large proteins, bind extremely well to ELISA plates at the pH range of 7.5 to 9.0. Purified antibodies are used, and since this is the only protein present during the binding step, there is no competition for sites. Excess, unbound antibody is removed, and the cytokine is then added. After incubation and washing away extraneous material, the enzyme conjugated antibody is added. Following incubation and washing, the only enzyme-conjugated antibody that will remain is that bound to the antigen. As can be seen in the figure, the tested cytokine is ‘sandwiched’ between two antibodies. For this reason, this type of ELISA is often referred to as a sandwich ELISA. The color is developed by the addition of the appropriate substrate (e.g. DeForge and Remick, 1991).

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There is a variation of both the direct and indirect ELISA that is often used in order to increase sensitivity. The detection antibody is conjugated to biotin rather than an enzyme. An additional step is then included in the assay, whereby avidin or streptavidin conjugated to an enzyme is added. The avidin–biotin interaction is very strong, and adding this second layer considerably enhances the sensitivity of the assay.

Advantages and pitfalls of immunoassays for cytokines Immunoassays have many advantages over bioassays. They are generally performed using commercially available kits, and thus are easy and quick to perform, with the turnaround time of hours, as opposed to days for bioassays. Immunoassays can be easily automated, and because they are based on antibody specificity, they can be used to distinguish cytokines with similar biologic activities (e.g. IL-1a and IL-1b or TNF-a and TNF-b), which are indistinguishable in bioassays. Nevertheless, immunoassays have disadvantages as well. Since both RIA and ELISA depend on an antibody recognizing a specific antigen or its epitopes, their performance is strictly dependent on the quality of antibodies used to build the assay. Not only should the antibody be specific for the cytokine measured, it should also have high affinity. Further, antibody titer is important. In general, monoclonal antibodies are preferable because of their specificity, although the sensitivity of detection may not be optimal. Polyclonal antibodies can react with other factors present in the biological samples to yield false-positive results. This is especially evident when structural homology exists between two cytokines, such as lymphotoxin and tumor necrosis factor (Pennica et al., 1984). Immunoassays are generally less sensitive than bioassays, being capable of detecting cytokines in the picogram to nanogram range. They also have a narrow dynamic range, and sample dilutions are necessary, if a cytokine is present at a high concentration. As indicated above, nearly all of the problems with immunoasays for cytokines are related to the quality of antibodies used for capture and detection of these molecules. The most commonly encountered problem is that antibodies incorporated in the assay might

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be detecting degraded peptide fragments, which are immunoreactive, but not biologically active. Thus, in certain circumstances, a lack of correlation between the biologic activity of a cytokine and its presence, as detected in an immunoassay, might become evident. The loss of bioactivity could be due to proteolytic cleavage or the presence of inhibitors (e.g. soluble cytokine receptors) in the biologic fluid. Another set of problems is related to variations in the primary structure of the antibody due, for example, to glycosylation or amidation, which might interfere with its binding to cytokine molecules. In this case, immunoassay results may be negative, while bioassay is positive. In addition, results of ELISA are frequently influenced by the ‘matrix effects’. This refers to nonspecific binding of proteins present in complex biologic fluids, which contributes to a high assay background and thus interferes with the result interpretation. The disparity of results observed when using ELISA kits made with antibodies from different sources is likely due to antibody characteristics such as its ability to bind to plastic or its affinity for the cytokine. Certain antibodies are able to detect soluble (secreted) as well as membrane-bound forms of the same cytokine, while others detect only soluble cytokines. Clearly, a discrepancy in results will be obtained in ELISAs incorporating these different antibodies. For example, IL-1 is known to be secreted as a 17 kDa fragment, but it also exists as a larger cellbound molecule (Dinarello, 1992). ELISA formats designed to measure 17 kDa IL-1b were reported to underestimate the 35 kDa membrane-associated cytokine (Herzyk et al., 1992). An antibody might be able to detect not only a free, soluble cytokine, but also the cytokine bound to its receptor or the cytokine-forming immune complexes in the circulation. In such a case, levels of the cytokine detected in the serum will be considerably higher than those measured in ELISA built with an antibody recognizing only the soluble form. Certain commercially available cytokine kits detect highly elevated levels of cytokines in normal human sera. It is possible that the antibodies used in these kits measure both free and bound cytokines (Paciotti et al., 1992). At this time, the biologic significance of such measurements is not known, and it may be important in such cases to resort to the simultaneous use of bioassays and immunoassays to confirm bioactivity of the analyte.

