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 He