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The presence in biologic samples of specific inhibitors of cytokines such as soluble cytokine receptors, cytokine antagonist molecules (e.g. IL-1b antagonist) or antibodies to cytokines might complicate detection of cytokines in biologic fluids (Engelberts et al., 1991; Gallay et al., 1991; Dinarello et al., 1998). Non-specific inhibitors present in human sera, such as albumin, a2-macroglobulin, lipids, tend to bind to cytokines, so that the assay might give false-negative results (James, 1990; May et al., 1992; Bargetzi et al., 1993). A variety of procedures have been tried to remove non-specific serum inhibitors of cytokines, including chloroform extractions (Cannon et al., 1993), treatment with polyethylene glycol (Larrick, 1989; Nemzek et al., 1999) and simple serial dilutions of specimens. It is unclear whether the assays employing these treatments provide biologically relevant information, however. The removal of specific cytokine inhibitors by using, for example, immunoaffinity columns for soluble cytokine receptors or immune complexes, might interfere with detection of cytokines and are not validated to date. Perhaps the most common pitfalls associated with cytokine detection stem from less than optimal handling of biologic specimens to be tested. The collection and storage of specimens for cytokine assays have to avoid or minimize losses of these proteins due to proteolytic degradation, denaturation or even binding to clotted serum components. Body fluids should be placed in the cold immediately after collection to avoid such losses, and the storage of samples at 70°C in small aliquots to avoid freeze–thaw cycles is recommended.

CYTOKINE PROFILES AND THEIR BIOLOGIC SIGNIFICANCE Measurements of a single cytokine in a biologic fluid or cellular supernatant are seldom informative. Activation of a cytokine cascade by a physiologic or pathologic stimulus usually leads to the production and release of several biologically related cytokines. For example, in evaluating immune responses of the host to antigens, it is desirable to distinguish between the presence of Th1-type (e.g. IL-2, IFN-c, TNF-a) versus Th2-type (e.g. IL-4, Il-5, IL-10) cytokines

(Romagnani, 1997a, 1997b). An inflammatory cascade induces a release of the so-called pro-inflammatory cytokines, and to confirm the inflammatory reaction, it may be advantageous to look for a proinflammatory cytokine profile rather than for individual components of the cascade, which may or may not be present at the detectable level. The preference for simultaneous measurements and discrimination of several different cytokines in biologic fluids has led to the development of several new formats of cytokine assays. These formats are generally referred to as ‘multiplex assays.’ One that has gained considerable popularity recently is a multiplex bead immunoassay designed to work in conjunction with the Luminex instrument (Carson and Vignali, 1999; Vignali, 2000). The technology utilizes distinct fluorescently labeled microspheres, each covalently linked to a cytokine-specific antibody. The combination of different beads in one tube allows for a simultaneous assessment of various cytokines. In the sandwich-type immunoassay similar in principle to ELISA, the capture antibody is immobilized on microbeads. It binds the cytokine present in the sample. Biotin-conjugated detection antibody specific for another epitope on the same cytokine is then added, and after extensive washing, fluorescein-labeled streptavidin is introduced. Fluorescence bound to beads is quantified in the Luminex instrument, a flow analysis system designed to perform simultaneous multi-analyte detection. The intensity of measured fluorescence is directly proportional to the concentration of the cytokine that was present in the original specimen. The currently available panels of microbeads allow for measuring a dozen or more of selected cytokines in a single 50 ll sample (Carson and Vignali, 1999; Vignali, 2000). All of the assay components (microbeads with immobilized capture antibody, detection antibody, recombinant cytokine standards, serum diluent, streptavidin-FITC, buffers and 96-well microtiter filter plate) are included in the assay kits available from an increasing number of vendors. Customized multiplex bead assay kits are commercially available in a form allowing for mixing pairs of capture and detection antibodies for detection of desired cytokines in a specimen. Multiplex assays have the capability to rapidly define a cytokine profile in very small biologic samples. For example, the Th1/Th2 panel measures IL-2, IFN-c, Il-4, IL-5 and

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IN VITRO PRODUCTION OF CYTOKINES

IL-10, while the pro-inflammatory panel quantitates human IL-1b, IL-6, IL-8, GM-CSF and TNF-a in a single sample. The multiplex cytokine assays offer flexibility, labor/time-saving and high throughput, with sensitivity and specificity that appear to be comparable to those of immunoassays for individual cytokines. An alternative procedure for rapid detection of cytokine profiles in biologic samples utilizes enhanced chemoluminescence (ECL). This technology is based on the use of magnetic beads coated with a biotinylated capture antibody and anti-cytokine detection antibody labeled with a ruthenium ion-containing reporter molecule (TAG). The light generated as a result of cyclical oxidation–reduction reaction initiated when TAG encounters an electrode is captured and measured (Deaver, 1995). Its intensity is proportional to the level of the TAG-labeled antibody bound to the cytokine captured on the magnetic beads. Somewhat similar in principle to the multiplex bead technology, and offering similar advantages of rapid, multiple-cytokine analysis in a single specimen, ECL seems to be less popular, largely because of the requirements for ruthenium-labeled antibodies and for special instrumentation, neither of which are widely available. Microplate-based assays which measure cytokinespecific mRNA are also available (Vukmanovic-Stejic et al., 2000; Gogos et al., 2000). These assays quantitate mRNA in cell lysates by using gene-specific biotin-labeled capture probes and digoxigeninconjugated detection probes. The cytokine RNA/ probe hybrid is captured on a streptavidin-coated microplate and following washing and the addition of the substrate (alkaline phosphatase conjugated to anti-digoxigenin antibody) the color is developed and read in a spectrophotometer. This method does not require mRNA purification, utilizes whole cells, is said to be more accurate than a Northern blot and has added advantages of being rapid, as well as adaptable to a high sample throughput. Another emerging technology features gene arrays to identify differentially expressed mRNA levels by comparing expression profiles of cytokines in different samples. This method can be used for the determination of cytokine genes up-regulated in response to physiologic or pathologic conditions, external signals or cellular stress. For example, using a side-by-

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side hybridization, it is possible to simultaneously determine the profile of cytokine expression during activation, proliferation or differentiation of various cell types. Although largely used as a research tool at this time, cytokine and chemokine gene arrays, applicable to screening of large numbers of biologic samples in a relatively cost-effective way, appear to be the technology of the future.

IN VITRO PRODUCTION OF CYTOKINES Measurements of plasma levels of cytokines are often not meaningful, as reviewed by Whiteside (1994a), except when studying pharmacokinetics of exogenously administered cytokines or in conditions associated with chronic cytokine release. In lieu of plasma assays, spontaneous or stimulated production of cytokines by PBMC or their subsets can be evaluated. The former is a measure of in vivo activation of these cells, while the latter can be viewed as a reliable measure of immune competence (Friberg et al., 1994). To measure spontaneous cytokine production, PBMC or a subset of purified cells are incubated in medium without any additional stimulation for 24–48 h, and the supernatant is then assayed for the presence of cytokines (Plate 61.3). This assay is based on the rationale that immune cells activated in vivo will spontaneously produce cytokines. However, any in vitro manipulation of cells prior to the assay will tend to give false-positive results and should be kept to a minimum. The stimulated cytokine production is a useful measure of the overall ability of tested cells to produce a cytokine. To be able to interpret the results correctly, it is necessary to establish normal ranges of cytokine production, using PBMC of normal volunteers (Friberg et al., 1994). We have previously determined that stimulated cytokine production by PBMC in response to anti-CD3 Ab or PMA/ionomycin is a stable trait for healthy volunteers (Rossi et al., 1993). Therefore, individuals with defects of cytokine production in response to ex vivo stimulation under uniformly controlled conditions can be identified using this approach. Furthermore, it has been demonstrated that stimulated cytokine production can be reliably performed on cryopreserved PBMC (Friberg et al., 1994), which is of practical importance. The

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obvious disadvantage of these assays is that the nature or identity of the cells responsible for cytokine secretion is unknown. These are assays for evaluating an overall potential of cellular populations to secrete cytokines in response to a particular stimulating agent (Plate 61.3). The advantage is that more than one cytokine can be simultaneously measured in the same cell supernatant, allowing for definition of a cytokine profile. Also the kinetics of cytokine production by various cell types can be determined using this assay format (Gonzales et al., 1994). A more sophisticated version of cytokine secretion assay, which is flow cytometry based, has become commercially available. The method permits detection and isolation of cytokine secreting cells, and it can be performed using whole blood or freshly separated PBMC (Oelke et al., 2000). The PBMC are stimulated with antigen for several hours to induce cytokine secretion by the antigen-responsive cells. A cytokine catch reagent, which is a conjugate of two different antibodies (anti-CD45 and anti-cytokine) is added and attaches to the surface of all leukocytes. During the subsequent incubation, a cytokine secreted by the antigen-specific cell binds to the catch reagent and this cell is visualized by a cytokinespecific detection antibody labeled with a fluorochrome. The labeled cells are then quantified in a flow cytometer (Munz et al., 2000). Alternatively, if isolation of cytokine-secreting cells is desired, magnetic labeling can be used in an additional step. Magnetic beads coated with anti-fluorochrome antibodies are added to the cells, and antigen-responding cells are positively selected in the magnetic field, using a special column (Manz et al., 1995). The cells recovered from the column can be used for flow analysis or cell culture. The cytokine secretion kits are commercially available in two different configurations suitable for detection only or for cell enrichment and detection. The enrichment reagents allow for recovery and analysis of antigen-specific, cytokine-secreting T cells, which are rare in the population (e.g. one in 105 cells). Because the enriched T cells are viable, they can be used for additional experiments. These antibodybased cytokine secretion assays have been largely used as means of separation of antigen-responsive T lymphocytes and establishing their frequency in tested cell populations.

DETECTION OF CYTOKINES IN CELLS AND TISSUES Cytokines are local mediators and their production or presence at the site of disease is likely to be more relevant to the ongoing pathologic processes than their systemic levels. Special methods have been developed for in situ detection of cytokines or for detection of cytokines in cells isolated from tissues. These methods include immunodetection of cytokine proteins or detection of cytokine mRNA using variously labeled probes.

Staining for cytokine proteins in tissues Immunohistochemistry for cytokines is an antibodybased method, and thus its success depends entirely on the selection of the antibodies to be used for cytokine detection (Sander et al., 1991; Chupp et al., 1998). Depending on the label conjugated to the antibody used in the assay (e.g. an enzyme or a fluorochrome), light or fluorescent microscope can be used for cytokine detection in tissues. Both direct and amplified (i.e. multilayer antibody) techniques can be selected, depending on the desired sensitivity of detection. Polyclonal or monoclonal antibodies to human cytokines are widely commercially available, but it is up to the user to select those that will perform optimally in the assay at hand. Thus, many antibodies that perform admirably in Western blots or immunoprecipitation for cytokine proteins are not suitable for immunostaining. The reason is that immunostaining has to be preceded by fixation, as without it, cytokines may be lost during washing of tissue sections. Even a mild fixation process might alter the conformation of a cytokine or cause its partial denaturation (Thorpe et al., 1992). Such altered cytokines will not be recognized by antibodies made to the cytokine in its native conformation. Therefore, only the antibodies able to recognize and bind to a cytokine after fixation should be selected for immunostaining. This may require prescreening of several different antibodies or even production of a new antibody. When appropriately selected and pretitered antibodies are used, immunostaining can provide useful information about tissue localization of cells expressing a particular cytokine and their relative fre-

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quency in the tissue microenvironment (Plate 61.4A). However, the interpretation of immunostaining for cytokines is not simple. The patterns of cytokine expression in different tissues vary, and they depend on the state of cellular activation, with activated cells staining more strongly than resting cells. This means that weakly or discretely stained cells might be missed. Non-specific staining has to be carefully controlled by the use of isotype control antibodies and checkerboard titration of anti-cytokine antibodies, as it may produce artefactual results. In examining tissue sections for cytokines, it is necessary to remember that a cytokine associated with a given cell may not be a product of this cell, but may be synthesized elsewhere. For example, cytokines found in the cytoplasm could be internalized products of other cells, and those associated with the cell surface could be occupying cellular receptors having been produced by another cell at a distant location. Only cytokines found in the Golgi zone of a cytokine-producing cell and often visualized as characteristic dot-like clusters can be considered as products of this cell. In immunostaining for cytokines, it is quite useful to stain for cytokine receptors in order to confirm the possible presence of an autocrine loop. Finally, it is not uncommon, particularly when using paraffinembedded specimens, to fail to detect a cytokine. Overall, immunohistochemical detection of cytokines in tissues is somewhat of an art, and even experienced pathologists have to devote considerable time and effort to become proficient in it.

Staining for cytokine proteins in single cells The staining methods for cytokine-positive single cells are antibody-based and thus similar to those described above. Cell permeabilization is necessary for detection of intracytoplasmic, as opposed to cell surface-associated, cytokines. A variety of such methods have been described (Labalette-Houache et al., 1991), and they all aim at a relatively gentle alteration of the cell membrane in order to allow the antibody to reach the cytokine inside the cell. Flow cytometry is the method of choice for analysis of cells stained for intracellular cytokines (Jung et al., 1993; Lewis and Campbell, 1995; Prussin, 1997; Chupp et al., 1998). Multiparameter flow cytometry allows for detection of

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more than one cytokine in the same cell or for the precise definition of the lineage of cytokine producing cells by using antibodies to surface markers. Successful staining for intracellular cytokines requires the use of: (1) a protein transport inhibitor, such as brefeldinA or monensin; (2) the appropriately gentle fixation and permeabilzation of cells; (3) anti-cytokine antibodies that bind to fixed cytokine proteins; (4) a stimulator protocol that generates suitably high frequencies of cytokine-producing cells. Like immunostaining in tissues, multicolor flow cytometry is applicable to analysis of hematopoietic and non-hematopoietic cells. Recently, this method has been widely applied to determinations of the frequency of antigen-reactive, cytokine-producing T cells in the peripheral circulation or in in vitro stimulated populations of cells (Picker et al., 1995; Waldrop et al., 1997). An example of the flow cytometry assay to determine the frequency of IFN-c producing T cells in response to an antigen is provided in Plate 61.5.

Methods detecting cytokine transcripts Production and expression of cytokine mRNA transcripts in cells and tissues can be accomplished by reverse transcriptase-polymerase chain reaction (RT-PCR), RNase protection assays (RPA) or in situ hybridization (ISH). The rationale for measuring cytokine transcripts rather than proteins is based on the notion that the former are detectable only in cells producing a given cytokine (ISH) and that in some pathologic conditions, cytokine messages may be present, but are not translated into proteins. On the other hand, RT-PCR identifies the presence of transcripts, but not the cell from which they are derived. The quality of tissue submitted for RPA, ISH or RTPCR remains the most critical aspect of this technology. The main and general disadvantage of the mRNA-based methods is the absolute requirement for an excellent quality of the nucleic acid, which requires rapid and special tissue/cell handling to prevent its degradation. To this end, it is obligatory to snap-freeze human tissues immediately (within minutes) after surgery in the operating room and never allow them to warm up for fear of activating endogenous RNases. The presence of mRNA for a cytokine in tissue is meaningful, but its absence is not, because the presence of undegraded RNA in tissue determines

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success. Also, mRNA transcripts for cytokines are especially short-lived, perhaps because of the dangers associated with excessive production of these powerful mediators. Thus the absence of cytokine transcripts could mean rapid processing or degradation rather than lack of their synthesis. Nevertheless, RT-PCR (Gilliand et al., 1990; Kruse et al., 1997; O’Garra and Vierra, 1992) and especially its newer version, real-time RT-PCR, have been widely used for cytokine message detection. The technique of RT-PCR determines the presence of a gene message using pairs of specific primers and a fixed number of amplification cycles. The method is highly sensitive and can be performed in a semiquantitative or quantitative format (Carding et al., 1992; Heid et al., 1996). Realtime qRT-PCR allows for accurate measurements of the copy number of a particular message relative to the expression level of a housekeeping gene or an endogenous reference, such as rRNA. The final value obtained represents the absolute number of mRNA copies per a selected number of rRNA copies, e.g. 108. The method enables the investigator to quantitatively compare cytokine gene expression between several different targets (Gibson et al., 1996). Once established, the real-time qRT-PCR is rapid and efficient, but the requirement for special equipment and stringent quality control tend to limit its use to special laboratories. The RPA system is a highly sensitive and specific method for simultaneous detection of up to 12 different mRNA species in a single sample of total RNA (Dent et al., 1997; Sallusto et al., 1998). The method utilizes specific riboprobes, which are generated using DNA-dependent RNA polymerases from the bacteriphages T7 or T3. The polymerases drive the synthesis of antisense RNA probes from DNA templates, each of distinct length and each representing a unique sequence in a specific mRNA species. The resulting linear probes are hybridized with target mRNA. After free probes and other single-stranded RNA molecules are digested with RNase, the hybridized probe/target RNA duplexes are resolved according to size on polyacrylamide gels and transferred to a nylon membrane. Quantification of the RNA fixed to the membrane by UV crosslinking follows, based on the label incorporated into antisense RNA probes (either 32P or biotin-16UTP). ISH for cytokine transcripts is less popular than

real-time RT-PCR and perhaps more difficult. Freshly harvested and quick frozen tissue biopsies are preferable over archival paraffin-embedded tissues for cytokine gene expression studies (Plate 61.4B). Comparative published data indicate that mRNA for cytokines is decreased or absent in paraffin-embedded specimens (Bromley et al., 1997). ISH can be performed as a radioactive procedure, in which the cytokine-specific oligonucleotide probes are labeled with, e.g. S35, or as a non-isotopic method with enzymatically labeled oligonucleotides (Vitolo et al., 1992, 1993, 1994; Naylor et al., 1995). Its main advantage is that it detects individual cells or clusters of cells expressing mRNA for a cytokine in normal tissue versus pathologic specimens (Zhang et al., 1994; Zhao et al., 1996). Furthermore, this detection is not dependent on the threshold level of cytokine production per cell. It should be remembered, however, that the presence of cytokine mRNA transcripts and protein do not always correlate (Wadhwa and Thorpe, 1998). The possibility for a combination of ISH with other methods, e.g. immunostaining, to identify or better characterize the producer cell is another advantage of this method (Naylor et al., 1995).

Frequency analysis by ELISPOT Methods based on cytokine expression or production by T lymphocytes stimulated in vivo or in vitro with a relevant antigen have become widely utilized in recent years. These methods have been especially useful for monitoring of responses to vaccines. Among these methods, the enzyme-linked immunospot assay (ELISPOT) has been developed to determine the frequency of precursor cells in fresh or cultured populations of lymphocytes capable of releasing cytokines in response to stimulation by a cognate antigen (Czerkinisky et al., 1988). ELISPOT has gained special popularity recently for several reasons. It is performed in 96-well plates and is adaptable to a high throughput, can be set up in several different formats either with unseparated PBMC or enriched subpopulations of lymphocytes and its results (i.e. the number of spots per well) can be determined in an unbiased way by image analysis. However, its most important feature is that it can provide the frequency of antigen-responsive cells among those plated (Herr et al., 1994, 1996, 1997). The principle of the assay

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is illustrated in Plate 61.6. ELISPOT is sensitive, capable of detecting one cytokine-producing cell per 105 plated, and it can be performed with cryopreserved PBMC (Asai et al., 2000; Bennouna et al., 2002). ELISPOT assays for IFN-c, IL-5, IL-10 and other cytokines can be set up by individual laboratories, but once again, the quality of capture and detection antibodies used to build the assay will determine its performance. We have evaluated ELISPOT as a monitoring assay for anti-cancer vaccines and find it to be useful for the assessment of CTL precursor frequencies prior to and after peptide-based vaccination in patients with cancer (Asai et al., 2000; Meidenbauer et al., 2000, 2002). Others have used it to study the frequency of viral-specific CTL (Lalvani et al., 1997; Doherty, 1998) in the peripheral circulation. In the research laboratory, this assay has almost replaced the use of more laborious cytotoxicity assays for the detection of antigen-specific T cells. The ELISPOT assay can be performed in several different formats, depending on the antigen used and the frequency of antigen-specific precursor cells in the peripheral circulation. The simplest format involves adding class-I MHC-restricted peptides to the appropriately HLAmatched PBMC for a duration of a 24 h assay and reading the number of spots, which directly reflects the frequency of the peptide-responsive T cells in the circulation (Scheibenbogen et al., 1997). When such a frequency is low, however, PBMC may be separated into CD8 and CD4 subsets which are then tested individually in the ELISPOT assay. In vitro sensitization (IVS) of T cells with the peptide(s) can be performed by culturing peptide-pulsed PBMC over a period of 1 or 2 weeks prior to the assay. Finally, with complex antigens or antigen–antibody complexes, presentation on dendritic cells (DC) may be necessary to induce T cell response. In such cases, the ELISPOT format presented in Plate 61.7 can be used (Bennouna et al., 2002). Newer modification of the assay allow for simultaneous detection of several cytokines (Okamoto et al., 1998). The interpretation of ELISPOT results is not simple. The most common problem is that of high background counts with responder T cells alone. The problem may become acute when IVS is used or when antigens are being presented on DC. The widespread practice of subtracting the number of background

from experimental spots is only admissible if the difference between the two is significant by a permutation test (Whiteside T.L., unpublished data).

GENOMICS FOR CYTOKINES The widening use of DNA chips or microarrays allows for the simultaneous analysis of many thousands of genes from various tissues or cells (Pollack et al., 1999). This methodology can be applied to screening for expression of cytokine genes in normal or pathologic tissues or cells (Foster, 2001). It involves mRNA extraction from the cells or tissues and its subsequent labeling and hybridization to the microarray. The hybridized products are visualized in a phosphorimager and analyzed using software capable of comparing control with test samples and of detecting differences in expression of cytokine genes in these samples. In this way, quantitative information on the level of gene expression, i.e. its induction or downregulation, is obtained. The microarrays are commercially available, and the opportunity for simultaneous and rapid screening of multiple samples for cytokine gene expression is likely to advance our understanding of the cytokine profiles associated with various human diseases or responses to therapies. In the experimental settings cytokine microarrays should help in understanding changes in cytokine cascades in specific tissues or cell types responding to external signals. Although it is too early to predict to what extent cytokine genomics will become diagnostically or clinically useful, it is safe to speculate that the future use of this technology will enormously expand our knowledge of cytokine biology.

PROTEOMICS FOR CYTOKINES Similar to gene arrays, new technological advances combining 2D polyacrylamide gel electrophoresis with mass spectroscopy allow for simultaneous analysis of many hundreds of proteins in body fluids or tissues (Anderson et al., 2000). This approach is based on the concept that pathologic changes in tissues or cells might be reflected in distinct patterns of cytokine proteins detectable in body fluids or cellular

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supernatants. Still in its infancy, particularly in respect to cytokine detection, this technology has been successfully used to map proteomic changes in sera to identify, e.g. patterns characteristic of ovarian cancer (Petricoin et al., 2002). The potential for capturing of distinct cytokine patterns or polarization in their production and relating them to a particular biologic event is clearly there, and it has an enormous experimental appeal.

SELECTION OF CYTOKINE ASSAYS With a broad array of cytokine assays available, the rational selection of the ‘right’ assay becomes an issue. In view of the fact that each cytokine acts on numerous target cells, inducing activation of not one but usually several genes, it has been all but impossible to ascribe a precise role to any single cytokine. At the same time, any cellular alteration is likely to result in the induction of not one but several cytokines, and their pleiotropic effects are also determined by the microenvironment in which they operate. Selection of which cytokine or how many cytokines to measure is therefore not a trivial decision (Whiteside, 1994a). The timing of cytokine measurements is also critical, because after induction, their concentration may peak rapidly and then drop to undetectable levels. The measurements of cytokines in the blood may not be optimal, while the availability of tissue biopsies or tissue exudates is limited. Cytokine production assays require separation of cells, and the RT-PCR-based methods call for mRNA extraction under rigorous conditions, as described above. The newer assays assessing cytokine polarization and cytokine profiles provide a wealth of information that is frequently difficult to interpret and correlate to the available clinical data. While the approach of performing a panel of cytokine assays, including those for cytokine receptors, may ultimately be validated, technologies for rapid screening of multiple cytokines are just beginning to be introduced. Today, no single method meets the criteria for a comprehensive, biologically relevant assessment of these soluble mediators. The average user is thus required to select a cytokine assay that is most likely to meet expectations, without completely understanding the complex biology of cytokines. It is

obvious that therapeutic administration of cytokines needs to be accompanied by monitoring of their plasma levels. Aside from this general recommendation, it is not possible at present to provide rational guidelines for cytokine measurements in disease. Perhaps the most useful application of cytokine assays today is in evaluation and quantitation of cellular responses to stimulatory signals. Assays such as ELISPOT, intracytoplasmic cytokine (ICC) assays for flow cytometry and flow-based cytokine secretion assays discussed above have become essential for monitoring of T cell responses to peptides or antigens both in research laboratories and in clinical trials. Overall, it has to be acknowledged that both the choice of a cytokine assay(s) and meaningful evaluation of assay results are difficult at this time. More than one assay might be necessary for correct data interpretation. The services of reference laboratories which specialize in cytokine testing might be preferable to the ad hoc use of commercial ELISA kits or individual bioassays, particularly when samples collected in the course of clinical trials are to be analyzed.

SAMPLE PREPARATION Collection and processing of samples for cytokine assays is an important component of testing if artifacts are to be avoided (Thavasu et al., 1992; Whiteside, 1994b). These occur for two main reasons: (1) cytokines present in the collected body fluids or supernatants are degraded or lost because of inappropriate specimen handling; or (2) specimens are collected in a correct way, but stored and assayed under conditions that are not optimal. In the first category, collection of body fluids into anti-coagulants such as EDTA or citrate might interfere with cytokine bioassays by removing divalent cations. In general, it is best to avoid coagulation, which might activate cells or result in removal of some cytokines with the clot, and use plasma rather than serum for cytokine assays. An important step is to place the specimen in the cold immediately after phlebotomy. In the second category of artifacts, prolonged storage in ambient temperatures, repeated freezing and thawing of samples or additions to the assays of mitogenic substances and cytostatic reagents, are all likely to interfere with cytokine testing. In ELISA, serum or plasma matrix,

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effects are frequently a reason for unacceptably high backgrounds. Also, the presence in body fluids of soluble receptors, cytokine antibodies or other cytokine binding proteins might interfere with the cytokine detection, and the removal of these inhibitors may be required (Cannon et al., 1988).

QUALITY CONTROL FOR CYTOKINE ASSAYS At this time, cytokine assays are not performed as routine clinical laboratory procedures. In fact, few laboratories have established standard operating procedures (SOPs) for cytokine measurements. Usually, a user selects a kit or reagent based on the recommendations of the manufacturer and performs the assay according to the procedure enclosed in the kit. Few users compare between kits to select the optimal performer and most forget that variations between lots of antibodies are likely to affect the assay results. As with other assays for biologic materials, it is essential that cytokine assays be correctly and uniformly standardized. The best way to assure that an assay is reliably detecting an analyte is to calibrate all home-grown cytokine procedures and commercial kits or reagents against the cytokine reference standards, regardless of assurances or calibration data available from manufacturers of cytokine kits. The Biological Resources Branch (NCI-FCRDC), the Division of Microbiology and Infectious Diseases (NIAID) and the National Institute for Biological Standards and Control (United Kingdom) make available reference reagents for human cytokines (Reynolds, 1997). These reagents can be obtained free of charge upon request. It is recommended that all users of cytokine kits or reagents, including laboratories working with cytokine bioassays, take advantage of this opportunity and set up QC programs based on these cytokine standards. To maintain the continuity of calibration with a limited quantity of the cytokine standard, an in-house reference sample can be identified, calibrated and frozen to be used each time the assay is performed. While a QC program for cytokine assays begins with obtaining and using the cytokine standards, it also includes the following components (Whiteside, 1994a; Aziz et al., 1998, 1999): (1) Availability in the laboratory of SOP for every cytokine tested. (2) Estab-

lishment of normal ranges for cytokine levels, using samples obtained from normal individuals. Each laboratory ought to define and use its own normal ranges for different cytokines in various biologic fluids. (3) Monitoring of inter-assay and intra-assay variability, using cytokine-rich supernatants prepared in bulk (e.g. by in vitro activation of normal lymphocytes or monocytes) every few months, stored at 80°C in small aliquots and included in every assay. Comparisons of different lots of such supernatants with the cytokine standard or with the previously calibrated lot is made before their acceptance. This type of strategy lends itself well to determinations of both inter-assay and intra-assay variability. (4) Determination of intraassay variability by incorporating blind duplicates of control or patient samples in the different positions each time the assay is performed. (5) ‘Spiking’ of samples with the known amount of the authentic cytokine followed by measurements of its recovery to rule out the presence of inhibitors, and (6) Participation in an external proficiency program, even one that is informally organized by several collaborating investigators, is highly recommended. For monitoring of serially collected samples, it is preferable to batch them, so that serial plasma samples from the same patient obtained in the course of a clinical trial and maintained at 80°C are thawed and tested together in a single assay. This avoids interassay variability. Results of cytokine assays should be reported in picograms or nanograms of cytokine protein per milliliter and not in arbitrary units. Collection and storage of samples for cytokines under conditions that prevent their degradation or a loss of activity are important and need to be rigorously followed, as discussed above.

CONCLUSIONS Cytokine measurements have been an important part of the process of discovery and understanding of the cytokine biology, although the role various cytokines play in health and disease remains incomplete, significant progress has been made in linking certain cytokines to pathologic changes in disease. This has encouraged efforts to monitor patients’ plasma and/or tissues for levels of cytokines and to try to correlate results of various cytokine assays with

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clinical endpoints. Cytokines or cytokine profiles are not yet ‘biomarkers’ of disease or disease activity. Interactions of one cytokine with another, with their soluble receptors or with various antagonists could, in part, explain the lack of correlation observed between systemic levels of cytokines and the clinical findings. Not only can an excess of one cytokine result in suppressed production of another, but an excessive utilization of a cytokine (i.e. its binding to cellular or soluble receptors) could result in its low plasma level. Even when both the cytokine and its soluble receptor levels are measured, the interpretation is often unclear, particularly with cytokines like TNF-a, which may exist and be active in a cell membraneassociated form and thus not be detectable in plasma. The presence of cytokine or cytokine receptor antagonists in human sera, as exemplified by IL-1Ra, which inhibits biologic activity of IL-1b by occupying its receptors on tissue cells (Dinarello and Wolff, 1993), is well documented. Viral cytokine or cytokine-receptor homologues, which bind to cellular receptors or to circulating cytokines, respectively, counteracting their biologic functions, are often present in human sera (Nicholas et al., 1997). Even in the absence of specific or non-specific inhibitors, excessive consumption of a cytokine versus lack of its synthesis could be responsible for negative assay results. Overall, the clinical usefulness of measuring cytokine levels in plasma remains unclear, and changes of the cytokine profile in the local microenvironment might be more meaningful, based on the principle that these changes reflect functional characteristics of the cells found at the disease site. The more recent use of cytokines as pharmacologic agents has been facilitated by an extremely rapid process of cytokine discovery, the ability to clone their genes and the availability of cytokine recombinant proteins. Also, cytokine gene therapies, which represent a novel strategy of cytokine delivery to the targeted sites, take advantage of the ability to transfer cytokine genes to cells, induce cytokine production and thus regulate essential cellular functions (growth, differentiation, apoptosis) via high affinity receptors expressed on cytokine-responsive cells in the tissue microenvironment (Aggarwal and Puri, 1995; Suminami et al., 1995). The newly emerging associations between the cytokine profiles and infectious or allergic diseases

provide the best examples of how cytokine determinations contribute to a better understanding of disease pathogenesis (Lucey et al., 1996; Romagnani, 1997a, 1997b; Grewe et al., 1998). These clinical observations argue for serial monitoring of cytokine levels in disease and during biotherapies and for continuing attempts to define changes in cytokine profiles that occur in disease or during therapies. Future measurements of cytokine levels should be accompanied by monitoring of selected cellular functions associated with the presence of a given cytokine. Hypothesisdriven cytokine monitoring might provide useful clinical information and strengthen the association between distinct cytokine profiles and disease. However, it is essential to bear in mind that only when validated and carefully controlled methods for cytokine detection are used can we expect to make progress in defining the role of cytokines in human disease. A critical importance of accurate and sensitive cytokine evaluation for the success of this objective is obvious. Application of cytokine genomics and proteomics in the near future is expected to rapidly advance the field of cytokine detection and to facilitate interpretation of cytokine patterns in relation to clinical events.

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CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION