Advances in Immunology Volume 77

ADVANCES IN

Immunology VOLUME 77

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ADVANCES IN

Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 77

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Copyright C 2001 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2001 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/01 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

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CONTENTS

ix

CONTRIBUTORS The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction

S. CELESTE POSEY MORLEY AND BARBARA E. BIERER

I. II. III. IV. V. VI.

Introduction Overview of T Cell Signaling Lipid Rafts Actin Cytoskeleton Actin Dynamics in Signal Transduction—General Principles Conclusion References

1 3 16 23 30 34 35

Raft Membrane Domains and Immunoreceptor Functions

THOMAS HARDER

I. II. III. IV.

Introduction Lipid Raft Concept: Bridging Biophysics to Biology Immunoreceptor Signaling and Raft Domains Outlook References

45 46 54 79 79

Human Basophils: Mediator Release and Cytokine Production

JOHN T. SCHROEDER, DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN

I. II. III. IV. V.

Introduction Basophil Growth and Maturation Cell Surface Markers Inflammatory Mediators Basophil Activation v

93 94 94 98 101

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VI. Signal Transduction and Pharmacological Control of Secretion VII. Basophils and Allergic Disease References

106 112 114

Btk and BLNK in B Cell Development

SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE

I. II. III. IV. V. VI.

Introduction Btk and B Cell Development Activation of Btk Downstream of Btk BLNK Connects Btk Activity to Downstream Effectors Conclusion References

123 124 129 137 142 150 151

Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2 s

MAKOTO MURAKAMI AND ICHIRO KUDO

I. II. III. IV. V.

Introduction Structures and Enzymatic Properties of sPLA2 s Expression and Functions of sPLA2 s sPLA2 Receptors Conclusion References

163 164 169 182 183 184

The Antiviral Activity of Antibodies in Vitro and in Vivo

PAUL W. H. I. PARREN AND DENNIS R. BURTON

I. II. III. IV. V. VI. VII. VIII.

Introduction Mechanisms of Neutralization Complement-Mediated Virolysis Antibody-Mediated Phagocytosis Antibody-Mediated Cytotoxicity Intracellular Neutralization Mechanisms of Antibody Protection in Vivo Mechanisms of Antiviral Antibody Activity in Established Infection IX. Observations with Nonviral Pathogens X. Conclusions References

195 196 225 226 226 227 227 241 244 244 248

CONTENTS

vii

Mouse Models of Allergic Airway Disease

CLARE M. LLOYD, JOSE-ANGEL GONZALO, ANTHONY J. COYLE, AND JOSE-CARLOS GUTIERREZ-RAMOS

I. Introduction II. Conclusion References

263 287 287

Selected Comparison of Immune and Nervous System Development

JEROLD CHUN

I. II. III. IV. V. VI. VII.

Introduction Major Cellular Components of the Nervous System Embryonic Divisions of the Nervous System Embryonic Development of the Cerebral Cortex Ventricular Zone Neuroblast Programmed Cell Death Nonhomologous End-Joining and DNA Rearrangement Conclusion References

INDEX CONTENTS OF RECENT VOLUMES

297 297 299 303 309 313 316 317 323 333

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Barbara E. Bierer (1), Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, 20892; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Yoshihiro Baba (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan Dennis R. Burton (195), Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Jerold Chun (297), Department of Pharmacology; Neurosciences Program; Biomedical Sciences Program; School of Medicine; University of California, San Diego, La Jolla, California 92037 Anthony J. Coyle (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Jose-Angel Gonzalo (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Jose-Carlos Gutierrez Ramos (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Thomas Harder (45), Basel Institute for Immunology, CH-4005 Basel, Switzerland Ichiro Kudo (163), Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Lawrence M. Lichtenstein (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 Clare M. Lloyd (263), Leukocyte Biology Section, Biomedical Sciences Division, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom Donald W. MacGlashan, Jr. (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 S. Celeste Posey Morley (1), Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, ix

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Maryland, 20892; and Committee on Immunology, Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts 02115 Makoto Murakami (163), Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Paul W. H. I. Parren (195), Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 John T. Schroeder (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 Satoshi Tsukada (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan Dai Watanabe (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan

ADVANCES IN IMMUNOLOGY, VOL. 77

The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction §

S. CELESTE POSEY MORLEY*, AND BARBARA E. BIERER*,# *Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; §Committee on Immunology, Division of Medical Sciences, and #Department of

Pediatrics, Harvard Medical School, Boston, Massachusetts

I. Introduction

The adaptive immune system is regulated in large part by the CD4+ helper T lymphocytes. Antigen-presenting cells (APCs) display peptide antigens in the context of major histocompatibility complex (MHC) molecules on their surface that can bind to the T cell receptor (TCR) on antigen-specific T cells. When bound, the TCR complex generates a complicated array of intracellular signals. The integrated outcome of these signals depends on the cellular context in which the signal is received and may result in T cell activation, anergy, or apoptosis. For instance, a developing thymocyte that binds to a self-antigen too avidly will be deleted in a process known as negative selection, a form of activation-induced cell death that is independent of CD95 (Fas) ligation. A mature T cell that binds to antigen in the periphery in the absence of appropriate cytokines or costimulation may be anergized, or rendered nonresponsive to future stimulation. A mature T cell that binds to a foreign antigen in the presence of appropriate cytokines, such as interleukin (IL)-12, and with the appropriate costimulation (e.g., CD28 ligation by CD80 or CD86), will be activated to mount an immune response appropriate for the eradication of the foreign antigen. A remaining question in immunology is the elucidation of the intracellular mechanisms by which the responding T cells arrive at the outcome of the TCRgenerated signal. How do the proteins within the cell phosphorylate, combine, dissociate, and/or translocate to alter, fundamentally, the physiology of the cell, determining the fate of the T cell? The answer appears to lie, in large part, in the way in which signaling components are spatially organized within the responding T cell (Germain and Stefanova, 1999). Traditionally, the field of T cell signal transduction employed a “billiard ball” model of intracellular signaling. Signaling cascades were, and frequently still are, modeled as linear flow charts from cell surface to cell nucleus. Although important to the early understanding of signaling cascades, this signaling paradigm has numerous (and obvious) limitations. It cannot explain how the same molecule— JNK, for example—can participate in signaling events that have diametrically opposite outcomes, such as T cell activation and T cell death (Dong et al., 1998; 1 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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Jacinto et al., 1998). It cannot explain the integration of signals from multiple receptors—anti-CD3 monoclonal antibody (mAb) stimulation alone results in anergy, while that of anti-CD28 and anti-CD3 results in activation. And it does not incorporate what is known about the cytoplasmic and structural organization of a cell. Only by a consideration of the three-dimensional network of proteins involved in signaling does the complexity and plasticity of signaling cascades become apparent. The organization of the cytoplasm by the actin cytoskeleton has long been an operational paradigm in cell biology, and it is becoming clear that dynamic changes in the actin cytoskeleton play a critical role in T cell signaling. The application of confocal microscopy to lymphocyte signaling has allowed the development of a visual image in real time of the supramolecular activation complex of T cells (Monks et al., 1998). Advances in digital video imaging of cellular movement has enabled the measurement of the rate at which the actin cytoskeleton reorients and moves toward the site of TCR engagement (Wulfing and Davis, 1998). Targeted gene disruption by homologous recombination in mice has permitted analysis of the specific contributions by proteins such as Vav (Fischer et al., 1998; Holsinger et al., 1998; Kong et al., 1998) and WASP (Snapper et al., 1998; Zhang et al., 1999a) to the regulation of both actin cytoskeletal dynamics and T cell signal transduction. Finally, the identification and characterization of Rho family proteins as regulators of actin and signaling have revealed new axes of signal transduction pathways. A new paradigm of T cell signaling has evolved in which the spatial and temporal organization of molecules, determined in part by the remodeling of the actin cytoskeleton, is as critical to the effectiveness of signal transduction as the identity of the molecules themselves. The ability to remodel actin, here termed actin dynamicity, is intimately involved in the current paradigm of the initiation of T cell signaling leading to T cell activation. The precise mechanism(s) by which actin dynamicity participates in organizing intracellular signaling components following TCR ligation remains an open question. Actin may play a critical role in the creation of the immunological synapse, the structured interface between the APC and the responding T cell (see below). Movement of actin cytoskeletal elements may recruit actin-bound signaling intermediates, such as CD3 ␨ , to the site of APC–T cell contact. Alternatively, movement of actin may recruit larger-order signaling structures, such as the recently defined lipid membrane microdomains, termed lipid rafts, that serve as platforms for the accumulation of numerous signaling molecules. This discussion reviews the recruitment and activation of tyrosine kinases and adapter proteins during TCR signaling, the structure and function of lipid membrane microdomains, and the regulation of actin cytoskeletal dynamics, focusing on experimental evidence that suggests dynamic, coordinate regulation between these three critical components of T cell signaling.

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II. Overview of T Cell Signaling

A signal is generated when a peptide in the context of an MHC molecule engages the TCR complex (reviewed in Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). This binding interaction sets off a cascade of membrane proximal events in T cell signal transduction that include activation (or inactivation) of enzymes (kinases and/or phosphatases), phosphorylation of substrates, and recruitment of adapter proteins that enable the formation of large signaling complexes. These early signaling events of tyrosine phosphorylation and protein–protein interactions enable the propagation of downstream signals that lead to calcium flux and the activation of downstream kinases, such as mitogen-activated protein kinases (MAPK). This in turn stimulates the activation of transcription factors such as nuclear factor of activated T cells (NF-AT) that are required for the up-regulation of IL-2 transcription and subsequent activation of the T cells (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). This cascade of events is discussed in detail in this section (Fig. 1). A. T CELL RECEPTOR COMPLEX The TCR complex expressed by CD4+ T helper lymphocytes contains the ␣/␤ TCR heterodimer noncovalently complexed to CD3 proteins (Clements et al., 1999; Germain and Stefanova, 1999). The CD3 complex itself consists of combinations of five different chains subdivided into two different families. One family consists of CD3 ␦, ε, and ␥ , and the other of CD3 ␨ and/or ␩ (Clements et al., 1999). One CD3 complex contains one ε/␥ pair, one ε/␦ pair, and either a ␨ /␨ homodimer or a ␨ /␩ heterodimer. All chains of the TCR–CD3 complex are transmembrane proteins and therefore contain extracellular, transmembrane, and intracellular regions. The extracellular regions of the TCR ␣/␤ chains contain the peptide–MHC binding site and grant the TCR its antigen specificity. However, the intracellular regions are short, have no intrinsic enzymatic activity, and appear not to serve as docking sites for downstream molecules. In contrast, the extracellular domains of the five CD3 chains are quite small and do not associate with the peptide–MHC complex, but the intracellular regions of these chains are crucial for appropriate signal transduction (Clements et al., 1999). Each of the CD3 ␦, ε, and ␥ chains carries one domain capable of being tyrosine phosphorylated, termed an immune receptor-tyrosine-based activation motif (ITAM), with consensus sequence (D/ExxYxxL/Ix7YxxL/I) (Chu et al., 1998). Each CD3 ␨ chain carries three (for a total of six for the homodimer). Tyrosine phosphorylation of these ITAM motifs upon TCR engagement allows for the downstream propagation of the intracellular signal. The mechanism by which peptide–MHC engagement of the TCR ␣/␤ heterodimer transmits a signal through the CD3 complex is unknown, but appears to depend upon

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FIG. 1. Recruitment and activation of tyrosine kinases and adapter molecules in T cell receptor (TCR)–mediated signal transduction. Ligation of either TCR-␣/␤ or CD3 results in the recruitment and activation of the tyrosine kinase p56Lck that in turn phosphorylates tyrosines in the intracellular tyrosine activation motifs (IT AMs) of the CD3 chains. Phosphorylation of the ITAMs creates binding sites for the recruitment of the tyrosine kinase ZAP-70. Once recruited, ZAP-70 is phosphorylated by p56Lck and thus activated to phosphorylate downstream adapter proteins, such as the linker of activated T cells (LAT). Phosphorylation of LAT leads to the recruitment and activation of other downstream signaling molecules, such as Grb2 and PLC␥ 1. Other critical T cell signaling molecules are also shown.

conformational and spatial changes within the individual complex and upon the ligation-dependent formation of multi-TCR associations (Clements et al., 1999; Germain and Stefanova, 1999). B. RECRUITMENT OF PROTEIN TYROSINE KINASES AND ADAPTER MOLECULES The phosphorylation of substrates by cytoplasmic protein tyrosine kinases (PTKs) creates docking sites for the binding of other proteins (Clements et al., 1999; Marie-Cardine and Burkhart, 1999). These substrates are frequently adapter proteins that have no intrinsic enzymatic activity but serve to bring together other proteins in large signaling complexes. The protein–protein interactions that hold these signaling complexes together are mediated by binding motifs on the partner proteins. Many of these motifs have been characterized, including Src homology 2 (SH2), Src homology 3 (SH3), phosphotyrosine binding (PTB), and pleckstrin homology (PH) domains (Marie-Cardine and Burkhart,

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1999). SH2 domains bind to a consensus Yxxx motif when the tyrosine residue is phosphorylated and different SH2 domains have different specificities for pYxxx motifs (Marie-Cardine and Burkhart, 1999). For instance, phosphatidylinositol 3-kinase (PI3K) binds preferentially to pYxxM, phospholipase C (PLC)-␥ 1 preferentially to pYVVL motifs, and Src kinase to pYxxV/I/L (reviewed in Fruman et al., 1998; Marie-Cardine and Burkhart, 1999). PTB domains also bind motifs containing phosphorylated tyrosine, but are biased toward the amino acids N terminal to the phosphorylated tyrosine. The PTB domain of Shc is biased toward the consensus motif NPXpY, while the PTB domain of Cbl prefers D(N/D)XpY (Lupher et al., 1996, 1997). SH3 domains bind to proline-rich regions. While PH domains appear to bind preferentially lipids, such as phosphatidylinositol 4,5bisphosphate (PIP2), and may mediate protein–membrane interactions (MarieCardine and Burkhart, 1999). When an appropriate MHC/peptide complex engages the antigen-specific TCR, the tyrosine residues of the CD3 ␨ ITAMs are phosphorylated by the Src family tyrosine kinase p56Lck (Germain and Stefanova, 1999). Constitutively associated with p56Lck, the coreceptor CD4 is engaged by the MHC molecule concurrently with the engagement of the TCR complex. It is believed that the coengagement of CD4 and the TCR complex by the same MHC/peptide complex brings the intracellular kinase p56Lck sufficiently close in proximity to the CD3 ITAMs that the Src kinase can phosphorylate the tyrosine residues contained within the ITAMs (Germain and Stefanova, 1999). Phosphorylation of the ITAMs creates binding sites for the SH2 domains of ZAP-70 (zeta-associated protein of 70 kDa), a member of the Syk family of tyrosine kinases. ZAP-70 is recruited to the signaling complex by binding the partially phosphorylated CD3 ␨ chain; associated with CD3 ␨ , ZAP-70 can then be phosphorylated by p56Lck and thus activated. Activated ZAP-70 itself phosphorylates downstream substrates, such as linker of activated T cells (LAT) and SH2 domain containing leukocyte protein of 76 kDa (SLP-76), that serve as the adaptor proteins necessary for creation of the signaling complex (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). The kinase activities of both Src and Syk family kinases are absolutely required for T cell signal transduction. Mice deficient in p56Lck have a severe block in thymic development, although the kinase p59Fyn can substitute in part for p56Lck in peripheral T lymphocyte function (Groves et al., 1996; van Oers et al., 1996a,b). T cell development is also arrested at an early stage of thymopoiesis in mice doubly deficient for ZAP-70 and Syk (Cheng et al., 1997; van Oers et al., 1996b). CD4/CD8 double negative (DN) thymocytes express an appropriately rearranged V␤ chain and the pre-TCR␣ chain, but cannot receive a signal through this pre-TCR complex to proceed to the CD4+CD8+double positive (DP) stage of development (Cheng et al., 1997; van Oers et al., 1996b).

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Jurkat T cells deficient in either p56Lck (J.CaM1 cells) or ZAP-70 (P116 cells) have been generated and characterized (Straus and Weiss, 1992; Williams et al., 1998). Both cell lines are deficient in the ability to respond to TCR or CD3 stimulation in that they fail to stimulate calcium influx, NF-AT activation, or IL-2 production. The importance of ZAP-70 in the human immune response has been confirmed by identification of one form of human severe combined immunodeficiency that is caused by a deficiency of this protein (Chan et al., 1994; Elder et al., 1994). 1. Linker of Activated T Cells LAT was originally identified as a heavily phosphorylated doublet (pp36/38) in lysates of stimulated T cells. The gene has recently been cloned (Zhang et al., 1998a) and is predicted to encode a transmembrane protein of 233 amino acids. Four of these amino acids are extracellular, 21 are transmembrane, and the remainder cytoplasmic. The cytoplasmic tail contains 10 different tyrosine amino acids, each of which has the potential for phosphorylation. Phosphorylation of several of these residues by ZAP-70 or Syk upon receptor stimulation creates binding sites for a variety of proteins, such as Grb2, PLC-␥ 1, SLP-76, and the p85 subunit of PI3K (Schraven et al., 1999; Zhang et al., 1998a). Two cysteine residues within LAT, proximal to the intracellular membrane at positions C26 and C29, are palmitoylated (Lin et al., 1999; Zhang et al., 1998b). Palmitoylation is required for the appropriate targeting of LAT to the lipid raft (Lin et al., 1999; Zhang et al., 1998b). Lipid rafts are membrane microdomains that serve as platforms for the recruitment of signaling molecules (discussed in detail below). The requirement for LAT in T cell signal transduction has been shown in a number of experimental systems. Overexpression of a mutated form of LAT in which two tyrosine amino acids had been mutated to phenylalanine (Y171F/ Y191F) inhibited TCR signal transduction, as assayed by transcriptional activation of AP-1 and NF-AT (Zhang et al., 1998a). Inhibition of transcriptional activation correlated with the failure to bind Grb2, PLC-␥ 1, and the p85 subunit of PI3K. The J.CaM2 and ANJ3 cell lines, both derived from Jurkat T cells, lack LAT expression (Finco et al., 1998; Zhang et al., 1999b) and are defective in T cell signal transduction. In J.CaM2, TCR ligation failed to stimulate the phosphorylation of PLC-␥ 1 and SLP-76. There was no calcium flux in response to TCR ligation and downstream MAP kinase activity was not stimulated. In consequence, there was no activation of NF-AT or AP-1 transcriptional activity (Finco et al., 1998). Tyrosine phosphorylation of PLC-␥ 1 and SLP-76 was reduced but not ablated in ANJ3 cells upon TCR stimulation, although calcium flux, extracellular signal-regulated kinase (ERK) activation, and transcriptional activation of NF-AT and AP-1 were completely deficient in this cell line (Zhang et al., 1999b). Reconstitution of both cell lines by overexpression

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of wild-type LAT reversed the signal transduction deficits (Finco et al., 1998; Zhang et al., 1998b). To further confirm the requirement of LAT in T cell signaling, mice with a targeted disruption of LAT were generated (Zhang et al., 1999c). Mice deficient for LAT protein had a complete absence of mature T cells. Thymocyte development was inhibited prior to the DP stage, despite normal rearrangement of the V␤ locus and normal expression of the pre-T␣ chain. The phenotype of the LAT null mice was virtually identical to that of mice deficient either for both Src-family kinases p56Lck and p59Fyn or for both ZAP-70 and Syk tyrosine kinases, consistent with a proximal signaling defect (Zhang et al., 1999c). Finally, LAT mutants that contained C26S/C29S substitutions, and that were therefore not palmitoylated nor localized to lipid rafts, could not reconstitute the signaling deficit of J.CaM2 cells (Lin et al., 1999). Thus, the adapter protein LAT and its appropriate membrane localization were necessary for appropriate T cell signal transduction (Lin et al., 1999; Zhang et al., 1999c). 2. SH2 Domain Containing Leukocyte Protein of 76 kDa (SLP-76) Another adapter protein that, with LAT, coordinately regulates the assembly of large signaling complexes is SLP-76, a cytoplasmic, 533–amino acid protein with a tyrosine-rich N-terminal region, a central proline-rich region, and a C-terminal SH2 binding (reviewed in Clements et al., 1999). The expression of SLP-76 is limited to cells of hematopoietic origin. Like LAT, SLP-76 is a substrate of ZAP-70 and is phosphorylated and recruited to the T cell signaling complex upon TCR or CD3 stimulation (Clements et al., 1999; Schraven et al., 1999). SLP-76 is required, along with LAT, for the appropriate activation of PLC-␥ 1, as PLC-␥ 1 phosphorylation, inositol phosphate production, calcium flux, MAPK activation, and NF-AT transcriptional activation were all ablated in a SLP-76 negative Jurkat T cell derivative (Yablonski et al., 1998). SLP-76 is also required for the appropriate recruitment and activation of Vav, a guanine nucleotide exchange factor (GEF) for Rac (Schraven et al., 1999) that is required for downstream cytoskeletal rearrangement, TCR cap formation, and calcium flux (Fischer et al., 1998; Holsinger et al., 1998). SLP-76, like LAT, is required for thymocyte development, and its absence blocks maturation of CD4/CD8 DN thymocytes at the CD25+CD44−stage of development (Clements et al., 1999). Finally, overexpression of SLP-76 can enhance the response to TCR stimulation (Motto et al., 1996) and can synergize with the overexpression of Vav to enhance NF-AT transcriptional activation in response to TCR engagement (Wu et al., 1996). Thus, early membrane proximal signaling events include the activation of p56Lck and p59Fyn, members of the Src family of PTKs; the recruitment and activation of ZAP-70 and Syk, members of the Syk family of PTKs; and the

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recruitment and phosphorylation of the adapter proteins LAT and SLP-76. LAT and SLP-76 cooperate to form large signaling complexes by nucleating the association of downstream effectors, such as PLC-␥ 1, Vav, or PI3K, and the association of other adapter molecules, such as Grb2, that in turn can recruit and activate downstream effectors, such as Ras (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). C. Ras/MAPK PATHWAY Ras is the prototype of the Ras superfamily of small GTP-binding proteins (Cantrell, 1996; Clements et al., 1999). When bound to GDP, Ras is inactive. Ras becomes activated when the nucleotide GDP is exchanged for GTP. Ras contains intrinsic GTPase activity, and slowly hydrolyzes the GTP to GDP, thereby becoming inactivated. When LAT is phosphorylated, it can recruit the adapter protein Grb2 to the signaling complex. Grb2 is a 217–amino acid protein that contains one SH2 and two SH3 domains. Grb2 constitutively associates with Sos and thus recruits Sos to the signaling complex. Sos serves as a GEF for Ras that catalyzes the exchange of GDP for GTP on Ras, and thus activates Ras. Ras activation in turn activates the kinase Raf, perhaps by stabilizing the membrane translocation of Raf. Raf is a serine/threonine kinase that phosphorylates MEK1, or MAPKK, that in turn phosphorylates and activates the extracellular signal-regulated kinases ERK1 and ERK2, also called MAPKs (mitogenactivated protein kinases). Activation of ERK1/2 is required for the activation of the transcription factor AP-1 and the downstream consequences of T cell activation such as up-regulation of CD69 and IL-2 production (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999). D. PLC-␥1 PATHWAY Phosphorylation and recruitment of PLC-␥ 1 activates PLC-␥ 1 to cleave its substrate, PIP2, into IP3 and diacylgycerol (DAG) (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999). Production of IP3 stimulates the IP3 receptor that triggers intracellular calcium release. Release of calcium from intracellular stores is sufficient to activate calcium release activated calcium (CRAC) channels in the plasma membrane of the T cell, resulting in oscillations of calcium flux across the cell membrane. Calcium forms a complex with calmodulin that then binds to and activates the serine/threonine phosphatase calcineurin. Calcineurin dephosphorylates the transcription factor NF-AT that, upon dephosphorylation, is activated and translocated to the nucleus. NF-AT and AP-1 form a complex required for the upregulation of IL-2 transcription. DAG and calcium also participate in the activation of various serine/threonine protein kinase C (PKC) isoforms, some of which, such as PKC␪, are critical to appropriate T cell signal transduction (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999).

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E. PI3K AND LIPID METABOLISM PI3K consists of a p85 adapter subunit and a p110 catalytic subunit (reviewed in Fruman et al., 1998). PI3K catalyzes the phosphorylation of the D-3 hydroxyl of the inositol ring, generating PI(3)P, PI(3,4)P2, and/or PI(3,4,5)P3 (Fruman et al., 1998). Generation of these lipids activates downstream effectors such as Akt/PKB, a protein that binds PIP3 by virtue of its PH domain (Franke et al., 1995). Akt is a serine/threonine kinase that phosphorylates and inactivates the pro-apoptotic protein BAD, thus promoting cell survival (Datta et al., 1997). The downstream effect of Akt explained the PI3K dependence of growth factor signaling to cell survival (Datta et al., 1997). Additionally, PI3K-mediated activation of Akt has been demonstrated to stimulate NF-␬B transcriptional activation (Romashkova and Makarov, 1999). Independently of effects on Akt, PI3K has been shown to activate Vav (Han et al., 1998) and the members of the Tec family of tyrosine kinases, such as Itk, Rlk, and Txk, that contain PH domains and bind PIP3 (Bunnell et al., 2000). Tec kinases contribute to the PI3K-mediated activation of certain PLC-␥ isoforms, calcium flux, and MAPK activation (Bunnell et al., 2000). F. Vav/Rac PATHWAY Rho GTPases are members of the Ras superfamily of small GTP-binding proteins (reviewed in Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell, 1998). They are activated when bound to GTP and inactivated when the GTP is hydrolyzed to GDP. Three classes of proteins are known to regulate the nucleotide binding of Rho family proteins. GEFs catalyze the exchange of GDP for GTP on the GTP-binding protein, thus activating the protein. GTPaseactivating proteins (GAP) accelerate the rate at which the GTPase cleaves its bound GTP to GDP, thus inactivating the GTPase. Guanine nucleotide dissociation inhibitors (GDI) stabilize the GDP-bound form of the GTPase, effectively inhibiting nucleotide exchange and thus the activation of the GTPase. The conformation of the GTPase depends on the nucleotide to which it is bound. When bound to GTP, but not to GDP, the GTPase is able to bind downstream effectors and transduce signals (Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell, 1998). Activated by membrane receptors, Rho family members link extracellular signals to cytoskeletal rearrangement (Hall, 1998). Different Rho family members exert strikingly differing effects on cell morphology. Activation of Rho by bombesin or lysophosphatidic acid in fibroblasts generates formation of stress fibers and focal adhesions (Ridley and Hall, 1992), activation of Rac by insulin, PDGF or EGF generates membrane ruffles or lamellopodia (Ridley et al., 1992), and activation of Cdc42 by bradykinin generates filopodia (Nobes and Hall, 1995). There is some interplay between the family members; activation of Rac

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is sufficient to activate Rho, possibly through the production of arachidonic acid (Peppenlenbosch et al., 1995), and activation of Cdc42 can activate both Rac and Rho (Nobes and Hall, 1995). The effects of the Rho family proteins on cell morphology are thought to be mediated by the ability of the Rho family members to generate de novo actin polymerization at specific locations and in specific formations (Mackay and Hall, 1998). In lymphocytes, Rac and Rho have been demonstrated to regulate, in part, the cytoskeletal alterations required for adhesion, spreading, and motility (D’Souza-Schorey et al., 1998; Verschueren et al., 1997). Rho family members have been implicated in cellular signaling processes beyond that of regulation of cytoskeletal morphology. Rac is required for oncogenic transformation by Ras, and both Rac and Cdc42 have been demonstrated to regulate JNK and p38 MAPK activity (Coso et al., 1995; Minden et al., 1995). Microinjection of constitutively activated forms of Rac, Rho, and Cdc42 can stimulate G1 cell cycle progression (Lamarche et al., 1996). In fibroblasts, the ability of Rac to promote cell cycle progression correlated with downstream actin polymerization, but not with induction of JNK activity (Joneson et al., 1996; Lamarche et al., 1996). Rac can also activate p67PHOX, a component of the NADPH oxidase complex in neutrophils (Diekmann et al., 1994). Other downstream effectors of Rac and Rho include p21-activated kinase (PAK), a Ste20related serine/threonine kinase, and PI(4)P-5 kinase (Reif and Cantrell, 1998). PAK may be a critical intermediate between Rac/Rho and the downstream effects of actin cytoskeletal rearrangement and JNK and MAPK activation (Bagrodia and Cerione, 1999). Regulation of PIP2 synthesis by Rac may be critical for the production of DAG and IP3 during signal transduction (Reif and Cantrell, 1998). In lymphocytes, Rac has been demonstrated to synergize with Syk to activate JNK (Jacinto et al., 1998) and both Rac and Rho have been implicated in lymphocyte apoptosis (Brenner et al., 1997; Lores et al., 1997; Moorman et al., 1996). Whether the participation of Rac/Rho in these lymphocyte systems is dependent on the downstream modification of the actin cytoskeleton is unclear. The proto-oncogene Vav is a GEF for Rac (Crespo et al., 1997) expressed exclusively in hematopoietic cells essential for effective T cell signal transduction (reviewed in Bustelo, 2000). Vav is a 95-kDa protein that contains a PH domain, a calponin homology domain, one SH2 and two SH3 domains, and a Dbl homology (DH) domain. Calponin homology domains are thought to potentially mediate binding to actin, and DH domains contain the GEF catalytic site. Vav is tyrosine phosphorylated in a p56Lck—and ZAP-70-dependent manner upon CD3 and CD28 stimulation, and translocates to the TCR complex upon phosphorylation (Bustelo, 2000; Salojin et al., 1999). SLP-76 and LAT are thought to be adapter molecules critical for the recruitment of Vav into the TCR signaling complex (Salojin et al., 1999; Wu et al., 1996). In turn, Vav is required for the recruitment

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of PKC␪ (Villalba et al., 2000). Recruitment of PKC␪ to the TCR signaling complex is dependent on Vav-stimulated actin polymerization (Villalba et al., 2000). Vav null mice have been generated and are viable, fertile, and appear grossly normal (Fischer et al., 1998; Holsinger et al., 1998). However, there were specific defects in the immune system. Thymic development was impaired by the Vav null mutation in the C57/B16 genetic backgrounds, with an accumulation in CD44−CD25+ DN thymocytes (Fischer et al., 1998). Thymocytes deficient in Vav expression were resistant to in vitro activation-induced cell death (AICD) stimulated by anti-CD3 and anti-CD28 mAb treatment and by peptide-specific TCR stimulation (Kong et al., 1998). Inhibition of AICD seemed to be dependent on defects in proximal TCR signaling events, including calcium flux, actin polymerization, and recruitment and activation of PKC␪. Inhibition of actin polymerization with cytochalasin D prior to stimulation of thymocytes also inhibited the activation of PKC␪ and subsequent AICD, suggesting that downstream effects of Vav are dependent on the actin rearrangement stimulated by Vav (Kong et al., 1998). Mature peripheral T cells exhibited deficits in calcium flux, IL-2 production, and proliferation, although early tyrosine phosphorylation events and the activation of MAPK and JNK were normal (Fischer et al., 1998; Holsinger et al., 1998). Despite the decrease in calcium flux, translocation of NF-ATc1 to the nucleus appeared to be normal, indicating that the decreased calcium flux of Vav null lymphocytes was still sufficient to stimulate NF-AT translocation and that Vav function was not required for nuclear translocation (Holsinger et al., 1998). Vav null lymphocytes were defective in the ability to polymerize actin and to form the TCR cap upon TCR stimulation (Fig. 2) (Fischer et al., 1998; Holsinger et al., 1998). The defects in cap formation, calcium flux, and IL-2 production could be mimicked by treating lymphocytes with cytochalasin D, concordant with the suggestion that downstream signaling of Vav is dependent on the regulation of the actin cytoskeleton by Vav (Fischer et al., 1998; Holsinger et al., 1998). Whether or not the ability of Vav to stimulate rearrangements of actin is dependent on GEF activity toward Rac, on the ability to function as an adapter molecule, or on another as yet unidentified function is unclear. The downstream effectors of Rho family members that trigger de novo polymerization are the subject of current study. The Wiskott–Aldrich syndrome protein (WASP) is thought to be a principle downstream effector of Cdc42 required for modification of the actin cytoskeleton by Cdc42 (Symons et al., 1996). Wiskott–Aldrich syndrome patients suffer from a severe immunodeficiency characterized by thrombocytopenia, impaired immunity, and eczema (Ramesh et al., 1999; Zhang et al., 1999a). T cells from Wiskott–Aldrich syndrome patients fail to proliferate normally in response to anti-CD3 mAb stimulation and have marked cytoskeletal abnormalities (Ramesh et al., 1999). Thymic

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FIG. 2. Formation of the T cell receptor (TCR) cap. Upon ligation of the TCR by either antigen or monoclonal antibody, the TCR–CD3 complexes move toward the engaged molecules. This TCR reorganization is dependent on a number of intracellular signaling molecules including Vav and WASP, among others, and on the actin cytoskeleton.

development and mature lymphocyte activation were inhibited in WASP-deficient mice (Zhang et al., 1999a). WASP-deficient thymocytes were delayed at an early stage of progression from CD44−CD25+to CD44−CD25− in the population of DN thymocytes, the same stage at which p56Lck −/− thymocytes were delayed in maturation (Zhang et al., 1999a). In mature WASP-deficient T lymphocytes, calcium flux, proliferation, and up-regulation of CD69 were inhibited in response to anti-TCR mAb stimulation. WASP-deficient lymphocytes were also defective in actin polymerization, cap formation, and receptor internalization following anti-TCR mAb stimulation (Fig. 2) (Snapper et al., 1998; Zhang et al., 1999a). These results support a model in which TCR-stimulated actin polymerization and cap formation generate the supramolecular activation complex (SMAC; reviewed below) required for sustaining the TCR signal (Snapper et al., 1998; Zhang et al., 1999a). Recent work has demonstrated a direct association between WASP and the Arp2/3 complex (Rohatgi et al., 1999). Seven subunits, including the actin-related protein (Arp)2 and Arp3, make up the Arp2/3 complex. This complex is the only known mediator of the nucleation of actin filaments (reviewed below) that can grow at the barbed end (Mullins,

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2000). The association between WASP and the Arp2/3 complex provides a clear mechanism by which Cdc42, via WASP, can trigger de novo actin polymerization and the resulting morphological changes (Rohatgi et al., 1999). In Vav null and WASP null lymphocytes, a deficiency in the ability to transduce the appropriate signal correlated with an inability to remodel the actin cytoskeleton in response to the receptor stimulus (Fischer et al., 1998; Holsinger et al., 1998; Kong et al., 1998; Snapper et al., 1998). Either rearrangement of actin cytoskeletal architecture and transduction of signals are mutually exclusive but parallel events, or the transduction of the signal is dependent on the ability to remodel actin. The latter possibility is suggested by studies in which the addition of exogenous compounds that modulate actin dynamics inhibited T cell signal transduction (Fischer et al., 1998; Holsinger et al., 1998). Dynamic changes in the actin cytoskeleton have been demonstrated to play a role in a variety of signal transduction pathways, but only recently have become the subject of investigation in lymphocytes. While it is now appreciated that actin cytoskeletal morphology is intricately involved in lymphocyte signal transduction, the understanding of the mechanisms by which it does so, and the mechanisms by which actin dynamics are regulated during signal transduction, remains incomplete. G. FORMATION OF THE SUPRAMOLECULAR ACTIVATION COMPLEX The involvement of the actin cytoskeleton in lymphocyte signal transduction was first suggested in 1973 when it was found that treatment of B lymphocytes with cytochalasin D, a fungal metabolite that caps F-actin and induces depolymerization, prevented receptor cap formation in response to anti-IgM (de Petris and Raff, 1973). Upon TCR recognition of specific peptide/MHC complexes, the area of contact between the T cell and the APC becomes enriched with other TCRs, generating a receptor cap (reviewed in Penninger and Crabtree, 1999), so termed because of the appearance of immunofluorescently labeled receptors on responding T cells. This interface is a highly complex structure of surface receptors, costimulatory molecules, and intracellular signaling proteins (Fig. 3). Fluorescent microscopy analysis of this interface revealed that the TCR clustered in a central area of the region of contact, and that this cluster is surrounded by a ring of the adhesion molecule LFA-1 (CD11a/CD18) (Monks et al., 1998). These two areas were mutually exclusive, as no appreciable LFA-1 was found in the central cluster while no TCR was demonstrated in the outer ring (Monks et al., 1998). Segregation of intracellular signaling molecules correlated with receptor segregation: talin, a cytoskeletal protein, was found exclusively in the outer ring while the ␪ isoform of protein kinase C (PKC) colocalized exclusively with the TCR in the central ring (Monks et al., 1997, 1998). This highly organized interface has been termed both the supramolecular activation complex, or SMAC (Monks et al., 1998), and the immunological synapse (Grakoui et al., 1999). Generation of the SMAC correlated with downstream lymphocyte effector function

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FIG. 3. Formation of the supramolecular activation complex (SMAC). In cross-section, antigen in the context of major histocompatibility complex (MHC) presented by an antigen-presenting cell (below) engages TCR and CD4 co-receptor on the surface of the responding T cell (above) in the central area of the SMAC. The area of TCR engagement is surrounded by a ring of adhesion molecules, such as LFA-1, engaged by ligand, such as CD54. LFA-1 is excluded from the central area of the SMAC, while engaged TCR is exclusively localized to the central area. The organization of some intracellular molecules mirrors the organization of these surface receptors; PKC␪ is exclusively localized to the central area of the SMAC, “beneath” the TCRs, while talin, an actin-binding protein, is exclusively localized in the peripheral ring of the SMAC, beneath LFA-1. Looking down on a SMAC, the organization of these molecules is schematically represented by a central circular area containing TCR and PKC␪ that is surrounded by a ring of LFA-1 and talin, respectively. (Adapted from Monks et al., 1998.)

(Grakoui et al., 1999; Monks et al., 1998), and interference with cap formation inhibited T cell signal transduction (Penninger and Crabtree, 1999). The cytoskeleton is also restructured in response to the TCR/MHC binding event. The microtubule-organizing center and actin microfilaments reorient toward the area of cell–cell contact (Penninger and Crabtree, 1999). Video microscopy has revealed a critical role for actin in costimulatory events (Wulfing and Davis, 1998) (Fig. 4). Briefly, beads coated with anti-CD54 (ICAM-1) mAb were used to monitor T cell cytoskeletal movement in a manner analogous to that in which fibroblast cytoskeletal motility is monitored. CD54 expressed on the surface of the APC can participate in T cell costimulation by binding to its ligand LFA-1, expressed on the responding T cell. However, CD54 on the surface of the T cell is not involved in T cell costimulation. T cell CD54 is linked to the actin cytoskeleton and can therefore be used to track actin cytoskeletal movement within the responding T cell. When a T cell bound to an APC carrying the appropriate peptide–MHC complex, the bead moved toward the region

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FIG. 4. Cytoskeletal movement in costimulation. In T cells, CD54 does not participate in the active T cell signaling complex, but is stably associated with actin cytoskeletal elements and can therefore been used to track cytoskeletal movement. Beads coated with anti-CD54 monoclonal antibody were used to visualize the surface movement of CD54, that should in turn parallel intracellular cytoskeletal movement. When T cells were allowed to adhere to antigen-presenting cells that expressed appropriate MHC with the specific antigen and appropriate co-stimulatory molecules, such as B7 (CD80/CD86), the coated beads were observed to move toward the area of cell–cell contact, implying that effective costimulation of T cells triggered cytoskeletal movement toward the region of contact. In the absence of appropriate co-stimulation, no such movement of the bead was observed. (Adapted from Wulfing and Davis, 1998.)

of contact, indicating that the cytoskeleton was reorienting towards the region of contact (Wulfing and Davis, 1998). Cytoskeletal movement was dependent upon effective costimulation through either CD28 or LFA-1 (Wulfing and Davis, 1998). The intracellular signaling of these costimulatory molecules was dependent upon PI3K activity and calcium. The mechanism by which cytoskeletal movement occurred was dependent on actin filament assembly/disassembly and myosin motor proteins (Wulfing and Davis, 1998). Based on these data and video fluorescence microscopy of T cells adhering to a coverslip coated with peptide-pulsed MHC and CD54, Grakoui and co-workers (1999) proposed a three-step model for SMAC formation (Fig. 5) in which the force created by actin cytoskeletal movement drives the rearrangement of surface receptors. In the first step, CD54 and LFA-1 binding form a junction that creates a fulcrum for cytoskeleton-based protrusive processes that in turn create a ring of T cell membrane that is in close proximity to the APC cell membrane. Close proximity allows for further TCR–peptide–MHC contact and recruitment of additional TCRs. If the TCR recognizes the complex with sufficient affinity,

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FIG. 5. Formation of the immunological synapse. Engagement of adhension molecules, such as LFA-1, has been hypothesized to be a critical initial step in synapse formation. Adhesion molecules associated with cytoskeletal elements have been proposed to serve as a “fulcrum,” enabling the generation of forced, close approximation of the surface of the T cell and the antigen presenting cell for sufficient duration for the antigen-specific TCR to sample the antigens presented in the context of MHC molecules (1). If an agonistic peptide is encountered, a signal is generated that results in the active movement of TCR to a central area of T cell–APC contact. Adhesion molecules are moved outside this central region (2). The newly formed SMAC, or synapse, is maintained by ongoing signaling processes through as yet undefined mechanisms (3). (Adapted from Grakoui et al., 1999.)

the second step of receptor transport occurs, in which TCRs associated with peptide–MHC complexes and nonengaged TCRs are moved into the central region of contact. The authors hypothesize but do not demonstrate that this is also a cytoskeletally mediated event. In the final step, the synapse is stabilized by an unknown mechanism. At this stage, the synapse is comparable to the previously described SMAC (Grakoui et al., 1999; Monks et al., 1998). The mechanism by which the actin cytoskeleton drives the rearrangements required to create the SMAC is unknown. III. Lipid Rafts

A. STRUCTURE Lateral spatial organization of the lipid membrane is a critical component of appropriate lymphocyte signaling (Germain and Stefanova, 1999). Differential partitioning of the lipids within the cellular plasma membrane has been defined by differential solubility in cold, nonionic detergents such as Triton X-100 (reviewed in Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen,

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1997). The lipids contained within cellular plasma membranes include glycerophospholipids, glycosphingolipids and sterols. Glycerophospholipids contain mostly unsaturated fatty acids and have a low melting temperature. In contrast, sphingolipids contain mostly saturated fatty acids and have a higher melting temperature. Sphingolipids and cholesterol have been hypothesized to stack in a more liquid-ordered (ℓo) phase to form platforms or rafts that move through the glycerophospholipids that in turn exist in a more liquid-disordered (ℓd) phase. These aggregates of (ℓo)-phase lipids have been called rafts and, because of their low buoyant density, can be isolated from Triton X-100 whole cell lysates by sucrose density centrifugation. The fraction in which the rafts are found is referred to as the Triton-insoluble fraction. That which remains in the higher density fractions of the sucrose gradient is referred to as the Triton-soluble fraction. Because of their differential solubility, lipid rafts are sometimes referred to as detergent-insoluble, glycolipid-enriched membrane microdomains (DIGs) or detergent-resistant membranes (DRMs). Because of their lipid constituecy, lipid rafts have also been referred to as glycosphingolipid-enriched membrane microdomains (GEMs) (Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen, 1997). Much work has been devoted to the question of the existence and function of these lipid rafts in physiological cell membranes. As the existence of these rafts was originally suggested by a detergent extraction method, concerns were raised that sphingolipids coalesced into rafts only as an artifact of detergent extraction (Brown and London, 1998b). However, this possiblity was considered unlikely because of studies in which varying ratios of different lipids were mixed and then extracted with Triton X-100. Detergent insoluble lipids were found only under conditions that allowed coalescence of lipids into the ℓd phase prior to addition of Triton X-100. In other words, the coalescence of some lipids into the ℓo phase (and thus into the defined lipid rafts) was not dependent on, and in fact was inhibited by, detergent extraction of other lipids. Further studies have ruled out the possibility that detergent extraction caused mixing of lipids from different phases, or contamination of one lipid phase with components of another (Brown and London, 1998b). However, as the relative detergent insolubility of lipid rafts is dependent upon maintenance of a cold (4◦ C) temperature, there are still concerns that rafts may not exist as such at physiological temperatures. Biophysical evidence from in vitro work using artificially created lipid membranes suggests the possibility of the existence of rafts (Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen, 1997), but biophysical evidence cannot confirm the existence of the raft. B. MICROSCOPIC ANALYSIS OF LIPID RAFTS In the absence of biophysical data, microscopic analysis of lipid membrane morphology has been used in the attempt to demonstrate the existence of lipid

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rafts (Harder et al., 1998; Varma and Mayor, 1998). One of the more convincing studies used anistropy measurements from homotypic fluorescence resonance energy transfer (FRET) to determine that GPI-linked folate receptor (GPIFR) was nonrandomly distributed on the cell surface, while transmembrane folate receptor was randomly distributed (Varma and Mayor, 1998). The nonrandomly distributed GPI-FR was estimated to be in aggregates approx ≈70nm across (Varma and Mayor, 1998). As this is below the resolution of light fluorescence microscopes (250–300 nm), this measurement offers an explanation of the uniform distribution patterns of GPI-linked proteins and other hypothetical raft markers observed with conventional fluorescence microscopy techniques (Jacobson and Dietrich, 1999). Harder and associates (1998) attempted to circumvent this limitation of microscopy by cross-linking raft markers with mAb, forcing their aggregation into patches large enough to be visualized by light microscopy. In this manner they demonstrated colocalization/copatching of raft markers, as determined by Triton X-100 insolubility, such as the GPI-linked proteins placental alkaline phosphatase (PLAP), Thy-1, and influenza virus hemagglutinin and the ganglioside GM1. Importantly, these patches excluded non-raft markers (proteins that were found in the Triton X-100–soluble fraction) such as transferrin receptor (TfR), the low-density lipoprotein receptor, and the vesicular stomatitis virus glycoprotein. Cross-linking of the non-raft markers also created patches, which were entirely distinct from the patches of cross-linked raft markers. A mosaic pattern of red and green with no overlapping yellow that covered the entire cell membrane was observed when TfR and PLAP were cross-linked and cells stained for these markers. This mosaic pattern indicated that there were no areas of overlap between the membrane domain that contained the GPI-linked PLAP and the membrane domain that contained TfR. The dependence of the existence of these separate domains upon lipid composition of the membrane was demonstrated by cholesterol extraction. When cells were treated with methyl-␤-cyclodextrin, a compound that depletes the membrane of cholesterol, patching of cross-linked TfR and PLAP was inhibited. This study thus offered strong evidence for the existence of two distinct, mutually exclusive membrane microdomains (Harder et al., 1998). C. FUNCTIONS OF LIPID RAFTS Lipid rafts have been implicated in a number of cellular functions, including intracellular trafficking (both biosynthetic and endocytic), apical sorting, regulation of membrane proteases, and signal transduction (Brown and London, 1998a; Jacobson and Dietrich, 1999; Simons and Ikonen, 1997). Two pathways exist for the endocytosis of proteins located on the apical surface, the clathrincoated vesicle pathway and another pathway dependent on lipid rafts. In many cell systems, the lipid rafts are associated with caveolin in membrane depressions

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called caveolae. Not all cell types contain caveolin, but the lipid raft–dependent endocytic pathway seems to function in these cells as well. Lipid rafts also appear to function as apical sorting platforms for the appropriate delivery of GPI-linked and N-glycan–containing proteins to the apical surface (Simons and Ikonen, 1997). Lipid rafts have been implicated in the regulation of both uPA and the coagulation cascade (Brown and London, 1998a). A number of different studies, detailed below, suggest the importance of the integrity of lipid rafts and their associated proteins for the induction and maintenance of appropriate singlaling. 1. Lipid Rafts in Signal Transduction Lipid modification of certain proteins is necessary and sufficient for their targeting to lipid rafts in the absence of activation (Kabouridis et al., 1997; Zhang et al., 1998b). The glycerophosphatidylinositol moiety that anchors GPI-linked proteins in the membrane targets these proteins to rafts. The Src family tyrosine kinases p56Lck and p59Fyn are doubly acylated (one myristoylation and one palmitoylation) at their N termini (Kabouridis et al., 1997). LAT is palmitoylated on two N-terminal cysteine residues (Lin et al., 1999; Zhang et al., 1998b). Importantly, these lipid modifications are required not only for localization to lipid rafts but also for the appropriate function of these molecules in lymphocyte signal transduction (Fig. 6) (Kabouridis et al., 1997; Lin et al., 1999; Zhang et al., 1998b). Xavier and colleagues (1998) demonstrated that the integrity of lipid rafts is required for efficient T cell activation. Using sucrose gradient centrifugation to isolate lipid raft components from T lymphocytes both before and after stimulation through the TCR, they demonstrated that the increase in tyrosine phosphorylation of proteins upon TCR stimulation is most dramatic in the lipid raft, as compared to proteins in the cytoplasm or non-raft plasma membrane. They further demonstrated that proteins critical to TCR signaling are either constitutively localized to lipid rafts, such as Lck, Fyn, Cbl, Syk, Ras, and Grb-2, or translocate to the rafts upon stimulation, as do Vav, Shc, ZAP-70, PLC-␥ 1, and CD3 ␨ (Fig. 6). Some Vav and PLC-␥ 1 are present in the raft prior to stimulation, but the amount is greatly enhanced upon anti-CD3 stimulation (Xavier et al., 1998). Zhang and co-workers (1998b) confirmed the localization patterns of Vav, PLC-␥ 1, Cbl, p56Lck , and Grb-2, though differed on the localization of ZAP-70; they found no evidence of ZAP-70 translocation to the lipid raft upon stimulation. However, this may be due to a difference in detergent extraction conditions (Xavier et al., 1998; Zhang et al., 1998b). The tyrosine phosphorylated forms of these proteins were observed primarily in the lipid raft fraction. Disruption of the detergent-resistant membrane compartment with nystatin and filipin disrupted TCR signaling, as assayed by tyrosine phosphorylation of PLC-␥ 1 and CD3 ␨ and by calcium mobilization (Xavier et al., 1998). Extraction

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FIG. 6. The T cell signaling complex in the lipid raft. A number of T cell signaling proteins are constitutively localized to lipid membrane microdomains, termed lipid rafts, or translocate to the rafts upon appropriate stimulation. The integrity of the lipid raft is required for efficient T cell signal transduction and activation.

of cholesterol with methyl-␤-cyclodextrin also prevented calcium flux in response to anti-CD3 stimulation. Finally, forced down-modulation of surface lipid raft components by treatment with exogenous gangliosides prevented TCR signal transduction, as assayed by calcium mobilization. These data strongly support the model in which lipid rafts serve as platforms for the association of signaling molecules, and that the integrity of these lipid platforms must be maintained for appropriate signaling (Xavier et al., 1998). Experimental support for a model of rafts serving as signaling platforms comes from the work of Janes and collaborators (1999). These investigators first demonstrated that p56Lck , LAT, and CD3 colocalized to rafts patched by cross-linking GM1 with the B subunit of cholera toxin (CTxB). The association of CD3 with the lipid rafts appeared to be of weaker affinity than that of p56Lck or LAT and was sensitive to extraction in 1% Triton X-100 (Janes et al., 1999), a finding that may explain the discrepancy of these results with others who have found no association of TCR-␣/␤ with lipid rafts (Kosugi et al., 1999). Most intriguingly, cross-linking of GM1 with CTxB was sufficient to stimulate tyrosine phosphorylation of substrates and calcium mobilization in Jurkat T cells, suggesting again

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that lipid rafts serve as platforms for the association of signaling molecules, and that the aggregation or coalescence of these rafts is sufficient to trigger the phosphorylation events necessary for signal transduction (Janes et al., 1999). Debate about the presence or absence of the TCR/CD3 complex in lipid rafts continues. Kosugi and associates (1999) detected CD3 ␨ , but not other CD3 chains or TCR-␣/␤ chains, in lipid rafts following TCR or CD3 stimulation. However, the translocation of CD3 ε, TCR-␣/␤, and CD3 ␨ to the lipid raft compartment was observed in another system (Montixi et al., 1998). The difference in observations may be due to a difference in detergents used to extract the lipid raft components. Although 1% Triton X-100 insolubility has been the defining extraction method for lipid raft constituents, lower concentrations of Triton X-100 (Xavier et al., 1998) and other detergents, such as Brij (Montixi et al., 1998), may maintain the association of proteins with weaker affinities for lipid rafts. The difference in observations could also be explained by the time course of the assays; Montixi and colleagues (1998) stimulated with anti-CD3 ε mAb for 5 min at 37◦ C, while most of the assays performed by Kosugi and associates (1999) stimulated cells with anti-CD3 ε mAb for 45 min at 37◦ C. Given the microscopic evidence (Janes et al., 1999) and the virtually universal observation that TCR/CD3 signaling requires the accumulation of signaling molecules at lipid rafts, it is likely that the TCR/CD3 complex does translocate or associate with the lipid raft, but that this association can be disrupted by extraction with 1% Triton X-100. Viola et al. (1999) recently presented data that suggested the involvement of raft redistribution in effective costimulation. They demonstrated that lipid rafts, as indicated by staining with CTxB–FITC, remained uniformly distributed when a T cell bound to beads coated with anti-CD3 mAb, but redistributed to “cap” at the area of bead–cell contact when the T cell bound to beads coated with both anti-CD3 and anti-CD28 mAbs. Redistribution of the CTxB–FITC– stained patches correlated with downstream activation of the T cell, as indicated by cell proliferation, tyrosine phosphorylation, CD3 down-modulation, and consumption of p56Lck . Passive clustering of the lipid rafts by cross-linking GM1 with CTxB or CD59 (a GPI-linked protein) was sufficient to costimulate T cells in combination with anti-CD3 mAb when immobilized on the surface of plastic tissue culture wells (Viola et al., 1999). These findings suggested that forced coalescence of lipid rafts was sufficient to transduce a signal, possibly by bringing raft constituent components in close proximity such that they become activated (Viola et al., 1999). 2. Lipid Rafts and the Actin Cytoskeleton The fact that the TCR complex and associated signaling proteins form an ordered SMAC at the T cell–APC contact (Monks et al., 1998), that lipid rafts

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colocalize to this area (Viola et al., 1999), and that the actin cytoskeleton forms a distinct cap at this contact site (Penninger and Crabtree, 1999) leads to the intriguing possibility that tyrosine phosphorylation of substrates, lipid raft coalescence, and actin cytoskeletal rearrangement are all intimately linked in forming the complex necessary for T cell signal transduction. Harder and Simons (1999) have demonstrated the colocalization of polymerized actin, tyrosine phosphorylated substrates, and lipid rafts in Jurkat T cells. Cross-linking of the GPI-linked protein CD59 with anti-CD59 mAb or the ganglioside GM1 with CTx followed by anti-CTx polyclonal antibody resulted in raft patching. These patches accumulated polymerized actin, as visualized by staining with FITC–phalloidin. When a transferrin receptor (TfR) construct that is specifically excluded from lipid rafts was cross-linked, no accumulation of actin was observed. Patching of lipid rafts induced by cross-linking of GM1 or CD59 also induced the recruitment of tyrosine-phosphorylated substrates to the patched lipid rafts, while cross-linking TfR did recruit tyrosine-phosphorylated substrates to the cell membrane. Inhibition of src-dependent tyrosine phosphorylation with the tyrosine kinase inhibitor PP1 prevented not only tyrosine phosphorylation in response to raft patching but also the accumulation of polymerized actin at these sites, suggesting that tyrosine phosphorylation was required for the actin cytoskeletal rearrangement. However, inhibition of actin polymerization by treatment of the cells with latrunculin did not prevent the accumulation of tyrosine-phosphorylated substrates induced by GM1 cross-linking, although the raft patches appeared to be less condensed and the fluorescent signal from the staining of tyrosine phosphorylated substrates was weaker. No specific substrates of tyrosine phosphorylation localized to the lipid rafts were examined, however, and therefore it remains possible that depolymerization of actin by treatment with latrunculin altered the identity of the phosphorylated, raft-associated proteins (Harder and Simons, 1999). The association of lipid rafts with the actin cytoskeleton has been suggested by other reports as well (Holowka et al., 2000; Moran and Miceli, 1998; Oliferenko et al., 1999). Oliferenko and colleagues (1999) demonstrated that CD44containing lipid rafts are anchored by F-actin, as assayed by both sucrose gradient isolation of lipid raft constituents (CD44) and fluorescence recovery after photobleaching (FRAP) in intact cells and in cells treated with the actin-depolymerizing agent latrunculin. Costimulation of T cells through the GPI-linked protein CD48 enhanced the translocation of CD3 ␨ to the insoluble fraction upon stimulation through CD3 (Moran and Miceli, 1998). This translocation correlated with enhanced IL-2 production and could be inhibited by pretreatment with either cytochalasin D or with methyl-␤-cyclodextrin, a compound that extracts membrane cholesterol. Thus, both intact lipid rafts and an intact actin cytoskeleton were required for appropriate signaling through CD3 and CD48 (Moran and Miceli, 1998). Costimulation of T cells through CD28 triggers both actin cytoskeletal redistribution and raft redistribution to the site

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of T cell–APC contact (Kaga et al., 1998; Viola et al., 1999), which again suggests coordination between the actin cytoskeleton and lipid raft regulatory mechanisms. In mast cells, cross-linking of FcεRI, which has been shown to associate with lipid rafts, resulted in the redistribution of the raft-associated proteins Thy-1 and Lyn to large patches coincident with the cross-linked FcεRI (Holowka et al., 2000). At 4◦ C, F-actin also redistributed to these patches but was dispersed when the cells were warmed to 37◦ C. Disruption of F-actin by treatment with cytochalasin D allowed greater association of raft components with cross-linked FcεRI and a prolongation of tyrosine phosphorylation in response to crosslinked FcεRI. These observations support a model in which F-actin regulates the association of proteins such as FcεRI with lipid raft components such as Lyn by segregating lipid rafts (Holowka et al., 2000). In brief, these studies demonstrated that proteins critical to signal transduction must either constitutively localize or translocate upon stimulation to the raft to function in the transduction pathway (Kabouridis et al., 1997; Lin et al., 1999; Zhang et al., 1998b), that cross-linking constitutive protein or lipid components of the lipid rafts is sufficient to transduce a signal (Janes et al., 1999; Viola et al., 1999), that the morphology and constitution of rafts are altered upon signal transduction (Janes et al., 1999; Viola et al., 1999; Xavier et al., 1998; Zhang et al., 1998b) and that these alterations in morphology and constitution are required for signal transduction (Xavier et al., 1998). Furthermore, there is an increasing amount of evidence that regulation of actin polymerization and of lipid rafts are tightly linked, and that alterations in one has profound effects on the other. IV. Actin Cytoskeleton

Microfilaments, microtubules, and intermediate filaments make up the cytoskeleton that maintains the intracellular architecture. Although there is extensive interplay between these three components, the role of microfilaments have been most extensively studied. Actin microfilaments are critical to maintenance of cell shape and adhesion, and are absolutely required for the rapid morphological changes such as ruffling that are required for cell motility. Microfilaments also play an essential role in cell division during cytokinesis, and inhibition of actin dynamics during proliferation generates multinucleate cells. A. STRUCTURE AND REGULATION OF POLYMERIZATION Microfilaments are composed of polymerized actin (Fig. 7) (reviewed in Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997). The actin monomer is a 43-kDa protein with a single nucleotide binding site for either ATP or ADP and a cation-binding site, which is thought to be magnesium (Mg2+) (Steinmetz et al., 1997). In its monomeric form, actin is referred

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FIG. 7. Actin polymerization. Actin exists in either a monomeric (G-actin) or polymerized (Factin) state. F-actin consists of a double-helical linear arrangement of monomers (below), but for simplicity is depicted as a signal array above. Actin contains intrinsic ATPase activity, and polymerization of actin is regulated by nucleotide binding.

to as globular actin, or G-actin. Filamentous actin, F-actin, consists of a parallel, double helical array of linearly assembled actin monomers. The actin filament is polarized; the two “ends” of an actin filament are not identical. These ends are referred to as the barbed end and the pointed end, due to their appearance in electron micrographs. Actin assembly, or polymerization, can occur at either end, but is much faster at the barbed end (Mitchison, 1992; Steinmetz et al., 1997). Polymerization of actin is regulated by ATP binding and hydrolysis (Fig. 7) (Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997). G-actin can exist either in an ATP- or ADP-bound form. ATP–G-actin has a higher affinity than ADP-G-actin for the ends of actin filaments, and thus nucleotide exchange of ATP for ADP on G-actin can stimulate actin polymerization. Actin monomers within the actin filament have intrinsic ATPase activity, and bound ATP is slowly hydrolized to ADP. Hydrolysis is slower than new polymerization, so a newly elongating filament will contain both ADP–actin (at the pointed end) and ATP–actin (at the barbed end). ADP-bound actin monomers depolymerize, or dissociate from the filament, from the pointed end. Polymerization can occur at the barbed end while the filament is depolymerizing at the pointed end; this cycle is referred to as treadmilling. Thus, regulating the rate of ADP/ATP exchange on actin monomers and regulating the ATPase activity of actin filaments can regulate the rates of actin polymerization and depolymerization (Kabsch and Vandekerckhove, 1992; Steinmetz et al., 1997).

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B. ACTIN-BINDING PROTEINS Regulation of the structure of the actin cytoskeleton occurs at multiple levels by different classes of actin-binding proteins (reviewed in Puius et al., 1998; Schmidt and Hall, 1998). A few representative proteins are summarized in Table I and in Fig. 8. Regulatory activities include actin monomer sequestration or buffering, nucleotide exchange, nucleation of actin filaments, capping and severing of existing actin filaments, cross-linking and bundling of filaments into higher-order networks, and cross-linking actin filaments to integral membrane proteins (Puius et al., 1998; Schmidt and Hall, 1998). Filament assembly/disassembly is in part regulated by actin-binding proteins that are thought to regulate ATP-related activities and to alter affinities for F-actin (Puius et al., 1998; Schmidt and Hall, 1998). Profilin binds to actin monomers. In addition to serving as a buffering protein, profilin has been

TABLE I BRIEF SUMMARY OF REPRESENTATIVE ACTIN-BINDING PROTEINS Protein Profilin

Activation and regulation Sequestration of actin monomers. Dissociates from G-actin upon PIP2 bindig. Nucleotide exchange.

Thymosin ␤4

Sequestration of actin monomer. Dissociates from G-actin upon PIP2 binding.

Capping protein

Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.

Gelsolin

Severing of actin filaments activated by binding of Ca2+. Capping of actin filaments. Dissociates from F-actin upon PIP2 bidning. Nucleation of actin filaments, enabling elongation at pointed end.

Villin

Severing of actin filaments in high concentrations of Ca2+. Cross-linking and bundling in low concentrations of Ca2+.

Fragmin, adseverin, scinderin

Severing of actin filaments activated by binding of Ca2+. Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.

Cofilin (ADF)

Disassembly of actin filaments, inhibited by serine phosphorylation. Sequestration of actin monomers. Dissociates from G-actin upon PIP2 binding.

␣-Actinin

Cross-linking and bundling of actin filaments. Activity is enhanced by PIP2 binding.

Filamin

Cross-linking and bundling of actin filaments. Activity is inhibited by PIP2 binding.

Spectrin, fimbrin

Cross-linking and bundling of actin filaments.

Talin

Nucleation of actin filaments at membrane.

Arp2/3 complex

Nucleation of actin filaments, enabling elongation at barbed end. Activated by WASP.

Ezrin, radixin, moesin

Cross-linking of F-actin to plasma membrane. Activated by PIP2 and by tyrosine and serine phosphorylation.

ADF, Acting-depolymerizing factor; PIP2, phosphatidylinositol 4,5-bisphosphate; WASP, Wiskott-Aldrich syndrome protein.

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FIG. 8. Examples of activities of actin-binding proteins. See text for details.

hypothesized to act as an ATP nucleotide exchange factor, enhancing the rate at which ADP is exchanged for ATP on actin monomers (Puius et al., 1998). The complex of ATP–G-actin with profilin has a much higher affinity for F-actin than does ATP–G-actin alone (Didry et al., 1998). Profilin can therefore enhance the rate of actin polymerization by increasing the affinity of actin monomers for actin filaments and by increasing the rate at which ADP–actin is recycled to ATP–actin (Mullins, 2000; Puius et al., 1998). Like many actin-binding proteins, profilin contains a binding site for PIP2 and dissociates from actin monomers when bound to PIP2 (Goldschmidt-Clermont et al., 1991). Cofilin, also called actin-depolymerizing factor (ADF), promotes actin filament disassembly and can, like profilin, buffer actin monomers (Schmidt and Hall, 1998). Cofilin binds to F-actin, preferentially ADP–actin within the filament, and induces a twist that can induce dissociation of the actin monomer from the filament (Bamburg, 1999). Through the promotion of actin assembly at the barbed end and of actin disassembly at the pointed end, profilin and cofilin can work in concert to remodel the actin cytoskeleton (Bamburg, 1999; Didry et al., 1998; Mullins, 2000). PIP2 binding induces dissociation of cofilin from actin (Schmidt and Hall, 1998). Recent work has demonstrated regulation of cofilin by serine phosphorylation by LIM kinase (Arber et al., 1998; Yang et al., 1998). Phosphorylation inactivates cofilin, preventing cofilin-mediated actin filament disassembly. Activation of LIM kinase therefore promotes actin polymerization, and is thought to serve as a downstream effector of Rac (Arber et al., 1998; Yang et al., 1998). Phosphorylation and nuclear translocation of cofilin has been

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demonstrated in activated T lymphocytes (Samstag et al., 1994, 1996), and costimulation of T cells promotes cofilin dephosphorylation and association with actin (Lee et al., 2000). Other activities that control actin dynamics are sequestration of actin monomers and nucleation. Thymosins are small G-actin binding proteins that are thought to “buffer” the pool of G-actin in the cytoplasm and thus regulate how much G-actin is available for polymerization (Mitchison, 1992). As mentioned previously, profilin and cofilin can also serve as buffering proteins (Schmidt and Hall, 1998). New actin filaments are generated from nucleation sites; two G-actin molecules must be brought into close proximity by a nucleating protein to generate a site for actin polymerization. The Arp2/3 complex (described above) is one of the most powerful nucleating complexes known to date (Rohatgi et al., 1999), capable of nucleating actin and generating new barbed ends for rapid polymerization (reviewed in Machesky and Insall, 1999; Schafer and Schroer, 1999). The Arp2/3 complex appears to be recruited into the TCR signaling complex via interactions with the adaptor molecule Fyn-binding protein (Fyb)/SLP-76– associated protein (SLAP) (Krause et al., 2000). Gelsolin (described in detail below) can also nucleate actin filaments, but generates pointed ends for polymerization (Kinosian et al., 1998; Wegner et al., 1994). Severing and capping proteins, such as gelsolin, perform at another level to regulate actin cytoskeletal remodeling. Gelsolin belongs to a family of severing proteins that includes villin, fragmin, adseverin, and scinderin. Severing proteins are generally activated by calcium binding (Puius et al., 1998). When bound to calcium they to bind the side of an actin filament and induce disassembly, generating two shorter actin filaments from one long one. Members of the gelsolin family can also cap severed filaments (Puius et al., 1998). Capping proteins, such as gelsolin and capping protein, bind to the barbed end of an existing actin filament, preventing further elongation (Machesky and Insall, 1999; Puius et al., 1998). PIP2 binding to either gelsolin or capping protein causes them to dissociate from the filament, “uncapping” the filament and allowing further polymerization at the exposed barbed end (Hartwig et al., 1995). Thus, severing and capping proteins control the number and length of actin filaments (Puius et al., 1998). Cross-linking proteins such as fimbrin, spectrin, ␣-actinin, and the ezrin/ radixin/moesin (ERM) family members combine actin filaments into higherorder structures to create the cellular actin architecture (reviewed in Puius et al., 1998; Tsukita and Yonemura, 1999). Fimbrin cross-links actin microfilaments and is inhibited by PIP2 binding. In contrast, PIP2 binding activates the cross-linking activity of ␣-actinin, a protein that also cross-links and bundles actin filaments (Puius et al., 1998). Other cross-linking proteins are listed in Table I (drawn from Schmidt and Hall, 1998). The ERM family members link actin microfilaments to the plasma membrane through interactions with integral membrane proteins (Tsukita and Yonemura, 1999). ERM proteins are localized

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to areas of distinct cell surface structures, such as microvilli, lamellipodia, and adhesion sites, where cortical actin must interact with the plasma membrane to maintain the surface structure (Tsukita and Yonemura, 1999). The bundles and networks of actin filaments created by these cross-linking proteins are highly stable and resistant to rapid polymerization and depolymerization (Puius et al., 1998). The regulation of actin cytoskeletal dynamics is thus extraordinarily complex, coordinated and modulated by an array of proteins with both overlapping and exclusive functions. While the cortical actin cytoskeleton can be maintained in a highly stable array of actin filaments, other pools of actin are undergoing constant polymerization and depolymerization under the control of a variety of these proteins. The constant flux could provide greater sensitivity to extracellular signals, allowing small perturbations in PIP2 and Ca2+ concentrations to result in rapid remodeling of actin. While the functions of two of the actin binding proteins described here—cofilin and the Arp2/3 complex—have been investigated in the context of T cell signaling (Krause et al., 2000; Lee et al., 2000; Samstag et al., 1994), the potential for the participation of the others—gelsolin, profilin, ERM proteins—remains an open question. C. EXOGENOUS AGENTS THAT MODIFY ACTIN Cell permeant compounds that modify the rates of actin polymerization or depolymerization have been invaluable in the investigation of actin-based pathways. Since genetic manipulation of actin is difficult in many eukaryotic cell systems, and since the molecular mechanisms of actin regulation by many actinregulatory proteins remain unclear, the identification of exogenous compounds with defined activities toward the actin cytoskeleton have been used to define the role of actin dynamics in various cellular processes. Cytochalasins are fungal metabolites that have long been used to induce actin filament destabilization (Carlier et al., 1986). Latrunculin is a more recently identified compound that binds actin monomers, resulting in dramatic depolymerization of actin (Ayscough et al., 1997; Spector et al., 1989). Jasplakinolide has an opposite effect on F-actin, binding to and stablizing existing actin filaments and in some cases driving increased actin polymerization (Bubb et al., 1994, 2000). The identification and mechanisms of these compounds are outlined in greater detail below. 1. Cytochalasin D Cytochalasins are fungal metabolites that have long been used to modify actin dynamics in vivo. Cytochalasin B, but not D or E, has also been demonstrated to inhibit glucose transport (Mookerjee et al., 1981). Cytochalasins B, D, and E cap the barbed end of actin filaments, preventing elongation of the filament (Carlier et al., 1986). Depending on the ionic environment, the cytochalasins can also bind actin monomers and promote ATPase activity, decreasing the pool of

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ATP-bound monomeric actin required for polymerization (Brenner and Korn, 1980). Interestingly, under certain conditions, such as high KCl and substoichiometric concentrations of cytochalasin, cytochalasin D can nucleate actin filaments by forming dimers of actin monomers (Carlier et al., 1986; Goddette and Frieden, 1986). The nucleation effectively promotes actin polymerization, which probably explains observations that under certain conditions cytochalasin D promoted actin polymerization in leukocytes (Rao et al., 1992). Over time, however, the depolymerizing activity of cytochalasin D predominates (Goddette and Frieden, 1986). The activity of cytochalasins toward lymphocyte activation is dependent on cytochalasin concentration; at low concentrations (<1 ␮M), cytochalasins augmented lectin-induced stimulation, while at high concentrations cytochalasins inhibited activation (Geppert and Lipsky, 1990; Grove et al., 1992). Depolymerization of actin with cytochalasin or C2 botulinum toxin has been shown to inhibit DNA synthesis downstream of mitogenic signals such as nerve growth factor or anti-IgM stimulation (Melamed et al., 1991, 1994, 1995). Treatment of the T cell line Jurkat with cytochalasin D prevented CD3-mediated signal transduction leading to growth arrest (Parsey and Lewis, 1993). Cytochalasin D has also been used to mimic defects in actin polymerization resulting from Vav deficiency (discussed above) (Fischer et al., 1998; Holsinger et al., 1998). Observations that cytochalasin treatment could promote gene transcription have been previously reported. Treatment of lymphocytes with cytochalasin D and PMA was sufficient to induce IL-2 receptor expression in primary lymphocytes. Neither drug induced expression in the absence of the other (Grove et al., 1992). Cytochalasin D treatment also promoted transcription of ␤-actin in murine erythroleukemia cells in an apparently serum response element (SRE)– independent manner (Sympson et al., 1993). More recently, cytochalasin D and jasplakinolide treatment have both been demonstrated to stimulate serum response factor (SRF) transcriptional activity (Sotiropoulos et al., 1999). Thus, cytoskeletal modification by cytochalasin implicates actin in signal transduction leading to gene transcription in lymphocytes and other cellular systems. 2. Latrunculin Latrunculin is derived from the marine sponge, Latrunculia magnifica, and has been demonstrated to alter actin cytoskeletal morphology in vivo (Spector et al., 1989). Latrunculin complexes with G-actin in a 1:1 ratio (Spector et al., 1989) and sequesters actin monomers in an assembly-incompetent complex by preventing nucleotide exchange (Ayscough et al., 1997). It does not promote F-actin severing or disassembly (Ayscough et al., 1997) and thus has a different mechanism of action than do the cytochalasins. Latrunculin has been used to probe the function of actin in a number of cellular processes, including calcium release (Patterson et al., 1999; Rosado et al., 2000), endocytic vesicle sorting

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(Cao et al., 1999), axonal transport (Reynolds et al., 1999), mitotic processes (Heil-Chapdelaine et al., 2000), and growth factor (Tsakiridis et al., 1998; Aplin and Juliano, 1999) and death receptor signaling (Subauste et al., 2000). Pertinent to the discussion of lymphocyte signaling, disassembly of actin by latrunculin was found to initially potentiate, and then inhibit, store-operated calcium release (Rosado et al., 2000). Disassembly of actin was also found to inhibit MAPK and p38 activation, DNA synthesis, and c-fos activation in response to insulin stimulation (Tsakiridis et al., 1998). Finally, disassembly of actin by latrunculin inhibited CD95 (Fas)–induced caspase-3 activation, thereby inhibiting CD95induced apoptosis (Subauste et al., 2000). 3. Jasplakinolide Jasplakinolide is a recently identified cyclodepsipeptide isolated from the marine sponge Jaspis johnstoni. It was originally identified in a screen for compounds with antifungal activity (Kahn et al., 1991; Scott et al., 1988) and later demonstrated to have antiproliferative activity towards the prostate cancer cell line PC3. The inhibition of proliferation correlated with a disruption of the actin cytoskeleton (Senderowicz et al., 1995). Jasplakinolide was later shown to induce actin polymerization and to compete with phalloidin for binding to F-actin (Bubb et al., 1994). More recently, jasplakinolide has been shown to increase nucleation of actin filaments, thus enhancing polymerization (Bubb et al., 2000). Unlike phalloidin, however, jasplakinolide freely crosses cell membranes, making it an ideal compound for in vivo modification of actin. Jasplakinolide has been used to investigate the role of the actin cytoskeleton in cell growth and differentiation (Fabian et al., 1995), enzymatic activation (Duncan et al., 1996), endocytosis (Shurety et al., 1998), and regulation of ion channels (Furukawa et al., 1995; Matthews et al., 1997; Patterson et al., 1999; Rosado et al., 2000). V. Actin Dynamics in Signal Transduction—General Principles

The actin-binding proteins, with their multiple and overlapping functions, allow for exquisitely precise temporal and spatial control of actin dynamics. The actin cytoskeleton not only provides a scaffold to maintain cell shape, but can be rapidly remodeled to allow cell motility, division, and growth. It is becoming increasingly clear that rapid remodeling of the actin cytoskeleton is also required for effective intracellular signal transduction (Acuto and Cantrell, 2000). The mechanisms by which actin participates in signal transduction include regulation of protein compartmentalization and/or localization, mechanical coupling, regulation of ion channels (reviewed in Janmey, 1998), and surface receptor patterning (Grakoui et al., 1999; Monks et al., 1998) and expression (Valitutti et al., 1995). The regulation of surface receptor patterning and expression will be discussed in the context of lymphocyte signal transduction.

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As a ubiquitous array of filaments networked throughout the cytoplasm, the actin cytoarchitecture is ideally suited to serve as a scaffold for signaling complexes (Janmey, 1998). A number of different classes of enzymes are regulated by their association with the actin cytoskeleton, including glycolytic enzymes, src kinases, abl kinase, protein kinase C isoforms, lipid kinases such as PI3K and phospholipases (Janmey, 1998). Translocation of these enzymes to the actin cytoskeleton upon cellular stimulation correlates with a change in their activities. For example, mechanical strain of fetal rat lung cells induced the cytoskeletal translocation and subsequent activation of pp60Src (Liu et al., 1996). PMA treatment of polymorphonuclear cells (PMNs) stimulated translocation of PKC-␣ and PKC-␤II to F-actin. Recent work has demonstrated that many isoforms of PKCs can be activated by stimulation of their association with F-actin (Slater et al., 2000). Translocation of the NADPH oxidase component p47phox and activation of oxidase activity correlated with the cytoskeletal translocation of these PKC isoforms (Nixon and McPhail, 1999). The integrity of the actin cytoskeleton has also been demonstrated to be required for the formation of complexes of adapter molecules, such as in the association of Shc with Grb-2 upon insulin stimulation of myoblasts (Tsakiridis et al., 1998). Stimulation of macrophages with colony-stimulating factor 1 also triggered the formation of a massive complex of signaling molecules that associated with F-actin, including STAT3, STAT5b, paxillin, Shc, SHP-1, and Grb-2 (Yeung et al., 1998). Thus, actin microfilaments can serve as scaffolding elements for signal transduction complexes. Mechanical coupling refers to the generation of a signal by a mechanical force, such as stretching or shear stress, that is transduced by an internal elastic structure to a potentially distant site where the mechanical strian is translated into a biochemical signal (reviewed in Janmey, 1998). In endothelial cells, shear stress generated by fluid flow can stimulate changes in cell morphology and gene transcription. Fluid flow over the hair cells of the inner ear generates the neuronal signaling that is translated as the sense of hearing. The application of elongational forces to collagen-coated beads adhered to the surface of an attached cell stimulated the formation of actin stress fibers, implying that mechanical tension applied to the cell membrane could translate to the activation of a Rho-related protein. As an internal, three-dimensional network spanning the cell, the actin cytoskeleton, along with microtubules and intermediate filaments, could serve as a mechanical coupler. The mechanisms by which tension applied to cytoskeletal structures results in signaling are still unclear (Janmey, 1998). The potential for mechanical coupling as a mechanism of signal transduction in T lymphocytes remains unexplored. Changes in actin have also been linked to control of ion channels. As detailed later, actin dynamics regulate the voltage-dependent calcium channels in murine hippocampal neurons in response to glutamate stimulation (Furukawa

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et al., 1995). Actin dynamics regulate L-type calcium channels in cardiomyocytes (Lader et al., 1999) and have been recently reported to regulate storeoperated calcium channels in smooth muscle cells and platelets (Patterson et al., 1999; Rosado et al., 2000). The activities of Na+ and Cl− channels have also been reported to be modulated by actin polymerization or depolymerization (Janmey, 1998; Jovov et al., 1999; Prat et al., 1999). The mechanism by which association with actin alters channel activity depends on the specific ion channel. Actin is thought to regulate store-operated calcium channels by mediating direct membrane coupling between the cellular plasma membrane and the endoplasmic reticulum, one of the main intracellular calcium storage compartments (Patterson et al., 1999; Ma, 2000). Cytoskeletal changes may indirectly affect channel activity by altering the delivery or clustering of channels on the membrane, or they may directly alter channel activity by altering the open probability of the channel or the channel’s conductance (Janmey, 1998; Jovov et al., 1999). Changes in actin dynamics can directly regulate gene transcription through as yet unknown mechanisms. This has been most elegantly demonstrated in a recent study of activation of SRF. Sotiropoulos and colleagues (1999) screened a VP16-tagged cDNA library using an SRE-CD8 reported construct and identified LIM kinase as an activator of SRF. When activated, LIM kinase phosphorylates and inactivates cofilin, promoting actin polymerization (Arber et al., 1998; Yang et al., 1998). Stimulation of actin polymerization with jasplakinolide was sufficient to activate SRF, and inhibition of polymerization with latrunculin inhibited LIM kinase–mediated SRF activation, implying that increased actin polymerization was both necessary and sufficient for SRF activation in this cell system (Sotiropoulos et al., 1999). The authors hypothesized that the cell “sensed” the size of the G-actin pool, and responded to a decrease in the pool by up-regulating SRF activity (Sotiropoulos et al., 1999). While the mechanistic details remain unclear, this work demonstrates that changes in actin dynamics can directly regulate cellular responses at the level of transcription. As further confirmation of a regulatory role for actin dynamics in cellular responses, regulation of cell morphology has been linked directly to transduction of a signal regulating gene transcription (Kheradmand et al., 1998). Binding of the integrin ␣5␤1 by soluble mAb is sufficient to induce cell rounding. The change in cell morphology was dependent on Rac and downstream actin cytoskeletal rearrangement. Integrin binding also activated NF-␬B via activation of Rac and downstream production of reactive oxygen species (ROS). Interestingly, treatment of cells with cytochalasin D was sufficient to round cells and to generate ROS that then activated transcription of the collagenase-1 promoter used as a readout for the assay. This observation implies that changes in the actin cytoskeleton sufficient to cause changes in cell shape are sufficient to regulate gene transcription (Kheradmand et al., 1998).

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A. ACTIN CYTOSKELETON IN T CELL SIGNAL TRANSDUCTION The actin cytoskeleton, and the molecules that regulate it, have been demonstrated to play an integral role in T cell signal transduction (reviewed in Acuto and Cantrell, 2000; Lanzavecchia et al., 1999; Penninger and Crabtree, 1999; Viola and Lanzavecchia, 1999; Xavier and Seed, 1999). Cross-linking of the T cell receptor results in a burst of actin polymerization that is required for downstream signaling (Brock and Chrest, 1993; Parsey and Lewis, 1993; Phatak and Packman, 1994; Selliah et al., 1995). The polymerization and remodeling of F-actin is thought to be required for the changes in surface receptor expression and patterning and for the organization of intracellular signaling complexes that are responsible for the transduction of activation signals. Evidence for the participation of actin in these changes and this organization include the observations that the ability of some critical signaling molecules, such as Vav, to participate in T cell signal transduction, is dependent on their ability to remodel actin (Fischer et al., 1998; Holsinger et al., 1998; Kong et al., 1998), and observations that modification of actin by exogenous compounds, such as cytochalasin D, inhibits T cell signaling (DeBell et al., 1992; Melamed et al., 1995; Parsey and Lewis, 1993; Valitutti et al., 1995). In addition to regulating the formation of SMACs and receptor recruitment to the site of T cell/APC contact (reviewed earlier), actin dynamics can modulate signal transduction by regulating receptor down-modulation and serial triggering (DeBell et al., 1992; Lanzavecchia et al., 1999; Valitutti et al., 1995). Serial triggering was proposed to explain the ability of a few peptide–MHC complexes on the APC to stimulate a sufficient number of TCRs for T cell activation. In this model, a peptide–MHC complex binds to a TCR specific to the complex with sufficient affinity to ‘activate’ the TCR. The activated TCR is then downregulated, or endocytosed, and another TCR moves into position to be triggered by the same peptide–MHC complex. In this manner, the few number of MHC molecules that carry the antigenic peptide can stimulate a large number of TCRs (Lanzavecchia et al., 1999; Valitutti et al., 1995). This down-regulation of the TCR is mediated by the actin cytoskeleton and can be inhibited with cytochalasin D (Valitutti et al., 1995). The actin cytoskeleton has also been implicated in organizing critical signaling molecules. As discussed previously, rearrangement of actin stimulated by Vav is required for the recruitment of PKC␪ to the SMAC (Villalba et al., 2000). Also, CD3 ␨ has been demonstrated to associate with actin microfilaments in both resting and activated T cells (Caplan et al., 1995; Rozdzial et al., 1995). In the studies described, “cytoskeletally associated” is defined by detergent insolubility. When cells are lysed in detergent-containing buffer, many proteins and some membrane compartments are released into the soluble phase, while some remain as particulate matter. Upon centrifugation, the soluble phase remains as the supernatant and the particulate matter forms a pellet. This pellet is described

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as the detergent-insoluble fraction and contains many cytoskeletally associated proteins (Caplan et al., 1995; Rozdzial et al., 1995). In resting T cells, a phosphorylated p16 form of CD3 ␨ is constitutively cytoskeletally associated (Caplan and Baniyash, 1996; Caplan et al., 1995). Upon T cell activation, there is an increase in phosphorylation of a noncytoskeletally associated p21 form and an increase in cytoskeletal association of an unphosphorylated form of p16 CD3 ␨ (Caplan and Baniyash, 1996). Caplan and Baniyash (1996) reported that the phosphorylated p21 form of CD3 ␨ remained in the soluble fraction, while Rozdzial and associates (1995) reported that the phosphorylated p21 CD3 ␨ translocated to the detergent-insoluble, so-called cytoskeletal fraction. To confirm that this translocation represented binding to cytoskeletal elements, Rozdzial’s group (1995) determined that CD3 ␨ and actin could be co-immunoprecipitated from the insoluble fraction. The translocation of phosphorylated p21 CD3 ␨ to the insoluble fraction could be inhibited by pretreatment with cytochalasin D, and was dependent on phosphorylation of the distal tyrosine amino acid of the third ITAM in the cytoplasmic region of CD3 ␨ (Rozdzial et al., 1995) that is mediated by p56Lck (Rozdzial et al., 1998). This association between CD3 ␨ and actin cytoskeleton correlated with IL-2 production, in that a point mutant of CD3 ␨ deficient in F-actin association was also unable to induce IL-2 production (Rozdzial et al., 1995). The difference in the observations of Rozdzial and Caplan could be due to different methods of stimulation; Caplan and associates (1995) used anti-CD3 ε mAb to stimulate, while Rozdial and colleagues (1995) cross-linked TCR-␣/␤. Though necessary for signal transduction, the function of the cytoskeletal association of CD3 ␨ has not been determined. It has been hypothesized to play a role in receptor stability, internalization and/or recycling (Caplan and Baniyash, 1996; Rozdzial et al., 1995). And though the distal tyrosine residue of the third ITAM is required for actin association, the association of CD3 ␨ with actin may not be direct. This association may be mediated by adapter proteins, the identities of which, if they exist, are currently undetermined.

VI. Conclusion

Maintenance of appropriate actin dynamicity is critical to the current paradigm of T cell signal transduction. No longer a “billiard ball” model, the prevailing paradigm relies on the formation of a highly ordered, three-dimensional network of proteins that create the supramolecular activation complex, the heart of the immunological synapse. TCR and co-receptor engagement stimulates the activation of cytoplasmic, receptor-associated tyrosine kinases. Tyrosine phosphorylation of substrates, such as Vav, results in the reorganization and increased polymerization of actin. Actin reorients and moves toward the region

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ADVANCES IN IMMUNOLOGY, VOL. 77

Raft Membrane Domains and Immunoreceptor Functions THOMAS HARDER Basel Institute for Immunology, CH-4005 Basel, Switzerland

I. Introduction

The cells of the immune system encounter and process antigens in different forms, as native structures, coated by antibodies, or as proteolytically derived peptides. To recognize and adequately respond to different antigens, immune cells express specific immune recognition receptors (IRRs) on their surface. Engagement of these receptors induces intracellular signaling cascades that lead to a physiological response. In addition, accessory molecules enhance, dampen, or otherwise modulate the response to antigen recognition. Immunoreceptors, accessory proteins, and consequently, early signal transduction events are embedded in the cell’s plasma membrane. Recent developments indicate that lateral compartmentalization of the plasma membrane into lipid domains is at the core of IRR signal transduction and signal integration. The current models of plasma membrane structure are founded in the fluid mosaic model by Singer and Nicolson (1972). This model describes cell membranes as a two-dimensional fluid bilayer of lipids forming a solvent for membrane proteins, which, without restrictions, diffuse in the plane of the membrane. While the fluid nature of the plasma membrane is well documented and now broadly accepted, further developments reveal that plasma membrane proteins do encounter restraints. Some proteins were found to move within confined zones, others were immobile—probably anchored to intracellular or extracellular proteins—and yet others exhibited a directed motion (Jacobson et al., 1995; Kusumi and Sako, 1996). Clearly, in many cases, confinement and nonrandom movements of membrane proteins can be attributed to protein–protein interactions. The view of plasma membrane structure was further refined by introducing concepts of lipid-based membrane domains that restrict the distribution and diffusion of membrane proteins (Karnovsky et al., 1982; Thompson et al., 1986; Welti and Glaser, 1994). Numerous cell biological, biochemical, and biophysical observations were integrated into the raft hypothesis, which postulates that lateral assemblies of cholesterol and sphingolipids, called lipid rafts, form a platform for numerous biological processes, ranging from cell signaling to polarized membrane transport (Anderson, 1998; Brown and London, 1998a; Simons and Ikonen, 1997). By accommodating specific proteins while excluding others, raft lipid domains were proposed to segregate membrane proteins and 45

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compartmentalize biochemical reactions in the plane of the membrane (Harder and Simons, 1997). The analysis of signaling in hematopoietic cells has been an important driving force for the development of the lipid raft concept (Hoessli and RunggerBrandle, 1985; Holowka and Baird, 1996; Stefanova et al., 1991). A picture of a highly dynamic raft/nonraft plasma membrane structure is emerging, exploited by IRRs to sense the engagement and translate it into downstream responses. In this chapter, I discuss the observations on which the concept of rafts and their involvement in immunoreceptor signaling are founded and how the functional role of rafts in immunoreceptor activation and the integration of multiple signals can be envisioned. II. Lipid Raft Concept: Bridging Biophysics to Biology

Cell membranes are highly complex mixtures containing hundreds of lipid species, and the concerted interactions among these lipids determine the physical behavior of lipid bilayers. The physical properties of lipids that are believed to convey a domain structure to cell membranes are briefly touched on here. Several excellent reviews cover this issue in detail and are recommended for further reading (Brown, 1998; Brown and London, 1998b; Rietveld and Simons, 1998). Glycerophospholipids have a glycerol backbone that is esterified by two fatty acids and a phosphorylated variable moiety, giving rise to the prevailing phosphoglycerides in mammalian cells: phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine. Many natural phosphoglycerides contain a polyunsaturated fatty acid at their sn-2 position (White, 1973). The kinked structure of unsaturated fatty acids impedes straightening and tight packing of acyl chains, and consequently, bilayers of natural phosphoglyceride are highly fluid and have low gel–to–liquid crystalline phase transition (melting) temperatures (Curatolo, 1987). The sphingolipids, sphingomyelin and glycosphingolipids (GSLs), are based on ceramide, which is formed by a sphingosine backbone coupled by an amide bond to a fatty acid moiety. Addition of phosphorylcholine to ceramide generates sphingomyelin, while GSLs are formed by the addition of carbohydrate sugar moieties. These carbohydrates are highly variable, yielding an enormous diversity of GSLs, ranging from monosaccharide moieties to more complex oligosaccharides such as sialic acid containing gangliosides (Hakomori, 1983). The hydrocarbon chains of the sphingosine backbone and the acyl group of sphingolipids are mostly saturated and can be tightly packed in the extended alltrans conformation (Curatolo, 1987). In addition, sphingolipids contain, close to the water–lipid interface, amide and hydroxyl groups that can function as hydrogen bond–accepting and –donating groups, respectively. Interactions between

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GSLs are additionally supported by hydrophilic interactions between the carbohydrate head groups. Hence, in contrast to phosphoglycerides, artificial bilayers of sphingolipids and particularly GSLs have high gel–to–liquid crystalline phase transition temperatures (Koynova and Caffrey, 1995). Cholesterol is the only mammalian steroid membrane lipid. It contains four hydrocarbon rings that compose the hydrophobic steroid ring system. These rings adopt a compact planar configuration. A hydroxyl group on the hydrophobic steroid rings forms a hydrophilic moiety that orients cholesterol toward the water phase. Cholesterol, intercalated into bilayers of phosphoglycerides and sphingolipids, straightens and orders their hydrocarbon chains and therefore has a major influence on the phase behavior of lipid bilayers (Almeida et al., 1992; Sankaram and Thompson, 1990b; Silvius et al., 1996). Numerous reports describe an interaction between sphingomyelin and cholesterol in artificial bilayers. This has been attributed to hydrophobic interactions of the planar sterol ring system with extended sphingolipid hydrocarbon chains and the formation of a hydrogen bond between cholesterol’s hydroxyl group and the ceramide amide moiety (Bittman et al., 1994; Sankaram and Thompson, 1990a). Sphingomyelin– cholesterol coupling in the cell plasma membrane was demonstrated by digesting plasma membrane sphingomyelin with sphingomyelinase. This caused an elevation of cholesterol contents in the endoplasmic reticulum, indicating backflow of cholesterol from the plasma membrane (Scheek et al., 1997; Slotte and Bierman, 1988). It is conceivable that cholesterol can likewise interact with other lipids or lipidated proteins that carry saturated hydrocarbon chains, for example, outer leaflet glycophosphatidylinositol (GPI)-anchored proteins or palmitoylated proteins inserted into the inner leaflet of the plasma membrane. These properties are thought to confer the role of cholesterol as a key lipid for the formation of raft-like membrane phases. A. LIPID DOMAIN FORMATION REFLECTS PHASE BEHAVIOR OF MEMBRANES Brown, London, and colleagues have proposed that raft/nonraft domain separation reflects a complex phase behavior of membranes. Basically, liquidordered (Lo) raft phases are surrounded by nonraft membrane in a liquiddisordered (Ld) phase (Brown and London, 1997). Cholesterol-induced coexistence of two liquid phases, a cholesterol-rich Lo phase and a cholesterol-poor Ld phase in lipid bilayers was first observed in binary mixtures of the nonnatural saturated glycerophospholipid dipalmitoyl phosphatidylcholine and cholesterol (Sankaram and Thompson, 1991). In the Lo phase, cholesterol is thought to induce higher acyl chain order by aligning with saturated hydrocarbon chains. However, instead of solidifying into a gel phase, lipids in the Lo phase retain rotational and lateral mobility (Almeida et al., 1992). In cholesterol-poor Ld phases, the acyl chains are less straightened and organized, and consequently, bilayers

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in Ld phases exhibit a high fluidity (Brown and London, 1997). The coexistence of Lo and Ld phases in single bilayers was demonstrated to occur at 37◦ C in artificial membranes containing a phosphatidylcholine analogue sphingomyelin, and cholesterol in physiological ratios, mimicking the conditions of the plasma membrane (Ahmed et al., 1997). Consequently, raft domains in cell membranes are now generally defined as cholesterol- and sphingolipid-rich membrane domains in an ordered lipid phase (Brown and London, 1998a). B. DETERGENT-INSOLUBLE CELL MEMBRANES The physical state of lipid bilayers critically determines their detergent solubility. Whereas membranes in Lo and gel phases are relatively resistant to solubilization by Triton X-100, Ld phases are easily solubilized (Brown and London, 1997; Schroeder et al., 1994). Brown and co-workers have demonstrated that GPI-anchored placental alkaline phosphatase (PLAP), constituted into artificial lipid bilayers in Lo and gel phases, is solubilized by Triton X-100. However, when PLAP is introduced into Ld phase membranes, it is easily solubilized, showing that it is not intrinsically Triton X-100 insoluble (Schroeder et al., 1994, 1998). Moreover, these investigators found that in artificial bilayers containing both Lo and Ld phases, Triton X-100 insolubility and the contents of a Lo phase correlated (Ahmed et al., 1997). Consistently, detergent-insoluble membranes prepared from artificial membranes are in an Lo membrane phase (Schroeder et al., 1994). Making use of the detergent resistance of Lo lipid bilayers, cell membranes resisting detergent extraction (using Triton X-100, NP40, or Brij 58) at low temperatures (4◦ C) have been used to define lipid raft domains biochemically. A detergent-insoluble light membrane fraction, here called the detergent-resistant membrane (DRM), is separated from other Triton X-100–insoluble cell material by density gradient centrifugation. This membrane fraction has also been referred to as the glycolipid-enriched membrane, detergent-insoluble glycolipidenriched membrane, Triton-insoluble floated fraction, Triton-insoluble membrane, and low-density detergent-insoluble fraction (Brown and London, 1997; Simons and Ikonen, 1997). DRMs have a specific lipid and protein composition, which is taken as the molecular composition of raft membrane domains in the cell. A large fraction of GSLs and sphingomyelin, as well as of cholesterol, reside in the DRM fraction, while, with the important exception of phosphoinositides, the fraction of phosphoglycerides in DRMs is reduced (Brown and Rose, 1992; Pike and Casey, 1996). The acyl chain saturation of DRM-associated phospholipids isolated from basophilic leukemia RBL–2H3 cells was analyzed by tandem mass spectroscopy. In accordance with the concept, this analysis showed a prevalence of saturated and monounsaturated acyl chains in DRMs (Fridriksson et al., 1999). Moreover, electrospin resonance measurements demonstrated that DRMs from RBL–2H3 cells adopt an Lo-like membrane

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phase in which lipids exhibit a high degree of order but retain substantial rotational freedom and lateral mobility (Ge et al., 1999). Despite its invaluable importance for studying the biochemistry of raft membrane domains, DRM analysis is a rather crude tool. First, weakly raft-associated membrane proteins may be lost from DRMs (Brown, 1998; Harder et al., 1998; Janes et al., 1999). Indeed, detecting DRM association for a particular molecule is quite sensitive to experimental variation and depends on the detergents used, membrane–detergent ratios, temperature, cell types, composition of the buffers used, and so on (Brown and Rose, 1992; Field et al., 1999; Parolini et al., 1999). Keeping the subtle balance of stringency and specificity is key when assaying the degree of DRM association of putatively weakly raft-associated molecules. Moreover, raft markers in the plasma membrane coalesce upon detergent extraction, and consequently, the DRM fraction has lost the structural organization of the rafts in the native plasma membrane (Mayor and Maxfield, 1995). Thus, it remains an important question to what extent two given raft markers reside in common membrane domains and how raft domains and raft-associated proteins interact with each other in cell membranes. C. LIPIDATED PROTEINS ASSOCIATED WITH DRMS The earliest descriptions of DRMs—taken as an indication of membrane domains—date back to 1973 (Yu et al., 1973). Work by Horejsi and by Hoessli showed that GPI-anchored proteins and Src family tyrosine kinases are associated with DRMs from T lymphocytes and proposed that these represent membrane protein–lipid complexes involved in T cell signal transduction (Hoessli and Rungger-Brandle, 1985; Horejsi et al., 1999; Ilangumaran et al., 2000; Stefanova and Horejsi, 1991; Stefanova et al., 1991). It was proposed that lateral assemblies of glycolipids play a role in the formation of transport vesicles in polarized membrane transport (Simons and van Meer, 1988). Indeed, influenza virus hemagglutinin (HA) and GPI-anchored proteins become detergent insoluble during their biosynthetic transport to the apical cell surface of polarized epithelial Madin–Darby canine kidney (MDCK) cells (Brown and Rose, 1992; Skibbens et al., 1989), indicating that they acquire a raft environment. With few exceptions, GPI-anchored proteins and fatty-acylated membrane proteins are the main membrane protein classes highly enriched in DRMs (Brown and London, 1998a; Melkonian et al., 1999). Hydrocarbon chains of GPI anchors are mostly saturated (McConville and Ferguson, 1993) and therefore are believed to mediate partitioning of GPI-anchored proteins into ordered lipid phases. Likewise, S-acylated DRM-associated transmembrane proteins contain mostly C16 saturated fatty acid palmitate—linked to cysteines by a thioester bond (Melkonian et al., 1999; Mumby, 1997; Resh, 1999). Influenza virus HA and the linker for activation of T cells (LAT) are well-studied examples of raftassociated transmembrane proteins. Both influenza virus HA and LAT harbor,

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proximal to their transmembrane region, three or two cytoplasmic palmitoyl acceptor sites, respectively, and both proteins require these S-acylation sites to be targeted into DRMs (Melkonian et al., 1999; Zhang et al., 1998b). In addition, specific amino acids in the transmembrane domain of influenza virus HA (mostly bulky amino acids in contact with the outer leaflet of the lipid bilayer) are important for its DRM association, showing that the polypeptide also contributes to DRM association (Scheiffele et al., 1997). However, to date, no amino acid consensus sequence of transmembrane regions has been identified that would specify DRM targeting. Importantly, some transmembrane proteins exist (e.g., the transferrin receptor) that are palmitoylated but are not targeted to DRMs. Cytoplasmic DRM-associated proteins are anchored in the inner leaflet of the cell membrane lipid bilayer via lipid moieties (Resh, 1999). This includes members of the Src protein tyrosine kinase family and certain types of G␣subunits of heterotrimeric G proteins as well as endothelial nitric oxide synthase. These proteins are targeted into the DRM fraction by N-terminal amide-bonded saturated C14 myristic acid and one or two palmitic acid moieties, thioester bonded to cysteines close to the N terminus (Resh, 1999). Other cytoplasmic proteins are targeted to DRMs via multiple palmitoylation, as found, for example, in specific types of G␣-subunits (Resh, 1999) and neuronal protein GAP-43 (Arni et al., 1998). Small GTPases of the ras superfamily contain a C-terminal prenyl group and a second membrane-binding determinant in their C-terminal tail, which composes either a stretch of basic amino acids or a palmitoylation site (Hancock et al., 1989; Lobell, 1998). DRM association of prenylated proteins such as small GTPases of the ras superfamily is relatively weak, possibly because of the branched and bulky structure of the prenyl group (Melkonian et al., 1999). However, H-ras has been detected in DRMs from Jurkat cells and from caveolae-containing fibroblast cells (see the next paragraph for a discussion of caveolae) (Mineo et al., 1996; Xavier et al., 1998; Yamamura et al., 1997). Also, Rac1 and RhoA, both targeted to membranes via a C-terminal prenyl group and a C-terminal stretch of basic amino acids, were recovered in caveolae-enriched DRMs from RAT-1 cells (Michaely et al., 1999). One major constituent of DRMs from many cell types, including epithelial and endothelial cells, is the caveolins—a family of palmitoylated cholesterol-binding proteins. Caveolins form oligomers that are thought to stabilize cholesterol-rich domains in cell membranes and to form platforms for numerous processes, including signaling, regulation of cholesterol homeostasis, mechanosensing, and polarized membrane transport (reviewed in Anderson, 1998; Kurzchalia and Parton, 1999; Okamoto et al., 1998). Caveolin oligomers are essential for the formation of caveolae, flask-shaped invaginations in the plasma membrane (Fra et al., 1995). Therefore, DRMs from caveolae-containing cells comprise caveolar and noncaveolar raft membrane domains. However, to date, neither caveolins nor caveolae have been detected in hematopoietic cells (Fra et al., 1994; Scherer

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et al., 1997). Therefore, if not stated otherwise, the DRMs discussed in this chapter represent noncaveolar raft membranes. What is required in order to form detergent-insoluble membrane phases in cells? Cholesterol depletion reduces the DRM association of all raft proteins studied so far, including Src-like kinases, GPI-anchored proteins, and transmembrane DRM protein influenza virus HA (Scheiffele et al., 1997). Therefore, cholesterol plays a central role in determining association of proteins with DRMs. Moreover, sphingolipid-deprived Chinese hamster ovary (CHO) mutants exhibit a reduced solubility of GPI-anchored PLAP (Hanada et al., 1995). DRMs have been demonstrated in cells devoid of GSLs (Ostermeyer et al., 1999). However, sphingomyelin expression is up-regulated in these cells, possibly to compensate for the loss of GSLs as structural components of raft-like Lo phases. Thus, it is possible that sphingomyelin, contributing saturated hydrocarbon chains, and cholesterol are sufficient for the formation of Lo DRMs in cells. Interestingly, exogeneously administered GPI-anchored protein CD59 rapidly integrated into the plasma membrane of U937 cells but partitioned into DRMs with a delay of 20 min (van den Berg et al., 1995). Likewise, newly synthesized Fyn Src-related tyrosine kinase, overexpressed in fibroblasts, is anchored into cell membranes (probably the plasma membrane) within 2–5 min via its myristoyl and palmitoyl fatty acylations, but acquires detergent resistance 10– 20 min after synthesis (van’t Hof and Resh, 1997). These surprisingly long delays may be explained as the times Fyn and CD59 acyl chains require to adopt an extended conformation, which would be accompanied by partitioning into ordered lipid domains. Within the ordered domain, the all-trans conformation of the acyl chains could be stabilized, keeping the protein stably raft associated. This underlines the potential elasticity of a dynamic Lo/Ld equilibrium in the plasma membrane in which acyl chain conformation may determine whether raft phases form or disintegrate in membranes and around proteins (Fig. 1). D. STRUCTURE OF RAFT DOMAINS IN THE PLASMA MEMBRANE Energy transfer and chemical cross-linking were used to measure the distance between GPI-anchored proteins in the plasma membranes of CHO cells. These studies indicate that the distance between GPI-anchored model proteins is relatively unaffected by their density in the plasma membrane, suggesting that they are concentrated in small membrane domains (Friedrichson and Kurzchalia, 1998; Varma and Mayor, 1998). Energy transfer and cross-linking between GPI-linked proteins were sensitive to cholesterol extraction and did not occur for transmembrane versions of the respective model proteins. In addition, analysis of self-association of a GPI-anchored green fluorescent protein (GFP) construct indicated a significant but relatively loose coupling of GPI-anchored proteins in the plasma membrane (De Angelis et al., 1998). Other energy transfer

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FIG. 1. Hypothetical model of lipid acyl chain conformation in different membrane phases. Lipids with extended saturated fatty acids form in Lo membrane phases, while lipids with unsaturated acyl chains constitute membrane domains in the Ld phase. Cholesterol straightens acyl chains. Acyl chains of glycophosphatidylinositol (GPI)–anchored proteins may adopt the all-trans extended ordered conformation in liquid-ordered domains, while a nonordered conformation formed by trans-gauche isomerization may be favored in liquid-disordered domains. The structure and composition of the inner leaflet of rafts remain to be defined. PC, Phosphatidylcholine; SM, sphingomyelin.

measurements using fluorescently labeled antibodies or the cholera toxin B subunit, however, showed no coupling of GPI-anchored proteins and ganglioside GM1 within microdomains, possibly reflecting the dilution of the raft markers in multiple microdomains or the dynamic exchange between the microdomains (Kenworthy et al., 2000). Using single-particle tracking, the thermal position fluctuation of raft-associated membrane proteins was studied. This demonstrated that in baby hamster kidney (BHK) and PTK2 cells, GPI-anchored proteins and influenza virus HA are stably associated with a basic cholesterol-dependent raftlike lipid assembly with an average radius 26 nm. Importantly, the GPI-anchored

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proteins were shown to diffuse in the plain of the membrane together with the associated raft-like membrane domain (Pralle et al., 2000). Moreover, ordered lipid domains were detected by single-molecule microscopy in the plasma membrane of smooth muscle cells. This study demonstrated confinement of a fluorescently labeled, fully saturated dimyristoyl phosphatidylethanolamine (DMPE) lipid probe to membrane domains that had an average size of 0.7 ␮m, and therefore were considerably larger than those of rafts suggested by the studies mentioned above. The authors described that these domains covered ∼13% of the smooth muscle cell plasma membrane. Moreover, these domains were stationary on the cell surface and thus somehow were anchored to a cellular matrix, possibly the actin cytoskeleton. Possibly, these domains did not represent the single raft domains but were assembled from elementary raft units (Schutz ¨ et al., 2000). These domains may correspond to relatively large confinement zones described for GPI-anchored proteins using single-particle tracking (Jacobson et al., 1995; Sheets et al., 1997). GPI-anchored proteins and DMPE can be considered the prototype of strongly raft-associated molecules. Indeed, many GPI-anchored proteins were shown to be associated with raft domains (Pralle et al., 2000), and DMPE resides in raft domains with a partition coefficient of 90 (Schutz ¨ et al., 2000). One may envision that other membrane proteins or lipids with lower affinity for raft lipid domains only transiently interact and dynamically exchange with such domains, while nonraft membrane proteins are largely excluded (Harder and Simons, 1997). E. STABILIZATION OF ORDERED-PHASE LIPID DOMAINS BY LATERAL CROSS-LINKING The current data indicate that raft domains in the plasma membrane are small and highly dynamic. However, large and stabilized raft-like membrane domains in a highly ordered lipid phase can be formed by lateral cross-linking of plasma membrane proteins and raft-associated lipids on the cell surface (Spiegel et al., 1984; Thomas et al., 1994). Independently cross-linked DRM-associated molecules coalesce into common patches, and this is inhibited by disruption of raft domains using cholesterol depletion or addition of exogenous GM1 (see Section III,B,4) (Harder et al., 1998; Mayor et al., 1994; Simons et al., 1999). In contrast, patched raft components and patches of transferrin receptor as a nonraft marker were sharply separated (Harder et al., 1998). The copatching of independently cross-linked proteins has been used as a test for raft association that appears to be less stringent than assaying detergent insolubility (Huby et al., 1999; Simons et al., 1999; Verkade et al., 2000). In some cases, partitioning of a non–cross-linked raft molecule into patches of a cross-linked raft marker has been described (Janes et al., 1999); however, this behavior is less pronounced than copatching of two independently cross-linked raft markers (Fra et al., 1994; Harder et al., 1998).

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It has frequently been observed that cross-linking of specific membrane proteins strongly enhances their detergent insolubility. First, resistance to detergent extraction of bona fide DRM-associated molecules increases. This was described for GPI-anchored protein PLAP and the GSL GM1, which were crosslinking by antibodies or by the pentavalent cross-linking bacterial cholera toxin B subunit, respectively (Hagmann and Fishman, 1982; Harder et al., 1998; Merritt et al., 1994). Several transmembrane proteins, including B lymphocyte surface protein CD20 (Deans et al., 1998), apical glycoprotein gp114 of MDCK cells (Verkade et al., 2000), and interleukin 2 (IL-2) receptor ␣—overexpressed in CHO cells (Field et al., 1999)—associate with DRMs following cross-linking. Likewise, signaling-independent association of IRRs to DRMs following ligation was described for the T cell antigen receptor (TCR) (Xavier et al., 1998), FcεRI (Field et al., 1997), and the B cell antigen receptor (BCR) (Cheng et al., 1999; Weintraub et al., 2000). Cross-linking–induced DRM association is a specific property of these membrane proteins, as other transmembrane proteins, such as the low-density lipoprotein receptor on epithelial MDCK cells (Verkade et al., 2000), vesicular stomatitis virus G protein in fibroblastoid BHK cells (Harder et al., 1998), IL-1 type I receptors on CHO cells (Field et al., 1999; Liu et al., 1996), and ␣4 integrins in RBL–2H3 mast cell lines (Field et al., 1999), remain Triton X-100 soluble following cross-linking. Thus, morphological and biochemical evidence indicates that lateral crosslinking of membrane proteins generates large and stable raft-like membrane domains. Cross-linking of bona fide raft-associated molecules may induce an increase in raft size by the coalescence of preexisting raft domains. Moreover, cooperativity of multiply aligned raft-anchoring determinants may further stabilize ordered membrane phases in patches of cross-linked raft components and increase their detergent insolubility. III. Immunoreceptor Signaling and Raft Domains

A large body of evidence indicates that lateral compartmentalization of the plasma membrane into raft lipid domains is required for IRR-signaling (Horejsi et al., 1999; Ilangumaran et al., 2000; Janes et al., 2000; Xavier and Seed, 1999). Proteins implicated to play a role in IRR signaling can be categorized by their different degrees of raft association. First, there are membrane proteins, which are strongly associated to DRMs: Src-related protein tyrosine kinases, the transmembrane linker protein LAT, and GPI-anchored proteins. Second, IRRs and several accessory proteins are weakly DRM associated; however, strong evidence speaks for their association with raft-like membranes, at least following their engagement. Finally, downstream signaling proteins accumulate in DRMs following IRR triggering, probably reflecting the formation of signaling protein complexes around DRM-associated proteins.

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A. CONSTITUTIVELY RAFT-ASSOCIATED SIGNALING MOLECULES 1. Src-Related Kinases Membrane Anchored by Multiple Acylations Src-related protein tyrosine kinases mediate the earliest phosphorylation events of immunoreceptor-elicited signaling cascades. Src kinases comprise a protein family of currently nine known members (Src, Lck, Fyn, Lyn, Yes, Fgr, Hck, Blk, and Yrk). All Src kinases have a common five-domain structure: an N-terminal domain unique for every member of the Src family, an Src homology 3 (SH3) domain, an SH2 phosphotyrosine-binding pocket, the SH1 phosphotransferase domain, and a C-terminal tail involved in the regulation of kinase activity (Rudd et al., 1993). The unique domain of Src kinases harbors two membraneanchoring determinants. The first is a myristoyl fatty acid moiety that is amide bonded cotranslationally to the N terminus of all Src-related kinases. Src and Blk kinases carry a second membrane-binding determinant that is a stretch of basic amino acids shown to interact with negatively charged phospholipids. Other Src kinases contain in their N-terminal peptide one or two cysteines that function as acyl (probably palmitoyl) acceptor sites (Koegl et al., 1994; Resh, 1994). Palmitoyl thioester bond formation on these cysteines occurs posttranslationally and, as demonstrated for Lck and Fyn, is reversible, leading to a dynamic turnover of the palmitic acid moieties on these tyrosine kinases (Paige et al., 1993; Wolven et al., 1997). Hence, dual-membrane targeting elements determine association of Src kinases to cell membranes. The unique N-terminal region of Lck contains two palmitoyl acceptor sites (Cys-3 and Cys-5). The presence of either palmitoylation site is sufficient to confer plasma membrane association of Lck in transfected fibroblasts and T cell lines (Bijlmakers et al., 1997; Kabouridis et al., 1997; Kwong and Lublin, 1995; Turner et al., 1990; Yurchak and Sefton, 1995). The N-terminal unique domain of Lck is sufficient to target heterologous proteins to the plasma membrane (Bijlmakers et al., 1997; Zlatkine et al., 1997). While Lck is mostly anchored in the plasma membrane, in some T leukemic cell lines and upon overexpression in HeLa cells an additional Golgi staining was observed (Bijlmakers et al., 1997; Ley et al., 1994). Overexpressed Lck lacking Cys-3 was distributed to the plasma membrane and Golgi region of fibroblasts while wild-type Lck was located in the plasma membrane (Bijlmakers et al., 1997). It was shown that newly synthesized Lck and CD4 associate in intracellular membranes of T cell lines and are transported in a brefeldin A–sensitive manner to the plasma membrane (Bijlmakers and Marsh, 1999). This suggests that newly synthesized CD4 may escort Lck to facilitate its transport to the cell surface. Fyn kinase requires the palmitoylation site at Cys-3 for membrane association and detergent insolubility, while an additional cysteine at position 6 appears to contribute little to membrane anchoring (van’t Hof and Resh, 1997). Fyn kinase resides intracellularly in a pericentriolar location in Jurkat T leukemic cells or

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T lymphoblastic cells (Ley et al., 1994), whereas it is localized in the plasma membrane when overexpressed in fibroblasts or HeLa cells (Shenoy-Scaria et al., 1994; van’t Hof and Resh, 1997; Wolven et al., 1997). Upon overexpression in CHO cells or fibroblasts, Fyn is completely membrane associated within 5 min after biosynthesis, probably targeted directly to the plasma membrane, while Lck requires 20 min for complete membrane anchoring and is possibly routed by biosynthetic membrane transport (Bijlmakers and Marsh, 1999; van’t Hof and Resh, 1997). The reasons for the differences in Lck and Fyn membrane association and subcellular localization remain an important open question. They may include differences in their membrane anchor, such as the number or spacing of palmitoyl moieties as well as the prevalence of basic amino acids in the unique domain of Fyn over the acidic amino acids in the respective region of Lck (Magee and Marshall, 1999). Differential subcellular localization of Lck tyrosine kinase may be an important mode of regulating access of these kinases to their IRR targets. In naive CD8+ cytotoxic T lymphocytes, a large portion of Lck is located intracellularly, separated from CD8 in the plasma membrane. Upon T cell activation and subsequent effector T cell formation, Lck translocates from these intracellular sites to the plasma membrane, where it associates with CD8 (Bachmann et al., 1999). Thus, developmental regulation of Lck subcellular localization may be a way to tune higher responsiveness of effector T cells to TCR engagement. Indeed, there are indications for the existence of an intracellular pool of raft markers in resting T cells (Viola et al., 1999). One may speculate that plasma membrane transport of Lck and other raft-associated molecules is blocked in naive T cells and turned on upon activation, raising the important possibility of developmental regulation of raft composition. 2. Functional Implications of Src Kinase Palmitoylation It was shown that the activated Y505F Lck mutant requires palmitoyl acceptor Cys-3 and Cys-5 for plasma membrane targeting, transformation of fibroblasts, and spontanteous induction of IL-2 secretion in a T cell hybridoma (Yurchak and Sefton, 1995). The JCaM1 derivative of the Jurkat T leukemic cell line lacks functional Lck (Straus and Weiss, 1992). Introducing Lck mutants lacking palmitoylation sites into JCaM1 cells showed that membrane anchoring of Lck by palmitoylation is required for efficient transduction of TCR-elicited signals (Kabouridis et al., 1997). The single Cys-3 and Cys-5 palmitoylation mutants were able to support early TCR signaling such as TCR ␨ -chain phosphorylation and induction of Ca2+ fluxes; however, subsequent TCR-evoked responses were impaired. An Lck mutant that lacked S-acylation sites was targeted to the plasma membrane as a transmembrane chimera. Importantly, this chimera was kinase active, although it did not mediate anti-CD3 antibody–elicited TCR signaling unless it was directly cross-linked to the TCR. Even though steric reasons for a

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reduced phosphorylation of TCR components by a Lck–transmembrane chimera cannot be excluded, these studies suggested that targeting of Lck to DRMs (i.e., to raft lipid domains of the plasma membrane) is important for its capacity to transduce TCR-evoked signals (Janes et al., 2000; Kabouridis et al., 1997). Specific mutations in the N-terminal region of Fyn reduce the acylation efficiency, that is, N-terminal myristoylation and palmitoylation of Cys-3. The decreased acylation of these Fyn mutants also correlated with a reduction in the ability of Fyn to bind to CD8–TCR␨ chimera (van’t Hof and Resh, 1999). Importantly, the tight association between CD8–TCR␨ and Fyn depended on the binding of the Fyn SH2 domain to the phosphorylated TCR␨ immunoreceptor tyrosine-based activation motifs (ITAMs). Moreover, CD8–TCR␨ /Fyn interactions were lost upon solubilization with the raft-disrupting detergent octylglucoside (Brown and Rose, 1992; van’t Hof and Resh, 1999). These data indicated that rafts and SH2 domain/phosphotyrosine-mediated protein–protein interactions contribute to the association of Fyn and CD8–TCR␨ . Intriguingly, reports showed that treatment of Jurkat T cells with 2-bromopalmitate inhibits palmitoylation of Lck, Fyn, and LAT in Jurkat cells (Webb et al., 2000). Moreover, incubation of cells with polyunsaturated fatty acids (PUFAs) results in a loss of Fyn association with DRMs (Webb et al., 2000). Loss of plasma membrane association of Lck and Fyn and displacement of LAT from DRMs are probably responsible for inhibition of TCR-evoked signal transduction in PUFA-treated Jurkat cells (Stulnig et al., 1998). These findings could point to the mechanism by which polyunsaturated fatty acids mediate immunosuppression and may lead to the development of novel immunosuppressive drugs. 3. Rafts and the Regulation of Src Kinase Enzymatic Activity The enzymatic activity of Src-related kinases is regulated by tyrosine phosphorylation, discussed here with Lck tyrosine kinase as an example. A negative regulatory tyrosine residue (Tyr-505 in Lck) is located in the C-terminal tail of Src kinases. Src and Hck kinase crystal structures revealed that this C-terminal phosphorylated tyrosine associates intramolecularly with the SH2 phosphotyrosine binding pocket, resulting in a conformation with reduced kinase activity (Sicheri et al., 1997; Williams et al., 1997, 1998; Xu et al., 1997). The tyrosine kinase Csk has been implicated in the phosphorylation of this negative regulatory site. The inhibitory effect of (in the case of Lck) Tyr-505 phosphorylation is balanced by its dephosphorylation by the tyrosine phosphatase CD45 (Trowbridge and Thomas, 1994). Consistent with this model, CD45-deficient T lymphocytes are severely impaired in proximal TCR signaling events (Justement, 1997; Koretzky et al., 1990; Pingel and Thomas, 1989), and CD45-deficient mice exhibit a block of T cell development in which CD4+CD8+ thymocytes accumulate (Byth et al., 1996; Kishihara et al., 1993). A mutant of Lck in which the regulatory Tyr-505 is replaced by a phenylalanine has deregulated activity (Abraham et al., 1991;

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Amrein and Sefton, 1988; Marth et al., 1988). Importantly, this deregulated Lck mutant can partially restore the block in T cell development in CD45-deficient TCR-transgenic mice. CD4+ T lymphocytes appeared in the periphery of these mice and responded to TCR stimulation with CD69 up-regulation, IL-2 production, and proliferation, indicating that TCR is functional in these cells (Seavitt et al., 1999). This supports the generally accepted view that CD45 dephosphorylates Tyr-505 of Lck and hence stimulates Lck kinase activity. In addition, phosphorylation of a positively regulating tyrosine (Tyr-394) in the active center of Lck kinase is required for phosphotransferase activity of Lck and hence dominates over the negative regulatory Tyr-505 (D’Oro et al., 1996). CD45 has also been implicated in dephosphorylating this site, assigning CD45 a role in negatively regulating Lck kinase activity (D’Oro and Ashwell, 1999; Thomas and Brown, 1999). Hence, CD45 probably has a complex function in a dynamic equilibrium of regulating Lck kinase activity by tyrosine phosphorylation. Regulation of Lck by tyrosine phosphorylation is tightly coupled to the raft/ nonraft equilibrium of the plasma membrane (Fig. 2). Csk tyrosine kinase, implicated in phosphorylating the negative regulatory Tyr-505 of Lck, binds to phosphotyrosine containing protein PAG (phosphoprotein associated with glycolipid-enriched membranes) or Csk-binding protein (Cbp) (Brdicka et al., 2000; Kawabuchi et al., 2000). PAG/Cbp is a ubiquitously expressed palmitoylated transmembrane protein and is tightly associated with DRMs. It has a short extracellular segment and an intracellular domain that contains 10 potentially phosphorylatable tyrosines. It shares low but significant homology with LAT (see below). Most likely, Lck activity in DRMs is down-regulated by recruitment of Csk to tyrosine-phosphorylated PAG/Cbp. Interestingly, PAG is the most strongly tyrosine-phosphorylated protein in resting T cells and therefore may down-regulate Lck-activity in these cells. Upon activation of resting T cells, the tyrosine phosphorylation of PAG and its association with Csk is reduced (Brdicka et al., 2000). Counteracting Csk activity in terms of Tyr-505 phosphorylation is the transmembrane phosphatase CD45, which has been identified as a nonraft protein: it is not associated with Triton X-100 detergent–insoluble membranes (Kabouridis et al., 2000; Rodgers and Rose, 1996; however, see also Parolini et al., 1996), and it segregates from patches of cholera toxin B subunit/antibody– cross-linked ganglioside GM1 (Janes et al., 1999). Hence, dephosphorylation of tyrosines may occur either at a weakly raft-associated pool of Lck or at the boundaries of raft/nonraft phases. Activating phosphorylation of Tyr-394 may be mediated by Lck autophosphorylation when several Lck molecules reside in a raft membrane microdomain (Shaw and Dustin, 1997). Two studies have demonstrated that Lck tyrosine kinase activity is very low in DRMs prepared by Triton X-100 solubilization of Jurkat T leukemic cells, while the Triton X-100–soluble Lck pool exhibits high phosphotransferase activity (Kabouridis et al., 2000; Rodgers, Crise and Rose, 1994). To account for

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FIG. 2. Balance of the tyrosine phosphorylation–mediated regulation of Lck embedded in the raft/nonraft structure of the plasma membrane. (A) Down-regulation of Lck kinase activity in rafts is mediated by recruitment of Csk to phosphorylated PAG/cbp, which is associated with raft domains. Csk phosphorylates Tyr-505, and Tyr-394 may be dephosphorylated by CD45, leading to a block in Lck kinase activity. (B) Kinase activation may occur by autophosphorylation of Tyr-394 as well as and Tyr-505 dephosphorylation by CD45, which preferentially resides outside raft membrane domains. cbp, Csk-binding protein; PAG, phosphoprotein associated with glycolipid-enriched membranes.

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these observations, it was proposed that Tyr-505 dephosphorylation by CD45 occurs in a nonraft environment, whereas the DRM-associated raft pool of Lck is enzymatically inactive as a result of Tyr-505 hyperphosphorylation (Rodgers and Rose, 1996). This concept is supported by the targeting of Csk to DRMs via phosphorylated PAG/Cbp. However, a different study showed that Lck was active in DRMs isolated from thymocytes (prepared by Brij 58 detergent extraction) and that Brij 58 DRM-associated Lck activity increased upon TCR triggering with anti-CD3 antibodies (Montixi et al., 1998). These differences may be caused by the use of different detergents or by analyzing different cell systems. Moreover, Arni and associates (1996) used a detergent-free method to isolate T lymphoma plasma membrane fragments that contained highly active Lck and Fyn as well as GPI-anchored proteins. Triton X-100 detergent treatment of these membranes stimulated the enzymatic activity of Lck and Fyn in a CD45-independent manner, suggesting that Lck/Fyn kinase activity is favored in an Lo lipid environment (Ilangumaran et al., 1999). We are left with a paradoxical situation: Lck requires membrane anchoring by fatty S-acylation (i.e., DRM targeting to exert its full activity), yet in Jurkat cells its Triton X-100 detergent–resistant pool appears kinase inactive. Moreover, phosphorylated TCR and signaling proteins strongly accumulate in raft-like membrane patches, suggesting that signaling occurs there (Janes et al., 1999). Clearly, Lck in DRMs is removed from a highly dynamic equilibrium of Lck phosphorylation and dephosphorylation that is embedded in the raft/nonraft environment of the plasma membrane. This “snapshot” may fail to resolve an activation of Lck kinase in rafts followed by rapid down-regulation by Csk or other regulators (Fig. 3). Moreover, it is not clear whether the Triton X-100–soluble active pool of Lck indeed resides in Ld nonraft membrane domains or whether it is more easily extracted by Triton X-100 from raft domains. The functional significance of segregating CD45 and PAG/Cbp/Lck in different phases of the plasma membrane remains an exciting issue to understand, and new information is likely to emerge soon, as the important molecules involved are identified. 4. LAT LAT, a substrate of the ZAP-70 protein tyrosine kinase, is a transmembrane protein expressed in T lymphocytes, natural killer cells, platelets, and mast cells (Weber et al., 1998; Zhang and Samelson, 2000; Zhang et al., 1998a). LAT is known to tightly associate with DRMs (Brdicka et al., 1998; Zhang et al., 1998b). It has a short extracellular domain and a cytoplasmic domain that harbors palmitoylation sites and nine tyrosines that are potentially phosphorylated and recruit signaling proteins (Zhang et al., 1998a). LAT forms complexes with numerous signaling proteins, including Grb2, phospholipase C␥1 (PLC␥1 ), Vav, the p85 subunit of phosphatidylinositol 3-kinase (PI3K), SLP-76, Cbl, and Grap (Zhang and Samelson, 2000; Zhang et al., 1998a). Grb2 binds Sos, a guanine nucleotide exchange factor for ras, leading to ras activation and the mitogen-activated

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protein kinase (MAPK) pathway. PLC␥1 hydrolyzes phosphatidylinositol-4,5bisphosphate (PIP2) yielding diacylglycerol, an activator of protein kinase C, and inositol-1,4,5-trisphosphate (IP3), which induces Ca2+ fluxes (Cantrell, 1996; Wange and Samelson, 1996; Weiss and Littman, 1994). In addition, LAT is complexed with SLP-76. SLP-76 forms a trimolecular complex with adapter protein Nck and Vav, a guanine nucleotide exchange factor for Rho family GTPases (Bubeck Wardenburg et al., 1998; Crespo et al., 1997). This complex is involved in activation of p21-activated kinase and stimulation of actin polymerization. Moreover, PI3K regulatory subunit p85 is bound to LAT, thus linking to the phosphoinositide signaling pathways. Cbl is part of a ubiquitination machinery involved in down-modulating receptor tyrosine kinase signaling (Joazeiro et al., 1999; Lupher et al., 1999; van Leeuwen et al., 1999). Therefore, LAT may form the core of signaling protein complexes that mediate and regulate early TCR signal transduction, including induction of Ca2+ fluxes, protein kinase C activation, activation of the ras–MAPK kinase pathway, and reorganization of the actin cytoskeleton (Zhang and Samelson, 2000). LAT expression in mast cells, platelets, and natural killer cells implies potential roles in the signaling of other IRR-related receptors. While LAT-deficient mice show a block in T cell development (at the CD4−CD8− stage), defects in natural killer cell function were not detected (Zhang et al., 1999b). However, phosphorylation of PLC␥ and platelet activation triggered by collagen receptor glycoprotein VI (GPVI) engagement is severely impaired in LAT-deficient mice (Pasquet et al., 1999). GPVI stimulation results in Fc receptor ␥ -chain phosphorylation and recruitment of Syk tyrosine kinase and is therefore highly reminiscent of Fc receptor signaling (Poole et al., 1997). Thus, LAT not only is a linker for TCR activation but also connects other IRRs to multiple signaling pathways. Two cytoplasmic cysteine palmitoyl acceptor sites (Cys-26 and Cys-29 for human LAT) close to LAT’s transmembrane region are responsible for targeting LAT into DRMs (Lin et al., 1999; Zhang et al., 1998b). The function of LAT palmitoylation was studied by reconstituting LAT into LAT-deficient Jurkat derivatives ANJ3 and JCaM2 (Lin et al., 1999; Zhang et al., 1999a). Both cysteines contribute to the DRM association of LAT; however, the membraneproximal Cys-26 (but not Cys-29) is sufficient for weak DRM association and signal transduction (Zhang et al., 1998b, 1999a). LAT mutants defective in DRM association cannot transduce TCR signals such as Ca2+ mobilization, activation of the ras pathway, and downstream signaling leading to nuclear factor of activated T cells (NFAT)-mediated gene activation and CD69 up-regulation (Lin et al., 1999; Zhang et al., 1999a). This shows that raft targeting is required for LAT function. Interestingly, a LAT construct in which the transmembrane domain and palmitoylation sites of LAT are replaced by the N-terminal membrane anchor of Lck is capable of transducing TCR signals, suggesting that LAT and Lck DRM targeting sequences are interchangeable for supporting LAT function (Lin et al., 1999).

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FIG. 3 Models for the involvement of raft domains in immune recognition receptor (IRR) activation. (A) Formation of large raft patches: cross-linking of raft-associated IRRs induces percolation of rafts. The increase in raft size around cross-linked IRRs causes critical concentration of detergentresistant membrane (DRM)–associated Src kinases and separation from negatively regulating tyrosine kinases such as CD45, leading to net phosphorylation of immunoreceptor tyrosine–based activation motifs (ITAMs) and signaling proteins. (B) Induced phase miscibility: engagement of the IRR triggers transition of the IRR membrane environment to form a liquid-ordered membrane phase miscible with the membrane environment of DRM-associated Src kinases (D-Src). Facilitated access of raft-associated Src kinases to engaged IRRs leads to increased phosphorylation of the IRR ITAMs.

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FIG. 3B (Continued )

5. Signaling via GPI-Anchored Proteins and Glycolipids In some instances, the signaling role of GPI-anchored proteins can be attributed directly to their direct association and cooperation with transmembrane receptors in a cis-signaling configuration. For example, the Fc␥ RIIA transmembrane receptor and GPI-anchored Fc␥ RIIIB are brought into close proximity upon binding to immunoglobulin G (IgG)–coated antigens, leading to synergistic signaling (Green et al., 1997; Zhou et al., 1995). However, in many cases, ligands for GPI-anchored proteins are not known. Still, it appears to be a general phenomenon that lateral oligomerization of GPI-anchored proteins or ganglioside

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GM1 can trigger signaling in hematopoietic cells (Brown, 1993; Gouy et al., 1994; Robinson, 1991). This has been a puzzle for a long time, considering that these molecules are anchored in the outer leaflet of the plasma membrane and hence lack cytoplasmic domains. Therefore, it was a seminal observation that GPIanchored proteins can be recovered together with Src-related tyrosine kinases in detergent-resistant membranes of hematopoietic cells (Brown, 1993; Cinek and Horejsi, 1992; Draberova and Draber, 1993; Horejsi et al., 1999; ShenoyScaria et al., 1992; Stefanova et al., 1991; Thomas and Samelson, 1992). The possible involvement of lipid domains in signaling via GPI-anchored proteins received further support by the finding that signaling via GPI-anchored CD59 and CD48 in Jurkat cells is inhibited by lowering the amounts of cholesterol (Stulnig et al., 1997). Using immunofluorescence microscopy, it was shown that patches formed by antibody–cross-linked GPI-anchored proteins accumulate cytoplasmic leaflet–associated Fyn tyrosine kinase in BHK cells (Harder et al., 1998). Likewise, in Jurkat cells, GM1 glycolipid—cross-linked by the choleratoxin B subunit—formed patches that accumulated intracellular Lck kinase and strongly concentrated tyrosine-phosphorylated proteins (Harder and Simons, 1999; Janes et al., 1999). The mechanism by which clustering of outer leaflet– anchored raft molecules accumulate inner leaflet–anchored Src kinases remains to be established. Possibly, accumulation of inner leaflet–signaling proteins in raft patches reflects a transbilayer coupling of lipids in the raft membrane, a phenomenon that has been demonstrated to occur in artificial membranes of sphingomyelin (Schmidt et al., 1978). However, transmembrane proteins that partition into GM1 patches could also support Lck accumulation and signal transduction in the patches—a scenario that received support from immunofluorescent studies, described next. It has been known for some time that cross-linking of GPI-anchored proteins and GM1 requires expression of the TCR in order to elicit a full signal, leading to proliferation and IL-2 expression in T cells (Gouy et al., 1994; Gunter et al., 1987). Janes and collaborators (1999) showed that TCR–CD3 complexes become concentrated in GM1 and CD59 patches, providing evidence that direct association of the TCR with raft-like patches may be the mechanism for induction of signaling by GM1 and GPI-anchored proteins. This led the authors to propose that TCR molecules that partition into raft patches find an environment of high–tyrosine kinase and low-phosphatase activity, leading to phosphorylation and induction of a signaling cascade. In addition, there is evidence for signaling pathways triggered by cross-linking of GPI-anchored proteins that occur independently of IRR expression (Deckert et al., 1995). While it is well established that cross-linking of GPI-anchored proteins activates T cells, the evidence for a functional importance of GPI-anchored proteins in T cell activation is somewhat conflicting. Several reports have described that T cell clones, hybridomas, and T lymphocytes, deficient in GPI anchor synthesis,

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weakly respond to TCR engagement and that reconstitution of GPI synthesis in these cells partially restored TCR signaling (Romagnoli and Bron, 1997, 1999; Yeh et al., 1988). On the other hand, conditional T lymphocyte–specific disruption of the Pig-a gene of the GPI-biosynthetic pathway did not affect T cell development. Peripheral T cells from these mice responded normally to various stimuli, including concanavalin A or CD3 ligation, despite the complete absence of GPI-anchored proteins (Takahama et al., 1998). How can these results be reconciled? It is possible that GPI-anchored proteins, which may be highly expressed on T cells, are important for providing raft building blocks or for recruitment raft domains into the TCR membrane environment (Killeen, 1997). One may speculate that peripheral T lymphocytes from conditional Pig-a gene– disrupted mice have compensated for the loss of GPI anchors. This may involve the selection of TCRs that do not require strong raft formation for triggering or could involve an increase in sphingolipid expression or fatty acid saturation in order to restore the necessary homeostasis in the raft/nonraft equilibrium in GPI-deficient lymphocytes. An inducible conditional disruption of the the Pig-a gene may circumvent these compensatory mechanisms and help elucidate the role of GPI-anchored proteins in TCR signaling. 6. Phosphoinositides It is the prevailing view that raft-associated lipids have a structural function as raft building blocks. One important exception, the signaling phosphoglyceride PIP2, which makes up 1% of the total phospholipid, is associated with DRMs from A431, MDCK, Neuro 2a, and Jurkat T leukemic cells (Czech, 2000; Hope and Pike, 1996). PIP2 has several functions in signaling and in the regulation of raft dynamics. First, PIP2 is a substrate of PLC␥ generating IP3 and diacylglycerol, which induce Ca2+ mobilization and activation of protein kinase C, respectively. Interestingly, following bradykinin and epidermol growth factor stimulation of A431 cells (which contain caveolae), the detergent-resistant pool of PIP2 is preferentially hydrolyzed by PLC␥ (Pike and Casey, 1996). Cholesterol depletion disrupts association of PIP2 with DRMs and blocks growth factor– induced PIP2 turnover in caveolae-containing A431 cells as well as in Neuro 2a cells, which are devoid of caveolae (Liu et al., 1998; Pike and Miller, 1998). In addition to its role as substrate for second-messenger generation, PIP2 mediates de novo actin polymerization by activation of actin-binding proteins (see Section III,E). Moreover, many signaling proteins contain pleckstrin homology modules that mediate binding to phosphoinositides and hence may be recruited to raft domains. An important example is Tec family tyrosine kinase Itk, which requires its pleckstrin homology domain, which is selective for phosphatidylinositol-3,4,5trisphosphate (PIP3), for association with DRMs (Bunnell et al., 2000). Tec kinases play an important role in immune cell activation, and the mode of DRM targeting will be important to elucidate (van Leeuwen and Samelson, 1999; Yang

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et al., 2000). The mechanism by which PIP2 is targeted to DRMs remains unknown. Raft targeting of PIP2 may be determined by specific interaction of the head group with other raft lipids or with raft-associated proteins. B. RAFT MEMBRANE DOMAINS IN IMMUNORECEPTOR SIGNALING Raft involvement has been studied for the signaling of the most prominent types of IRRs: the Fc receptors—in particular the high-affinity IgE receptor FcεRI, the TCR, and the BCR. Most IRRs are multichain receptors in which specific subunits are responsible for ligand binding, while others couple receptor engagement to the initiation of signaling cascades. These receptors lack an intrinsic kinase activity and rely on protein tyrosine kinases of the Src family Lck, Fyn, Lyn, and Blk for their activation. Receptor engagement induces phosphorylation of paired tyrosine residues in a motif called ITAM (Flaswinkel et al., 1995). Phosphorylated ITAMs recruit Syk family tyrosine kinases, which become activated by phosphorylation and mediate downstream signaling such as Ca2+ mobilization or ras activation, eventually leading to physiological response of the activated cell. 1. Fc Receptors: FcεRI, Fc␥ RIIA/Fc␥ RIIIB, and Fc␣R The high-affinity IgE receptor FcεRI, highly expressed on mast cells and basophils, triggers histamine or noradrenaline secretion, prostaglandin and leukotriene synthesis, and cytokine expression, leading to immediate allergic reactions and inflammatory responses. FcεRI is a heterotetrameric complex composed of FcεRI ␣-subunits and FcεRI ␤-subunits and a dimer of the FcR ␥ -chain. FcR ␥ -chain also functions in the signal transduction of other IgG receptors, such as Fc␥ RI, Fc␥ RIII, and Fc␣RI, and by GPVI, a collagen receptor involved in platelet activation (Beaven and Metzger, 1993; Daeron, 1997; Poole et al., 1997). FcεRI is sensitized by IgE molecules that tightly associate to the FcεRI ␣-subunit via their Fc segment. Binding of oligomeric antigens to receptor-bound IgE induces lateral oligomerization of FcεRI molecules and triggers tyrosine phosphorylation and signaling cascades. Following oligomerization, paired tyrosines in the ITAMs of the FcεRI ␤-chains and FcR ␥ -chains become phosphorylated by Src family tyrosine kinase Lyn. Syk kinase binds to phosphorylated ITAMs of FcR␥ via SH2 phosphotyrosine binding domains. Syk becomes activated, leading to induction of Ca2+ fluxes. In addition, the ras– MAPK pathway is turned on, which induces cell proliferation, PLA2 activation, and leukotriene/prostaglandine synthesis (Kinet, 1999). Pioneering studies by Baird and Holowka using fluorescence microscopy provided first evidence that patches formed by antigen-mediated cross-linking of FcεRI represent membrane domains in a highly ordered lipid phase. They showed that IgE receptor patches accumulate the fluorescent lipid dye DiIC16 in the mast cell line RBL–2H3. DiIC16 has two fully saturated C16 alkyl chains

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and is thus believed to preferentially partition into raft-like ordered phases of lipid bilayers (Spink et al., 1990; Thomas et al., 1994). Lateral mobility of DiIC16 was strongly reduced in the patches of cross-linked FcεRI, underlining the highly ordered state of the lipids in FcεRI patches. Consistently, FcεRI clusters do not enrich lipid dye FAST DiI, which, because of its unsaturated alkyl chains, partitions into more fluid regions of the plasma membrane (Thomas et al., 1994). Further work demonstrated that patches of IgE receptors codistributed with cross-linked Gd1b and GM1 gangliosides, considered to be tightly raft-associated membrane lipids (Pierini et al., 1996; Stauffer and Meyer, 1997). In cytochalasin D–treated RBL–2H3 cells, patches of IgE receptor colocalized with Lyn tyrosine kinase targeted to DRMs via myristoyl and palmitoyl fatty acylations. Depletion of cholesterol from RBL–2H3 cells by methyl-␤-cyclodextrin (M␤CD) extraction strongly reduced DRM association of Lyn, colocalization of Lyn with FcεRI patches, and tyrosine phosphorylation of FcεRI ␤-chains and FcR ␥ -chains. Therefore, DRMs and cholesterol-dependent interactions are important for coupling of Lyn with cross-linked FcεRI (Holowka, et al., 2000). Supporting the notion that FcεRI patches represent foci of downstream signaling, GFP—fused to paired SH2 domains derived from PLC␥ and Syk— transiently associates with IgE receptor patches (Stauffer and Meyer, 1997). In addition, these patches were shown to recruit Vav, a GTP/GDP exchange factor for the small GTPases rac, rho, and cdc42 (Arudchandran et al., 2000). Recruitment of Vav may be important for changes in the actin cytoskeleton that were proposed to regulate the interaction of raft components with patches of FcεRI. Not every type of FcεRI engagement induces sustained signaling. This was shown by the finding that latex beads coated with ligands for IgE–FcεRI receptor complexes were not capable of inducing sustained Ca2+ fluxes and degranulation in RBL–2H3 cells. This correlated with a depletion of Lyn tyrosine kinase, and DiIC16-stainable plasma membrane domains from the bead–cell contact area. The actin cytoskeleton was suggested to play a role in the clearance of raft markers from the latex bead–cell contact area (Pierini et al., 1996) (see Section III,E). Following antigen-mediated cross-linking, a large fraction of FcεRI and in particular tyrosine-phosphorylated FcεRI ␤-chains and FcR ␥ -chains associate with DRMs (Field et al., 1997). The association of ligated FcεRI with DRMs is relatively weak. It is preserved only by using a Triton X-100 concentration one tenth of that used to detect DRM association of Lyn kinase (Field et al., 1995, 1999). Detergent insolubility can be detected prior to tyrosine phosphorylation (Field et al., 1997). It was shown that DRM association of FcεRI did not require FcεRI ␤-chains or the cytoplasmic domain of the FcR ␥ -chain and hence did not require the signaling ITAMs (Field et al., 1999). These observations implicate that the transmembrane domains of FcεRI ␣-chains and FcR ␥ -chains are important for DRM association of ligated FcεRI. In addition, FcεR ␤-chains and

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FcR ␥ -chains have been reported to be fatty acylated, and possibly these modifications may participate in the association of ligated FcεRI with raft membrane domains (Kinet et al., 1985). It is noteworthy that CD20, which elicits signals in B lymphocytes following antibody-mediated cross-linking, shares sequence homology with FcεRI ␤-chain and rapidly acquires detergent insolubility upon ligation (Deans et al., 1998). In CD20, a short, membrane-proximal cytoplasmic section has been shown to play a role in cross-linking–induced DRM association (Polyak et al., 1998). Raft domains have also been implicated in the signaling of IgG receptor FcR␥ RII, which transduces signals via a single ITAM in its cytoplasmic tail. Fc␥ RIIA and GPI-anchored Fc␥ RIIIB are coexpressed on primate polymorphonuclear leukocytes (Unkeless et al., 1995). Binding of IgG-coated immunocomplexes by Fc␥ Rs on these cells induces a respiratory burst reaction. Coligation of GPI-anchored Fc␥ RIIIB with Fc␥ RIIA or CR3 complement receptor synergizes in respiratory burst induction (Zhou et al., 1995). When Fc␥ RIIA and Fc␥ RIIIB were constituted into Jurkat T leukemic cells, coligation of both receptors effectively induced Ca2+ fluxes of higher amplitude and duration than cross-linking of each receptor alone. In the Jurkat cell system, this was also observed upon coligation of other GPI-anchored proteins, showing that synergism in signal transduction is a function of the GPI anchor. It is conceivable that under physiological conditions, immune complexes coligate Fc␥ RIIA and Fc␥ RIIIB and support Fc␥ RIIA triggering by forcing raft membrane domains into the Fc␥ RIIA plasma membrane environment (Green et al., 1997). The IgA receptor Fc␣R mediates phagocytosis, respiratory burst, and degranulation in neutrophils and monocytes. Engagement of Fc␣R is coupled to FcR ␥ -chain phosphorylation, leading to the initiation of downstream signaling cascades (Daeron, 1997). Raft involvement in signaling for this FcR was indicated by showing that antibody–cross-linked Fc␣R cocapped with raft-associated ganglioside GM1 independently of FcR ␥ -chain expression. Interestingly, cocapping of Lyn tyrosine kinase with Fc␣R required FcR ␥ -chain expression, suggesting that it is at least partially mediated by protein–protein interactions. Moreover, Fc␣R ligation induced recruitment of Tec family tyrosine kinase Btk into DRMs (Lang et al., 1999). The amounts of Src-related protein tyrosine kinases have been observed to increase in DRMs upon activation of Fc receptors. This increase was described following stimulation of FcεRI in RBL cells and Fc␣R-expressing B cell transfectants for Lyn kinase, and for Fgr kinase following Fc␥ RIIA ligation from polymorphonuclear leukocytes (Field et al., 1995; Lang et al., 1999; Zhou et al., 1995). In Fc␣R-expressing B cells, the increase of Lyn in DRMs depended on FcR ␥ -chain expression. Hence, raft association of the kinases may be stabilized by signaling-induced protein–protein interactions, as discussed above.

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Alternatively, association of Lyn/Fgr to rafts or the rafts themselves may be stabilized possibly by a change in acyl chain conformation. It is noteworthy that the fraction of polyunsaturated fatty acids in DRMs increases following FcεRI ligation in RBL cells, probably reflecting a change in the composition of raft-like ordered membrane phases (Fridriksson et al., 1999). A further possible mechanism is a specific increase in the palmitoylation of Lyn/Fgr as described for specific G␣-subunits of heterotrimeric G proteins following ␤-adrenergic receptor activation (Mumby, 1997). However, evidence for regulation of S-acylation in the Src kinase family has until now not been described. 2. BCR Activation of the BCR stimulates naive B cells and provides a signal for survival, proliferation, and antibody production. The BCR is composed of an antigenbinding IgM molecule and a heterodimer of Ig␣ and Ig␤ chains, both of which harbor ITAMs. Src tyrosine kinases Lyn, Fyn, and Blk have been implicated in phosphorylating BCR ITAMs, leading to syk tyrosine kinase recruitment and activation. The BCR is stimulated by lateral oligomerization by antigens binding to the IgM chains (DeFranco, 1997; Kurosaki, 1999; Weiss and Littman, 1994). Following cross-linking by antigen or anti-Ig antibodies, BCR is translocated into DRMs. This occurs rapidly (within 6 sec) after cross-linking at 4◦ C and is independent of tyrosine phosphorylation by Src kinases (Cheng et al., 1999; Weintraub et al., 2000). Hence, it appears improbable that an ATP-consuming process within the cell is responsible for DRM targeting. Possibly, lateral crosslinking or a change in conformation stabilizes BCR–raft association. It is important to mention that BCR forms higher-order oligomers in the plasma membrane, and this may facilitate the formation of large clusters in response to ligation (Reth and Wienands, 1997). Additionally, the BCR was observed to be rapidly internalized after activation together with ganglioside GM1. While it is not definite whether GM1 is preferentially endocytosed over bulk plasma membrane lipids, these data showed that the stimulated BCR resides in an environment of GSLs (Cheng et al., 1999). Upon BCR signaling, multiple tyrosine-phosphorylated proteins accumulate in DRMs together with the BCR. Moreover, it was shown that filipin-mediated cholesterol sequestration blocks BCR-elicited Ca2+ fluxes in the A20 B cell line (Aman and Ravichandran, 2000). Most interestingly, targeting of the BCR into a detergent-insoluble fraction is blocked in an anergic B cell population in which response to BCR ligation is strongly reduced (Weintraub et al., 2000). The reason for this difference between anergic and nonanergic B lymphocytes is not known, and its elucidation may lead the way to an understanding of how immunoreceptors are targeted to DRMs following their ligation as well as an understanding of immune tolerance.

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3. TCR The TCR, expressed on the surface of T lymphocytes, triggers signaling cascades, which lead to responses of activated T cells such as lysis of target cells, proliferation, and cytokine secretion. The TCR is triggered upon binding to a cognate peptide–MHC complex on the surface of antigen-presenting cells (APCs). In addition, T cell activation requires multiple accessory molecular interactions between T cells and APCs. The TCR is a multimeric transmembrane protein complex composed of a variable clonotypic Ti␣␤ (or Ti␥ ␦) heterodimer, the CD3 ε␥ ε␦ heterodimers and a dimer of TCR␨ (or related TCR ␩–␩/␩–␨ dimers). Ti␣␤ binds to cognate peptide–MHC class I or class II complexes presented on the surface of an APC. CD3 ε-,␥ -, and ␦-chains as well as TCR␨ dimer contain signal-transducing ITAM motifs. TCR engagement induces phosphorylation of ITAMs by Lck and, to lesser extent, by Fyn protein tyrosine kinases, leading to ZAP-70 tyrosine kinase recruitment and its activation by tyrosine phosphorylation (Chan and Shaw, 1996). ZAP-70 phosphorylates LAT, a transmembrane protein that couples TCR activation to multiple signaling pathways, including induction Ca2+ fluxes and activation of the ras pathway (Cantrell, 1996; Wange and Samelson, 1996; Weiss and Littman, 1994; Zhang and Samelson, 2000). Using fluorescence microscopy, Janes and co-workers (1999) analyzed patches of the TCR—formed by cross-linking with anti-CD3 antibodies—on the surface of Jurkat cells. These patches accumulated raft-associated Lck tyrosine kinase (visualized as green fluorescent protein chimera) and GPI-anchored protein CD59, suggesting that TCR patches represent large, stabilized raft-like membrane domains. Supporting this, TCR patches are strongly stained with the lipid dye DiIC16, which preferentially partitions into raft-like lipid phases (T. Harder and L. Kuhn, unpublished observations). In converse experiments, patches of raft lipid ganglioside GM1 were formed by cholera toxin B subunit–mediated cross-linking. These patches accumulate Lck and tyrosine-phosphorylated proteins (Harder and Simons, 1999; Janes et al., 1999). Interestingly, TCR/CD3— without being cross-linked—partitioned into large raft-like patches formed by GM1, and this did not depend on active Lck/Fyn tyrosine kinases, suggesting that the non–cross-linked TCR per se has an affinity for raft domains (Janes et al., 1999). Montixi and associates (1998) studied TCR association to DRMs prepared from thymocytes using extraction with the detergent Brij 58. Following triggering of the TCR with anti-CD3 antibodies, a significant proportion of TCR subunits Ti␣, CD3ε, and TCR ␨ -chain (and in particular its highly phosphorylated p23 form) partitioned into Brij 58 DRMs. In addition, Brij 58 DRMs strongly accumulated tyrosine-phosphorylated proteins. Importantly, the recruitment of TCR subunits into the Brij 58 DRMs was sensitive to the inhibition of Lck and Fyn using the Src kinase inhibitor PP1. This showed that signaling-dependent

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mechanisms were responsible for Brij 58 detergent insolubility of activated TCR components and suggested that Brij 58 may preserve TCR signaling complexes in membrane domains. It is important to note that cross-linking–induced stabilization of FcεRI or BCR in Triton X-100–insoluble membranes, discussed above, was signaling independent and may therefore represent a different, probably initial, step in DRM association of the cross-linked IRRs. Xavier and colleagues (1998) compared enrichment of the TCR ␨ -chain in DRMs, detergent-soluble membranes, and cytosolic fractions before and after TCR activation of Jurkat cells. They found that phosphorylated TCR ␨ -chain is enriched in Triton X-100 DRMs following TCR activation. In contrast, others have reported the absence of TCR/CD3 subunits in Triton X-100–insoluble membranes (Janes et al., 1999; Leyton et al., 1999; Zhang et al., 1998b), possibly as a result of the different representations of the DRM fraction by the different studies. Accumulation of TCR/CD3 in patches of cross-linked GM1 is sensitive to Triton X-100 extraction, suggesting that a weak association of TCR with raft membrane domains is lost during the preparation of Triton X-100 DRMs (Janes et al., 1999). Many cytoplasmic signaling proteins are recruited to DRMs following TCR activation, probably by association with DRM-associated phosphorylated LAT. Accordingly, following TCR activation, a significant fraction of Vav, PLC␥ , and grb2 partitions into DRMs (Zhang et al., 1998b). Moreover, TCR triggering induces translocation of SHC into DRMs and DRM association of PI3K isoforms, possibly by binding to the LAT-bound p85 regulatory subunit of PI3K (Xavier et al., 1998). Recruitment of ZAP-70 into DRMs described by Xavier and collaborators (1998) most likely occurs via association with DRM-associated tyrosine-phosphorylated TCR ␨ -chain. This is corroborated by fluorescence microscopy, which showed that SH2 domains of ZAP-70 fused to GFP accumulated in membrane patches formed by cross-linking of TCR/CD3 or raft glycolipid GM1 (Janes et al., 1999). 4. Effects of Raft Perturbation on IRR Signaling Several strategies have been followed to study the effects of raft disruption on IRR signaling. Formation of raft-like Lo phases in lipid bilayers depends on cholesterol, and therefore different strategies have been used to perturb cholesterol in immune cells. Cyclic glucose heptamer M␤CD extracts cholesterol from membranes by complexing it into a hydrophobic pocket, rendering it soluble in the aqueous medium (Klein et al., 1995). Cholesterol depletion from RBL cells inhibited FcεRI ␤-chain and FcR ␥ -chain tyrosine phosphorylation and blocked colocalization of Lyn tyrosine kinase with patches of FcεRI in cytochalasin D– treated RBL–2H3 cells (Sheets et al., 1999a). Likewise, cholesterol extraction with M␤CD blocked the Ca2+ fluxes in Jurkat cells in response to TCR/CD3

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engagement (Moran and Miceli, 1998; Xavier et al., 1998). Moreover, polyene antibiotics nystatin and filipin sequester cholesterol in cell membranes (Bolard, 1986). Treatment with these drugs was also shown to inhibit anti-CD3– induced Ca2+ fluxes and tyrosine phosphorylation in Jurkat cells (Xavier et al., 1998). Consistently, filipin treatment strongly reduced association of Fyn tyrosine kinase with a CD8 transmembrane chimera carrying TCR ␨ -chain ITAMs (van’t Hof and Resh, 1999). Importantly, a careful examination revealed that early effects of cholesterol perturbation by M␤CD depletion and filipin treatment in Jurkat cells were not as simple (Kabouridis et al., 2000). Specifically, loss of LAT and Lck DRM association occurred within the first 15 min of M␤CD extraction and was accompanied by an increase in tyrosine phosphorylation and activation of the ras pathway, leading to phosphorylation of ERK1 and ERK2 MAPKs. Hyperactivation of the ras pathway was also observed upon cholesterol depletion in RAT-1 fibroblasts (which contain caveolae) (Furuchi and Anderson, 1998). In contrast, induction of Ca2+ fluxes was inhibited in M␤CD-treated Jurkat cells, possibly caused by disrupting raft association of the PLC␥ substrate PIP2 (Kabouridis et al., 2000; Pike and Miller, 1998; Xavier et al., 1998). In 3T3 fibroblasts, cholesterol perturbation, either by M␤CD extraction or by overexpression of a dominant negative mutant of cholesterol-sequestering protein caveolin1, inhibited transforming activity of activated H-ras but not of activated K-ras (Roy et al., 1999). Intriguingly, both forms of ras have C-terminal prenylation common to the ras family proteins but differ in their second membrane-binding determinant: H-ras is membrane-anchored via a palmitoyl anchor, while K-ras contains a stretch of basic amino acids that mediate association to phospholipids (Lobell, 1998). It appears likely that the outcomes of cholesterol perturbation for the different activated ras transfectants are due to the palmitoyl membrane anchor, which targets H-ras into cholesterol-rich microdomains. These domains may mediate signaling downstream of activated H-ras, leading to oncogenic transformation of NIH–3T3 cells. An additional strategy to disrupt raft domains is exogenous application of high amounts of ganglioside GM1 (Simons et al., 1999). In GM1-treated Jurkat cells, GPI-anchored proteins are partially internalized and anti-CD3 antibody–elicited Ca2+ fluxes and tyrosine phosphorylation are inhibited (Xavier et al., 1998). Treatment of Jurkat cells with PUFAs has been used to disrupt the interaction of Lck and Fyn with raft membrane domains. It was shown that treatment with PUFAs inhibited protein tyrosine phosphorylation and Ca2+ fluxes in response to ligation of the TCR and GPI-anchored CD59 (Stulnig et al., 1998). PUFA treatment caused an increase in PUFAs in the phospholipids of the Jurkat cells, suggesting an increase in membrane fluidity and possibly disruption of the rafts (Stulnig et al., 1998). However, an alternative explanation for this phenomenon was given by the observation that bromopalmitate and PUFAs inhibit protein

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palmitoylation and hence cause displacement of proteins such as LAT, Lck, and Fyn from DRMs (Webb et al., 2000). Integration of the data on the effects of raft perturbation shows a relatively broad spectrum of cellular events. Raft domains may have a role that extends beyond the formation of a platform that concentrates signaling proteins in membrane domains while excluding negatively regulating phosphatases. Instead, rafts may also function by keeping critical signaling proteins (e.g., kinases and their substrates) separated, leading to deregulated interactions upon generation of a single plasma membrane phase by cholesterol depletion. C. RAFT DOMAINS AND THE MECHANISM OF IMMUNORECEPTOR TRIGGERING A wealth of data has accumulated in support of a key role of raft domains in IRR triggering, and several models have been put forward to explain the mechanism by which this occurs. In the first first model, it is assumed that IRRs are associated with raft domains. Percolation of raft domains into large patches by IRR cross-linking may be translated into receptor activation. Accordingly, raft-associated signaling proteins become critically concentrated and activated in the vicinity of the engaged receptors, while nonraft CD45 phosphatase is segregated from the IRR patches. Taking the example of TCR signaling, dephosphorylation of Lck’s Tyr394 (which needs to be phosphorylated in order for Lck kinase to be active [D’Oro et al., 1996]) by CD45 would be reduced by this separation, resulting in enhanced autophosphorylation of stimulatory Tyr-394 (Holowka et al., 2000; Janes et al., 2000) (Fig. 2). Possibly, substrates other than Lck are also protected from CD45-mediated tyrosine dephosphorylation, such as components of the TCR or ZAP-70. In support of this model, patches of GM1—cross-linked by the cholera toxin B subunit—accumulate TCR/CD3 and exclude CD45 (Janes et al., 1999). In addition, the kinase activity of Lck and Fyn may be favored in an ordered membrane environment (Ilangumaran et al., 1999). It is important to note that formation of large patches of FcεRI and TCR is not required for triggering and that low-level oligomerization into dimers or trimers is a sufficient stimulus for their signaling (Boniface et al., 1998; Cochran et al., 2000; Davis et al., 1998; Germain, 1997; Holowka and Baird, 1996; Metzger, 1999). As discussed above, the size of raft domains harboring GPI-linked proteins is in the range of 50 nm in diameter, covering a surface 2000 nm2 (Pralle et al., 2000). Moreover, the inter-receptor spacing between two IgE receptor molecules in FcεRI lattices formed by anti-IgE antibody cross-linking is 10 nm, and each IgE receptor unit occupies a surface of ∼350 nm2 (Ryan et al., 1986). Therefore, a raft domain could accommodate five cross-linked FcεRI receptor molecules without having to increase in size. Considering the dynamic nature of raft domains, low-order oligomerization of an immunoreceptor does

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not necessarily lead to an increase in their size. Triggering would have to be induced by a small increase in the concentration of Src-related protein tyrosine kinases and a low degree of segregation from nonraft regulatory phosphatase CD45. Indeed, a very subtle increase in net phosphorylation must then be sufficient to surpass a threshold for triggering. A fundamentally different model suggests that engagement of IRRs causes a qualitative change in the phase of its plasma membrane environment (Sheets et al., 1999b). Accordingly nonengaged IRRs have a weak raft affinity and largely reside in an Ld membrane phase that is, however, close to the transition to the Lo phase. Cooperative action of weak raft affinities or an ordering effect of multiple aligned transmembrane helices could cause, even at a low extent of cross-linking, an increase in the Lo phase in the plasma membrane environment of engaged receptors. In support of this, coligation of IRRs with GPI-anchored proteins or ganglioside GM1 synergistically potentiates signaling and tyrosine phosphorylation, possibly as a consequence of increasing the amount of their raft plasma membrane microenvironment (Green et al., 1997; Leyton et al., 1999; Moran and Miceli, 1998; Viola et al., 1999). The Lo phase environment of engaged receptors is miscible with the membrane environment of permanently raft-associated signaling proteins such as DRM-associated Src kinases or LAT. This would allow these molecules to gain access to the IRRs. Accordingly, rafts negatively regulate signaling by reducing undue interaction of raft-associated Src-related kinases with their unengaged IRR targets. The threshold for triggering is set by the degree of IRR engagement required to induce phase transition. It is important to note that, in addition to raft-facilitated access, protein–protein interactions are required for ongoing signaling. For example, increased tyrosine phosphorylation following ligation of CD3 and GPI-linked CD48 is blocked in T cell hybridoma cells expressing activated Lck mutants that lack a functional SH2 domain (Lewis et al., 1997; Moran and Miceli, 1998). Likewise, Ca2+ fluxes and activation of the ras pathway in RBL–2H3 cells elicited by FcεRI ligation was shown to require DRM targeting by a dually acylated Lyn membrane anchor as well as SH2 domains (Honda et al., 2000). The crux of defining the role of raft membrane domains in the triggering of IRRs is to understand the molecular composition and physical properties of IRRs’ immediate membrane environment before and after triggering. D. CD4 AND CD8 CORECEPTORS FOR THE MAJOR HISTOCOMPATIBILITY COMPLEX CD4 and CD8 are expressed by helper or cytolytic T lymphocytes, respectively, where they function as coreceptors for major histocompatibility complex (MHC) class II and MHC class I on the surface of APCs. CD4 and CD8 associate with the raft-associated signaling proteins Lck and LAT. CD8 binding to both LAT and Lck depends on the specific cysteine CXC motif on CD8␣ (Bosselut

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et al., 1999). Thus, by simultaneous binding of the MHC to the TCR and to CD4/CD8, Lck and LAT are guided into proximity of the TCR. High concentrations of monomeric soluble MHC class I molecules stimulated Ca2+ mobilization in CD8+ T lymphocytes, suggesting that CD8–TCR heterodimerization by MHC class I can be a trigger for TCR activation (Delon et al., 1998). However, in a comparable experimental setup using CD4+ T cell lines, monomeric engagement by soluble peptide–MHC class II was not able to trigger the TCR, despite the presence CD4 (Boniface et al., 1998; Cochran et al., 2000). CD4 and CD8 are required for T cell activation with weaker peptide–MHC ligands but are dispensible for activation by optimal peptide–MHC TCR ligands and stimulation by anti-TCR/CD3 antibodies (Viola et al., 1997). Interestingly, CD4 (Cerny et al., 1996; Millan et al., 1999; Parolini et al., 1996; Xavier et al., 1998) as well as CD8 (Horejsi et al., 1999) have been recovered in DRMs from T cell lines. Interestingly, CD4–Lck association is disrupted following protein kinase C activation (Parolini et al., 1999). Important questions for future studies are whether binding to Lck and LAT affects DRM association of CD4 and CD8 and to what extent raft domains control the functions of CD4 and CD8. E. ACTIN CYTOSKELETON IN CROSS-TALK WITH RAFT MEMBRANE DOMAINS The actin cytoskeleton plays an important role in signaling via immunoreceptors and in the activation of T lymphocytes. Several reports have indicated that actin may modulate raft structure and composition. In Jurkat T leukemic cells, actin filaments accumulate at patches formed by cross-linking of DRMassociated GPI-anchored proteins (Deckert et al., 1996) and ganglioside GM1 in a manner dependent on tyrosine phosphorylation (Harder and Simons, 1999). In contrast, nonraft membrane patches formed by cross-linking of transferrin receptor did not accumulate actin and tyrosine-phosphorylated proteins (Harder and Simons, 1999). As discussed above, phosphoinositides have been implicated to reside in raft domains and may mediate local actin polymerization. Phosphoinositides are specifically bound by pleckstrin homology domains, which are found on many proteins involved in signaling and actin polymerization (Czech, 2000). One key pathway of actin polymerization is the PIP2-dependent activation via the pleckstrin homology domain containing Wiskott–Aldrich syndrome protein (WASP), the small rho family GTPase cdc42, and the actin-nucleating Arp2/3 complex (Higgs and Pollard, 1999; Rohatgi et al., 1999). Overexpression of phosphatidylinositol 4Pi 5-kinase was shown to up-modulate the amounts of PIP2 in NIH–3T3 and REF52 cells and induced the formation of actin tails on intracellular vesicles in a WASP–Arp2/3–dependent manner. Actin comets were observed on vesicles containing classical raft markers such as GM1 or influenza virus HA but not on nonraft transferrin-positive vesicles (Rozelle et al., 2000). Thus, the specific concentration of PIP2 may activate actin nucleation and

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polymerization at raft domains. Moreover, PIP2 has been shown to activate proteins involved in actin–membrane interactions. For example, it stimulates the binding of members of the ezrin–radixin–moesin protein family to CD44—a broadly expressed protein that resides in DRMs (Hirao et al., 1996; Ilangumaran et al., 1998; Neame et al., 1995; Oliferenko et al., 1999). Phosphorylation of PIP2 by PI3K generates PIP3. PIP3 is a strong activator of Vav GDP/GTP exchange activity for small GTPases of the rho family, hence leading to extensive reorganization of the actin cytoskeleton following IRR activation (Fischer et al., 1998; Han et al., 1998). Vav is targeted into DRMs following TCR and FcεRI activation in Jurkat cells and RBL–2H3 cells, respectively, possibly mediated by complex formation with DRM-associated LAT (Arudchandran et al., 2000; Zhang et al., 1998b). Indeed, GFP-tagged Vav translocates to the plasma membrane upon FcεRI aggregation in RBL cells and partially colocalizes with FcεRI patches (Arudchandran et al., 2000). Intriguingly, cytochalasin D–induced inhibition of de novo actin filament formation enhanced colocalization of FcεRI patches with Lyn tyrosine kinase, GPIlinked Thy-1, and ganglioside GD1b in RBL–2H3 cells (Holowka et al., 2000). This correlated with an increased strength and duration of protein tyrosine phosphorylation following FcεRI triggering in cytochalasin D–treated cells. It was proposed that the actin cytoskeleton is involved in clearing raft-associated signaling proteins from clusters of FcεRI as a mode of down-modulating signaling via FcεRI. Supporting this, actin filaments transiently accumulate at the contact zone between FcεRI binding antigen-coated beads and RBL–2H3 cells (Pierini et al., 1996). Raft and raft-associated signaling proteins are cleared from this contact zone—probably the reason for the failure of these beads to stimulate sustained Ca2+ fluxes and degranulation. Hence, raft–actin interaction may negatively regulate FcεRI signaling by controlling the colocalization of raft-associated signaling proteins to clusters of FcεRI and may explain enhanced and sustained FcεRI signaling in cells treated with cytochalasin D and latrunculin (which depolymerizes actin) (Frigeri and Apgar, 1999; Pierini et al., 1997). Actin has been shown to associate directly with TCR ␨ -chain in a Lck tyrosine kinase–dependent manner (Rozdzial et al., 1995, 1998). Coligation of the TCR with GPI-anchored CD48 was shown to support interactions of actin TCR ␨ -chain (Moran and Miceli, 1998). It is possible that the actin cytoskeleton is involved in stabilization of the TCR in raft membrane microdomains. In contrast to the stimulatory effects actin perturbation has on FcεRI signaling, cytochalasin D treatment inhibits activation of T cells (Valitutti et al., 1995). This is most likely linked to the requirement of reorganizing the actin cytoskeleton in order to elicit T cell activation, discussed in the next section (Penninger and Crabtree, 1999).

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F. ARCHITECTURE OF T CELL ACTIVATION Rearrangement of cytoarchitecture is a hallmark of T lymphocyte activation. T lymphocytes polarize toward the site of activation, for example, with an APC or a bead coated with TCR-activating antibodies. This polarization is reflected in movement of the microtubule-organizing center toward the APC or stimulating bead and is accompanied by actin polymerization at the site of engagement (Lowin-Kropf et al., 1998; Sedwick et al., 1999). It was shown that actin was required for sustained signaling of a T cell line by an APC, possibly by stabilizing APC–T cell contacts (Valitutti et al., 1995). Disruption of the genes for Vav and WASP severely affected actin-dependent capping following TCR stimulation by antibodies. Early TCR-elicited signaling events such as tyrosine phosphorylation were relatively weakly affected in WASP- and Vav-deficient T cells, while TCR activation–induced proliferation was strongly reduced (Cantrell, 1998; Fischer et al., 1998; Snapper and Rosen, 1999; Snapper et al., 1998). It was proposed that the actin cytoskeleton induces higher-order oligomerization of the TCR—reflected in cap formation—which may contribute to the sustained signaling required for T cell activation (Fischer et al., 1998; Turner et al., 1997). T lymphocyte polarization may also impose a transport of raft domains to the source of T cell activation. Engagement of CD28 by B7 antigens on the APC surface provides a strong costimulatory signal for T cell activation (Slavik et al., 1999). Interestingly, it was found that, depending on CD28 engagement, raft-associated ganglioside GM1 became concentrated at the pole of a naive T cell facing a polystyrene bead coated with TCR/CD28-stimulating antibodies, suggesting that raft domains moved to the site of TCR/CD28 engagement (Viola et al., 1999). Upon CD28 coengagement, the decay of tyrosine phosphorylation on proteins following inhibition of Lck/Fyn tyrosine kinase activity using PP1 was considerably slower than for TCR/CD3 engagement alone (Viola et al., 1999). This CD28-dependent retardation of protein tyrosine dephosphorylation can be caused stabilized kinase activities in the T cell–bead contact zone and exclusion of nonraft phosphatases from raft-associated TCR signaling assemblies. TCR stimulation together with CD28 or LFA-1 coengagement induced an actin– myosin motorprotein-dependent flow of plasma membrane constituents to the contact site (Wulfing ¨ and Davis, 1998). In natural killer (NK) cells, a polarization of patches formed by cholera toxin B subunit–cross-linked GM1 toward a target cell has been described. Movement of these patches to the contact area between the NK cell and the target cell was blocked when NK cell inhibitory receptors were engaged by the MHC on the target cell (Lou et al., 2000). Multiple molecular interactions coordinate the formation of APC–T lymphocyte conjugates, which form a highly defined contact zone termed the immunological synapse (Kupfer and Singer, 1989; Paul and Seder, 1994). Signals received by the T cell by engagement of the TCR and other surface molecules are

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integrated in the immunological synapse and translated into T lymphocyte responses (van der Merwe et al., 2000). The lateral segregation of membrane proteins in the immunological synapse was first described in fixed APC–T cell conjugates (Monks et al., 1998). Very similar structures form when T cells are activated on planar lipid bilayers loaded with GPI lipid–anchored cognate peptide–MHC and ICAM-1 (Grakoui et al., 1999). The mature immunological synapse successively forms within 5–10 min of APC–T cell contact and exhibits a clear definition of membrane domains that are thought to support lateral compartmentalization required for the activation of T cells. Its key feature is the segregation of a central supramolecular activation cluster (cSMAC) from a concentrically surrounding ring termed peripheral SMAC (pSMAC), which is further divided into an inner and an outer adhesion ring. TCR/CD3 and its MHC ligands, CD28–B7.2/CD80 pair, cytosolic signaling proteins Lck and Fyn, and protein kinase C␪ are enriched in the cSMAC. CD2 and its CD48/CD58 ligands are concentrated in the inner adhesion ring of the pSMAC, while LFA-1–ICAM pairs, actin, and talin reside in the outer part (van der Merwe et al., 2000). CD43 is a large, highly glycosylated protein that is excluded from the APC–T cell interface (Sperling et al., 1998). Moreover, CD45 is excluded from the central region in conjugates of a human T cell line with an APC (Leupin et al., 2000). In line with the formation of membrane domains of high-kinase, low-phosphatase activity in the immunological synapse, phosphotyrosine-containing proteins are strongly concentrated at the interface between APC and T cells (Muller et al., 1999). TCR and its peptide–MHC ligand, as well as CD2 and CD48/CD58 in the central region of the immunological synapse are relatively small molecules that span a gap between the APC and the T cell of ∼15 nm. In contrast, integrin LFA-1 and its ligand ICAM, CD43, as well as CD45 span >50 nm. Hence, it was proposed that lateral segregation of these molecules is supported by parallel apposition of T cell and APC plasma membranes in which protein pairs are aligned according to their size in a process that may generate signaling subdomains involved in TCR triggering (Shaw and Dustin, 1997). The enrichment of Lck/Fyn and the TCR complex in the central zone of the immunological synapse implies that raft domains reside in the cSMAC. Indeed, the passive recruitment of GPI-anchored CD48 into the contact zone of a T cell hybridoma and an APC-supported TCR ␨ -chain phosphorylation, the association of TCR with the actin cytoskeleton as well as downstream responses such as IL-2 secretion (Moran and Miceli, 1998). The density of peptide–MHC ligands in the cSMAC was shown to reach up to 350 molecules per square micron for strong agonists. Assuming that densities reached by antibody–cross-linked patches of FcεRI (2000 molecules per micron) resemble those of antibody–cross-linked TCR, this is considerably lower than the density of TCR in antibody–cross-linked patches. Hence, it is important

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to define whether the densities of engaged TCR molecules reached within the cSMAC are sufficiently high to induce a percolation of the raft into a continuous patch or whether raft domains are dispersed within the immunological synapse. This question may be answered by analyzing bona fide raft markers and their distribution in the immunological synapse. It remains to be defined whether rafts are directly involved in the definition of SMAC–membrane domains and in T lymphocyte activation by an APC or whether the role of rafts is restricted to the initial TCR triggering that precedes formation of the immunological synapse. IV. Outlook

The concept of a lateral compartmentalization of the plasma membrane into membrane domains of different phases has now accumulated a wealth of experimental support. Membrane components are thought to specifically partition into raft domains dictated by inherent partition coefficients. While protein–protein interactions can be characterized by crystal structures, mutational analysis, the study of binding kinetics, and so on, the nature of raft-mediated interactions between membrane proteins are not yet defined. It is not clear whether rafts function by keeping together two proteins in one membrane domain or whether rafts may serve as the meeting point of dynamically exchanging membrane components and proteins mediating stable interactions. Raft dynamics are shifted toward larger, stabilized rafts when proteins are oligomerized. It is an attractive hypothesis that triggering of immunoreceptors involves a change in the raft membrane environment, either by increasing the size of raft domains around IRRs or by a qualitative acquisition of an Lo membrane phase. Future studies will use novel methods that directly address the molecular environment of resting and activated IRRs. We will then need to understand how rafts are involved in the modulation of immune cell activation and how costimulatory or inhibitory signals are perceived and integrated. ACKNOWLEDGMENTS I am grateful to Raul Torres, Klaus Karjalainen, Derek Toomre, and Kai Simons for their critical review of the manuscript. I thank Shasha Tarakhovsky and Burkhard Schraven for communicating results prior to publication. The Basel Institute for Immunology was founded and is supported by Hofmann La Roche Ltd.

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ADVANCES IN IMMUNOLOGY, VOL. 77

Human Basophils: Mediator Release and Cytokine Production JOHN T. SCHROEDER, DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224

I. Introduction

The binding of antigen to receptor-bound IgE on the surface of basophils (and mast cells), resulting in the release of potent inflammatory mediators such as histamine, leukotrienes, and prostaglandins, has long been recognized as the hallmark response contributing to the signs and symptoms associated with allergic disease. The role of the basophil in early studies investigating the mechanisms underlying this reaction was primarily one of a surrogate by which to better understand those of the less accessible tissue mast cell. Indeed, functional similarities between the two cell types were suggested some 30 years ago, when it was shown that in vitro histamine release from basophils to various allergens predicted the severity of the respiratory symptoms experienced by the donor when exposed to that allergen (Lichtenstein et al., 1968). The comparison between basophils and mast cells, however, has since been abandoned, as there is mounting evidence indicating that the basophil, which is far more responsive to a variety of stimuli and cytokines, plays a more significant role in the late responses following allergen exposure rather than the early events that seem most attributed to the mast cell. To extend on this belief, studies have shown that human basophils themselves are cytokine-secreting cells, producing cytokines originally described in a subset of mouse T lymphocytes (Mossman et al., 1986). Although this finding stemmed from work done in murine mast cell lines (Plaut et al., 1989; Wodnar-Filipowicz et al., 1989), there is some doubt that isolated human mast cells possess similar capabilities. Basophils, however, readily generate large quantities of interleukin 4 (IL-4) and IL-13—two of the so-called T helper 2 (Th2) cytokines that are found in tissues during allergic inflammation and are thought to contribute to the overall pathogenesis of disease. This discovery, which is still very much in its infancy, along with the fact that basophils selectively infiltrate allergic lesions along with eosinophils and lymphocytes, raises an important concept: namely, the belief that these cells can contribute to disease by modulating the biological responses of other cell types. Thus, some 130 years since their first description in humans, we are continuing to evaluate the potential of the basophil and its role in immune responses. This chapter focuses on existing as well as more recent information pertaining to the biology of these cells, with particular emphasis on the parameters, pharmacological control, and mechanisms regulating the generation of IL-4 and IL-13 from these cells. 93 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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II. Basophil Growth and Maturation

Under normal circumstances, basophils develop in the bone marrow and are thought to be released into circulation as mature cells, as which they represent <1% of the leukocytes found in blood. For comparison, it seems quite certain that mast cell development involves the release of an intermediate precursor from bone marrow, which somehow targets organ tissue and skin, where its final maturation into a mast cell is under the influence of stem cell factor and perhaps other cytokines (Galli, 1990). Despite early beliefs, the overwhelming consensus at this time is that circulating basophils do not give rise to tissue mast cells. Studies have shown, in fact, that basophils more likely share a common precursor with eosinophils and the frequency of these progenitors is increased in atopic individuals (Denburg, 1998; Denburg et al., 1985a,b). In vivo and in vitro studies show that IL-3 has a profound influence on the maturation of basophils. The numbers of both cell types and their progenitors were shown to increase dramatically after the infusion of recombinant IL-3 or granulocyte–monocyte colony–stimulating factor (GM-CSF) into nonhuman primates, and cells having ultrastructural characteristics of both basophils and eosinophils have been described (Dvorak et al., 1989). Furthermore, numerous in vitro studies have shown that cells resembling basophils, both morphologically and functionally, develop from CD34+ cells after 2–3 weeks of culture in media containing IL-3 (Kirshenbaum et al., 1992). Several reports, in fact, have described culture protocols using IL-3 for obtaining basophil-like cells from cord blood precursors for use in functional studies (Kepley et al., 1998a; Valent et al., 1990). It is unclear how long basophils survive in circulation, although early studies suggest that their life span is on the order of days to weeks (Murakami et al., 1969). More recently, in vitro studies have suggested that mature basophils thrive in IL-3 and that this cytokine, at picomolar concentrations, can maintain the viability of these cells for several weeks (MacGlashan et al., 1998). Thus, it seems reasonable to believe that, in vivo, basophils in a microenvironment containing IL-3 can also survive for weeks. As discussed in more detail below, the function of basophils under the influence of IL-3 is dramatically up-regulated, indicating that this cytokine has pro-inflammatory characteristics in addition to growth and anti-apoptotic properties. III. Cell Surface Markers

A. ADHESION MOLECULES Dual flow-cytometric techniques that utilize characteristics of cellular light scatter and immunofluorescence have made it possible to phenotype rare cells in mixed leukocyte suspensions. For the human basophil, this has enabled the elucidation of a variety of cell surface molecules important for adhesion and cell activation (Bochner et al., 1997). The basic approach is to distinguish basophils

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from other leukocytes using an antibody specific for its unique determinant— IgE—that is conjugated with one fluorochrome (e.g., fluoroisothiocyanate), and to use a second antibody conjugated with a second fluorochrome (e.g., phycoerythrin) that is specific for the molecule in question. With the mounting evidence that basophils selectively infiltrate allergic lesions, it is not surprising that they express a variety of molecules that enable them to migrate from the circulation across the endothelial barrier via a complex network of adhesion–deadhesion events. The adhesion molecules found on basophils include selectins, integrins, and molecules belonging to the immunoglobulin (Ig) family. The major selectin, L-selectin (CD62L), binds the ligands GlyCAM-1, CD34, and MAdCAM-1, and its expression declines on basophils exposed to certain chemoattractants (Bochner and Sterbinsky, 1991; Wimazal et al., 1999). Selectins are thought to play a role in the early rolling, or “tethering,” that occurs on endothelium. In contrast, the expression of the ␤2-integrins, CD11b/CD18 (Mac-1) and CD35 (CR1), but not CD11a/CD18 (LFA-1) is increased in response to the same activating stimuli (Bochner and Sterbinsky, 1991; Bochner et al., 1988, 1989, 1990). These, along with the ␤1-integrins and intercellular adhesion molecules (ICAMs) (or Ig-like molecules), are thought to play a role in the actual transendothelial migration process. In particular, the ␤1-integrin, very late antigen (VLA)-4 (␣4␤1), found on basophils, eosinophils, and T lymphocytes but not neutrophils, binds the ligand, vascular cell adhesion molecule-1 (VCAM-1), and this interaction plays a critical role in the selective trafficking of these cells into allergic lesions. Moreover, the expression of VCAM-1 on endothelium is up-regulated by IL-4 and IL-13 (Bochner et al., 1995; Schleimer et al., 1992). As noted, activated basophils secrete both of these cytokines, suggesting that they may facilitate their own migration, as well as that of other cell types, by regulating the expression of specific adhesion ligands, such as VCAM-1. B. CYTOKINE RECEPTORS The inflammatory nature of basophils can also be attributed to the fact that these cells express a variety of receptors that bind specific cytokines. Those identified on mature human basophils include IL-1 through IL-5 and IL-8; high-affinity receptors are found only for IL-3, IL-4, and IL-8 (Valent, 1994). Most significantly, the IL-3 receptor, which is composed of a specific ␣-chain and a common ␤-chain (also shared with the IL-5 and GM-CSF receptors), is retained on mature basophils even after their development from precursor cells. In fact, based on recent flow-cytometric studies, its expression is far greater on basophils than on any of the other major leukocytes, including neutrophils, eosinophils, monocytes, and lymphocytes, suggesting that it might be used as a marker by which to distinguish basophils from these cell types (Sarmiento et al., 1995; Yamada et al., 1998). Interestingly, rare dendritic cell populations found

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in blood also express high levels of IL-3 receptor (Olweus et al., 1997). Although basophils differ by lacking the expression of human leukocyte antigen (HLA) class II molecules, it is intriguing to think that the two cell types may share a common origin. Basophils also express receptors for several of the so-called chemokines (or chemotactic cytokines), of which there are four major families currently comprising a total of some 40 proteins (Nickel et al., 1999). However, basophils primarily express receptors only for those that belong to the CC or ␤-family (i.e., those that have no intervening amino acid between their conserved cysteine residues). The one exception is IL-8, which is a member of the CXC family (whose members have an amino acid between their conserved cysteine residues) and is more commonly associated with neutrophil trafficking. High levels of CCR3 are also found on basophils, and this receptor has overlapping specificity for several CC chemokines, namely, the monocyte chemotactic proteins (MCPs) 4, 3, and 2 as well as eotaxin and RANTES (Uguccioni et al., 1997). All of these chemokines have been shown to promote basophil migration in vitro and several, particularly eotaxin and RANTES, are well-known chemoattractants for eosinophils and lymphocytes, which partially accounts for the fact that basophils are similarly recruited to allergic lesions along with these other cell types. As discussed below, some of these chemokines have been reported to affect mediator release and cytokine secretion from basophils, suggesting that they not only are responsible for cell migration but also may modulate cellular activation. C. MARKERS OF ACTIVATION The high-affinity (but not the low-affinity) receptor for IgE (FcεRI) has been the hallmark determinant found on basophils as well as mast cells. Studies in the 1980s suggested that three subunits make up this receptor; molecular cloning and transfection studies validated this view by revealing that they are expressed in the membrane as an ␣␤␥ 2 tetramer (Kinet et al., 1988). IgE binding is mediated through the ␣-subunit, and this has an extremely high affinity (K a > 1010 ). The x-ray crystal structure of the ␣-subunit has recently been resolved to 2.4 A. Within the portion of the subunit that confers specificity for IgE are found four tryptophan residues organized in an unusual loop that provides for a hydrophobic environment and likely accounts for the high affinity for IgE binding (Garman et al., 1998, 1999; Hulett et al., 1999). For basophils, it has been shown that the number of FcεRI receptors can vary between 5000 and 1 million and is very much dependent on the donor (MacGlashan et al., 1983). Studies performed in the late 1970s showed evidence that the expression of FcεRI on circulating basophils correlates with the IgE antibody levels in serum (Malveaux et al., 1978). This led to the hypothesis that the IgE concentration “drove” the number of IgE receptors on basophils and mast cells. Only recently has this concept been confirmed in humans in work made possible with the development of anti-IgE

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therapy to prevent allergic disease (Fick, 1999). The intravenous administration of a humanized monoclonal anti-IgE antibody (E25) to subjects allergic to dust mite allergen caused a >90% reduction in their serum IgE levels, which resulted in a dramatic reduction in the ability of antigen to induce histamine release from basophils isolated from these donors (MacGlashan et al., 1997). Most interestingly, the drop in serum IgE levels was accompanied by the loss of FcεRI␣ expression. Upon completion of the anti-IgE therapy, serum IgE levels eventually returned, as did the expression of the receptor and the ability of antigen to induce histamine release (Saini et al., 1999). In vitro studies confirmed these data, elucidating the kinetics for the down-regulation and up-regulation of FcεRI␣ and demonstrating that this regulation could be attributed to the absence or presence of IgE (MacGlashan et al., 1998, 1999). A comprehensive analysis pertaining to the mechanisms and consequences of IgE antibody–dependent regulation of FcεRI expression is beyond the scope of this chapter but has been provided elsewhere (MacGlashan et al., 2000). Although the expression of FcεRI was originally thought to be limited to basophils and mast cells, several studies have indicated that a variety of other cells can express this receptor. Most convincing are those demonstrating that Langerhans cells (Bieber et al., 1992; Wang et al., 1992), monocytes (Maurer et al., 1994), and dendritic cells (Maurer et al., 1996) express FcεRI␣. Interestingly, the ␤-subunit, which is believed to amplify the signaling through FcεRI, has not been identified on these cells. Other reports have suggested that FcεRI␣ is also expressed on eosinophils (Gounni et al., 1994; Rajakulasingam et al., 1998), although there has been intense debate regarding these findings (Kita and Gleich, 1997; Kita et al., 1999). There is evidence, however, that an intracellular pool of the ␣-subunit is found in eosinophils (Seminario et al., 1999). It seems possible that this reservoir may be responsible for the immunodetection of FcεRI␣ in many of the studies reporting cell surface expression. It is important to note that IgE concentration does not appear to up-regulate FcεRI␣ expression on eosinophils, as it does for mast cells and basophils. There is presently no clear explanation, with regard to function, for the expression of FcεRI on cells other than basophils and mast cells, although a role in parasitic immunity has been suggested. Human basophils express several activation-linked markers other than FcεRI. With regard to other Ig receptors, only Fc␥ RII (CD32) has been identified and is responsible for the binding of various subclasses of IgG antibody. Although its role on basophils is not fully understood, there is evidence suggesting that it relays intracellular signals that work to oppose those initiated with FcεRI crosslinking (Daeron et al., 1995). Thus, cross-linking of Fc␥ RII/IgG complexes may prevent basophil activation for mediator release. This is not to be confused with the evidence that anti-IgG antibody can induce histamine secretion by crosslinking IgG–IgE complexes bound to the IgE receptor (Lichtenstein et al., 1992).

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Basophils also express CD40, an activation-linked antigen more commonly found on B cells. CD40 has sequence homology with the tumor necrosis factor (TNF) receptor family, which includes TNF-␣ and nerve growth factor (NGF). Interestingly, human basophils as well as and mast cells have been shown to express the cell surface ligand for CD40 (CD40L) (Gauchat et al., 1993). This antigen is found on a limited number of cell types, including activated T cells, endothelium, and platelets. The interaction between CD40 and CD40L has been shown to constitute an important step for IgE synthesis such that, in the presence of IL-4 or IL-13, it provides a necessary co-stimulus for B cell synthesis of this Ig. In fact, by secreting IL-4 and IL-13 and expressing CD40L, basophils alone can provide the necessary signals for B cell production of IgE, which were classically thought to be provided only by activated T cells (Gauchat et al., 1993; Yanagihara et al., 1998). Three monoclonal antibodies have recently been characterized, and all appear immunologically specific for proteins expressed only by basophils or their progenitors. Antibody 2D7 detects a 72-kDa granule-associated protein that is released upon activation (Kepley et al., 1995). BB1 is also reported to detect a cytoplasmic molecule, which, upon degranulation, is also secreted and thought to represent a novel mediator (McEuen et al., 1999). A third antibody, 97A6, is thought to identify a surface antigen found on basophil progenitors in addition to mature cells (Buhring et al., 1999). While these antibodies detect proteins having no known function, they are currently being used for identifying basophils in sites of allergic inflammation (see below).

IV. Inflammatory Mediators

A. HISTAMINE Basophils and mast cells synthesize significant amounts of histamine in a reaction resulting from the decarboxylation of L-histidine. It is stored in the cytoplasmic granules of basophils complexed with the highly charged proteoglycan, chondroitin sulfate, as opposed to heparin in the mast cell. The amount of histamine stored in basophils is remarkably consistent among donor populations, amounting to ∼1 pg per cell, and both IL-3 and GM-CSF have been shown to have an important role in its increased synthesis during the latter stages of basophil maturation. Its release from activated cells has been well characterized ultrastructurally for several different modes of activation (Dvorak, 1998). The physiological consequences of histamine release result from its role as a potent smooth muscle spasmogen and its ability to cause vascular leakage by dilating terminal aterioles and constricting postcapillary venules. There are data that histamine can modulate specific immune responses. In particular, cytotoxic T lymphocyte (CTL) responses are down-regulated by histamine binding to H2

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receptors on CD8+ T cells, which is accompanied by increases in intracellular cAMP levels (Plaut et al., 1973; Rocklin and Habarek-Davidson, 1984). H2 antagonists have been shown to prevent increases in intracellular cAMP and to reverse the inhibitory effects on CTL activity (Griswold et al., 1986; White and Ballow, 1985). More recently, the production of IL-12 and interferon ␥ (IFN-␥ ) by monocytes has been shown to be inhibited by histamine, while IL-10 secretion is augmented by this amine (Elenkov et al., 1998; Lagier et al., 1997). In light of this evidence, it seems possible that histamine may function to down-regulate Th1-like activity while promoting Th2-like responses. This hypothesis, in fact, seems consistent with the knowledge that basophils secrete IL-4—a cytokine that is known to promote the development of Th2-like responses (see below). B. LEUKOTRIENE C4 Unlike histamine, leukotriene C4 (LTC4) is not stored in basophils but is synthesized within minutes after activation by the metabolism of arachidonic acid through the lipoxygenase pathway. Both phosphatidycholine and phosphatidylinositol likely provide for the arachidonic acid in this reaction, and these phospholipids are themselves thought to be metabolized through the enzymatic activity of phospholipase A2. It has been known for some time that the amount of LTC4 generated per basophil (10–100 fg) is far less than the picograms-per-cell quantities of histamine found in these cells. On a molar basis, however, LTC4 is some 6000 times more potent than histamine in contracting smooth muscle (Bochner, 1995). Along with its metabolites, LTD4 and LTE4, the three leukotrienes have a profound bronchoconstrictive and mucus-producing effect when released in the respiratory system and induce a prolonged wheal-and-flare reaction when secreted in the skin. In fact, there is the belief that the pathophysiology of asthma is mediated, to a large extent, by the actions of these mediators. The recent clinical introduction of LT inhibitors demonstrates this to be the case (Calhoun et al., 1998; Kane et al., 1996). C. CYTOKINES For many years, basophils were thought to release only preformed histamine and newly synthesized LTC4 after activation by a variety of stimuli. As noted above, there is now firm evidence from many laboratories that basophils are also a major source of IL-4 and IL-13 (Brunner et al., 1993; Gibbs et al., 1996; Li et al., 1996a; MacGlashan et al., 1994; Ochensberger et al., 1996; Schroeder et al., 1994b). Both cytokines are found at sites of allergic inflammation, and the immunomodulatory properties mediated by each are recognized as pivotal in the pathogenesis of allergic inflammation and disease. In particular, IL-4 and IL-13 are the only two known cytokines that are capable of inducing an Ig isotype switch in B lymphocytes from IgM to IgE (Defrance et al., 1994;

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de Vries and Zurawski, 1995; Punnonen et al., 1993; Vercelli and Geha, 1993). For both cytokines, this is initiated through receptor-mediated activation of the transcription factor STAT6, which produces germ-line cepsilon transcription (Hill et al., 1999; Kaplan et al., 1996). The subsequent interaction of CD40 on the B cell with its ligand, CD40L, found on several cell types including the basophil, results in transcriptional signals followed by the secretion of IgE. As noted, both IL-4 and IL-13 activate the endothelium for increased expression of VCAM-1 and this adhesion molecule promotes the selective transendothelial migration of eosinophils, basophils, and T lymphocytes (Bochner et al., 1995; Hemler, 1990; Moser and Fehr, 1992; Schleimer et al., 1992; Thornhill et al., 1990). The expression of major histocompatibility complex (MHC) class II antigens (i.e., HLA-DR) is also up-regulated on antigen-presenting cells following exposure to IL-4 or IL-13 (Defrance et al., 1994). Most significantly, the development of the Th2 phenotype in so-called Th0 lymphocytes has been shown to be dependent on IL-4 exposure (Abehsira-Amar et al., 1992; Swain et al., 1990). Thus, in order for T cells to produce IL-4 and other Th2 cytokines (e.g., IL-13, IL-5, and IL-6), they must first be exposed to IL-4. This has spawned a number of theories as to what is responsible for the initial secretion of IL-4, and a variety of cell types have been suspected, including specific T cell populations (i.e., NK1.1 cells in the mouse), mast cells, basophils, and eosinophils) (reviewed in Romagnani, 1998). Whereas IL-13 does not share this Th2-promoting property of IL-4 (IL13–specific receptors have not been identified on T cells), this cytokine does appear to have a novel role in collagen deposition (Chiaramonte et al., 1999) and mucus production (Grunig et al., 1998; Wills-Karp et al., 1998), both of which are prominent features in chronic inflammatory diseases such as asthma. To date, there is no evidence showing that basophils secrete Th1-like cytokines or any other Th2-like cytokines. One report has shown evidence that the chemokine macrophage inflammatory protein 1␣ is secreted by basophils, suggesting an additional role in cell recruitment (Li et al., 1996b). The production of other chemokines by basophils has not been reported, although there are unpublished studies suggesting that they might. While the seminal studies linking FcεRI activation with IL-4 and IL-13 generation were performed with mouse mast cell lines (Plaut et al., 1989), in humans there is strong evidence that basophils are perhaps the predominant source of these cytokines. We first demonstrated this in blood by showing that both protein and mRNA for IL-4 correlated with the presence of basophils (MacGlashan et al., 1994). Others have since shown that basophils are the predominant source of IL-4 and IL-13, even in mixed leukocyte cultures (1–2% basophils) receiving specific allergen as stimulus (Devouassoux et al., 1999a; Kasaian et al., 1996). This has been a novel finding, since antigen-specific T cells were commonly thought to be the primary source of these cytokines in response to allergen. However, the explanation for these findings is rather straightforward: the frequency of

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antigen-specific basophils (i.e., those expressing antigen-specific IgE) far outnumbers that of allergen-specific T cells, which, at best, is on the order of 1 in 1000–5000 naive T cells. Thus, it has been shown that even a small percentage of basophils can secrete up to 10-fold greater levels of IL-4 and IL-13 compared to those produced by mixed lymphocytes (Devouassoux et al., 1999a). This may mean that basophils represent the major source of these cytokines in late-phase allergic reactions (see below), despite reports that they constitute a relatively small percentage (∼2–5%) of the cellular infiltrate. The technical problems of identifying and isolating basophils infiltrating allergic lesions have made it difficult to determine whether these cells contribute to the production of IL-4 and IL-13 at these sites. As noted below, we have recently shown that basophils obtained by bronchoalveolar lavage following segmental allergen challenge in the lungs are capable of producing IL-4, thus providing the first evidence that these cells secrete this cytokine at sites of allergic inflammation. In contrast, studies using immunohistochemical staining techniques have suggested that T cells, mast cells, and eosinophils produce IL-4 and other cytokines in biopsies taken from allergic lesions (Moller et al., 1996; Pawanker et al., 1997; Ying et al., 1997). However, the in vitro evidence supporting the secretion of IL-4 by isolated tissue mast cells has not been confirmed since its original description (Bradding et al., 1992). Thus, it seems possible that mast cells require a tissue component in order to generate IL-4 or that the techniques utilized in their isolation render them nonproductive. Alternatively, this discrepancy may reflect technical issues between the assays utilized to detect IL-4 (i.e., immunohistochemistry versus enzyme-linked immunosorbent assay). Nonetheless, the parameters and requirements for the in vitro generation of IL-4 and IL-13 by basophils are well established. In contrast, little information is available regarding how these cytokines are made by mast cells (or by eosinophils), despite numerous reports describing their expression in these cells (Ebisawa et al., 1995; Gibbs et al., 1997; Jaffe et al., 1995; Moqbel et al., 1995; Nakajima et al., 1996). V. Basophil Activation

A. IgE DEPENDENT As noted above, the IgE-dependent secretion of mediators and cytokines from basophils and mast cells, in its simplest form, is initiated when IgE antibody bound to FcεRI is cross-linked by specific antigen. In actuality, this interaction is quite complex, with the overall response being very much dependent on the sensitivity of the cell (i.e., the number of receptors needed for aggregation in order to achieve 50% of maximal secretion) (MacGlashan, 1993; MacGlashan et al., 1986). In fact, the sensitivity among donor basophils has been determined for

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mediator release and is quite variable, requiring as few as 200 and up to 30,000 receptor aggregations in order to induce a half-maximal response (MacGlashan et al., 1983). Because of the technical difficulties involved in their isolation, the sensitivity of mediator release from mast cells is not known but is generally thought to be similar to the basophil response. Since basophils and mast cells can be sensitized with IgE to a multitude of antigens, the consequences of cell sensitivity become complex, yet very important (reviewed in MacGlashan et al., 2000). Furthermore, much of the information pertaining to sensitivity has been derived from studies investigating basophil histamine release; thus, it is difficult to say whether similar parameters will apply for the generation of LTC4 and cytokines. Studies have suggested, however, that there are only subtle differences with regard to sensitivity for the release of these three classes of mediators (MacGlashan and Schroeder, 2000). It seems, however, that the number of receptor aggregates necessary for a half-maximal response of histamine will also produce a 50% response in cytokine and LTC4 secretion. The basophil response follows a classic bell-shaped curve when cells are challenged with a wide range of antigen concentrations. The same is true when crosslinking is induced by anti-IgE antibody, which is commonly used as an in vitro stimulus of basophils because of its ability to mimic, to some extent, the stimulation mediated by antigen and its ability to induce release from cells obtained from most donors, both allergic and nonallergic. Under optimal conditions, the IgE-mediated release of preformed histamine is nearly complete by 20 min. The generation and release of LTC4 follow a time course similar to those seen with histamine. In contrast to mediator release, cytokine secretion is considerably slower. Induced levels of IL-4 are first detected by 60–90 min, the response is half-maximal by 120 min, and it is essentially complete by 240 min after activation (MacGlashan et al., 1994; Schroeder et al., 1994b, 1998a). There is evidence that small quantities of IL-4 protein (<10 pg/106 basophils) can be detected within 5–10 min after stimulation; however, these levels represent only a small fraction (∼1%) of that commonly generated over the course of 4 hr (Schroeder et al., 1998b). Unlike IL-4, the generation of IL-13 follows a slower yet more prolonged time course (Fig. 1). Most studies show that IgE-mediated secretion of IL-13 begins several hours after IL-4 is initiated and peaks some 20 hr after activation (Li et al., 1996a; Redrup et al., 1998). A few studies, however, have reported little difference, if any, between the time course of IL-4 versus IL-13 secretion; there is presently no known explanation for such divergent findings (Ochensberger et al., 1996). The kinetics for IgE-dependent IL-4 and IL-13 mRNA accumulation clearly support the evidence that these two cytokines differ in their time course. First, it is important to note that we and others have consistently detected, by competitive reverse transcriptase–polymerase chain reaction and Northern hybridization, small amounts of IL-4 mRNA expressed in resting basophils, amounting to about

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FIG. 1. Relative time course for mediator release and cytokine secretion by human basophils in response to immunoglobulin E(IgE)–dependent activation; comparison with the interleukin 13 (IL-13) secreted following IL-3 stimulation. LTC4, Leukotriene C4.

10 copies per cell (MacGlashan et al., 1994; Patella et al., 1999; Schroeder et al., 1998a). While the significance of this constitutively expressed message is not known, it may, as noted above, account for the low levels of IL-4 protein secreted when cells are placed in medium alone. With activation, however, increases in IL-4 mRNA can be detected within 30 min and levels peak by 90–120 min. Interestingly, the mRNA levels for IL-4 are near baseline by 180 min following activation (J. T. Schroeder, 1998). Although less information has been published on the generation of IL-13 mRNA, the evidence available shows its accumulation to be much later than that for IL-4. Increases are barely detectable at 2 hr postactivation but are pronounced by 4 hr (Ochensberger et al., 1996; Redrup et al., 1998). What is not fully known at this time is the duration of its expression. Our preliminary data suggest that IL-13 mRNA is down by 18 hr after activation, and this likely accounts for the waning in the secretion of protein for this cytokine observed at this time. There is evidence that so-called viral superantigens exist that are capable of nonspecifically interacting with IgE on the surface of basophils and mast cells, resulting in mediator release resembling that associated with FcεRI cross-linking (Patella et al., 2000). For example, the gp120 protein of the human

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immunodeficiency virus (HIV) has been shown to activate basophils for IL-4 and IL-13 release by interacting with the variable heavy domain 3 (VH3) region of IgE (Patella et al., 1999). B. IgE INDEPENDENT It has been known for many years, and recently is becoming even more apparent, that basophils react to a growing list of diverse stimuli that act independent of cross-linking (Schroeder et al., 1995b). In fact, this is a property not shared by mast cells, suggesting that the basophil is a far more excitable cell. With the additional information that many of these so-called IgE-independent stimuli are generated during allergic reactions, it seems reasonable to suggest that they have a role in amplifying or prolonging inflammation by directly inducing mediator release from basophils infiltrating the lesion. The anaphylatoxins C5a and C3a and the bacterial peptide f–Met–Leu–Phe (FMLP) were among some of the first substances discovered to induce histamine release from basophils in a reaction not requiring FcεRI cross-linking (Siraganian and Hook, 1976, 1977). For FMLP, this release is half-maximal within 15 sec, severalfold more rapid than the time required for IgE-mediated release. As discussed below, this suggests that FMLP, a univalent stimulus that mediates activation through a G protein–coupled receptor, utilizes intracellular pathways different from those involved in FcεRI cross-linking. Under the correct conditions, both C5a and FMLP are also able to trigger the generation of LTC4 (Warner et al., 1989), although C5a-induced secretion most often requires prior exposure to IL-3. Furthermore, these stimuli, when used alone, generally do not induce the secretion of IL-4 or IL-13 from basophils (Li et al., 1996a; Ochensberger et al., 1996; Redrup et al., 1998; Schroeder et al., 1994a,b). In fact, with the exception of IL-3 and histamine-releasing factor (HRF) (see below), the inability to directly activate basophils for cytokine generation seems to be common feature among the IgE-independent stimuli, suggesting that the generation of IL-4 and IL-13 is primarily a consequence of signaling mediated through FcεRI cross-linking. Many of the IgE-independent stimuli that activate basophils for mediator release are derived from other immune cells. Platelet-activating factor and major basic protein, which are released from activated platelets and eosinophils, respectively, are two substances that have been reported to induce basophil histamine release (Okuda et al., 1988; Thomas et al., 1989). To date, there are no reports as to whether these substances provoke the generation of IL-4 and IL-13 in basophils. As predicted from early studies investigating so-called HRFs, it is now known that many cytokines have the capability of activating basophils. In fact, several belong to the CC chemokine family, which means that they also promote the trafficking of many different cell types, including basophils, into reaction sites. Members of the MCP family, particularly MCP-1 and MCP-3,

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are recognized as some of the more potent inducers of basophil histamine release, causing exocytosis at nanomolar concentrations and in the absence of any co-stimulus (Dahinden et al., 1994; Stellato et al., 1997; Uguccioni et al., 1997; Weber and Dahinden, 1995). Other chemokines, such as eotaxin, RANTES and macrophage inflammatory protein 1␣, which appear more important for basophil and eosinophil chemotaxis, have also been shown to induce marginal histamine release when used alone, primarily from cells isolated from allergic individuals (Kuna et al., 1992). These proteins, however, produce a more vigorous response when used with cells first “primed” by IL-3. It has recently been proposed that basophils isolated from allergic individuals are, in fact, in a primed state, which likely accounts for the ability of chemokines and other cytokines to directly induce histamine from these cells (Lie et al., 1999). None of these CC chemokines has been reported to directly stimulate IL-4 or IL-13 secretion from basophils. One recent study has shown evidence that eotaxin and other CC chemokines binding to CCR3 do prime basophils for increased IL-4 induced by antigen (Devouassoux et al., 1999b). Finally, IL-8 is the only CXC chemokine cited as possessing histamine-releasing activity, although it is far more effective at doing this from primed rather than unprimed cells (Krieger et al., 1992). While the histamine-releasing activity of the chemokines discussed above is not dependent on IgE expression, there is a class of proteins for which the expression of IgE does appear to play a role in basophil activation. In the initial description of what is now known as HRF, this molecule was defined by its ability to directly induce histamine release from basophils expressing a specific type of Ig, referred to as IgE+(MacDonald et al., 1987). By definition, cells not directly responding to HRF were thought to express so-called IgE−. While the nature of this IgE heterogeneity remains an enigma, there is mounting evidence that HRF does not directly bind IgE+, but rather exerts activity through a specific, yet unidentified, receptor that is found on several cell types, not just basophils (Bheekha-Escura et al., 1998). Ultrastructural studies additionally support this belief by showing quantitative differences during degranulation induced by HRF compared to those events typically seen with FcεRIcross-linking agents (e.g., anti-IgE antibody) (Dvorak et al., 1996). Recombinant HRF not only induces histamine release from basophils expressing IgE+ but also stimulates the generation and secretion of IL-4 (MacDonald et al., 1995; Schroeder et al., 1996). Interestingly, the secretion of IL-13 has not been detected from cells activated with this protein (J. T. Schroeder, 1998). However, its ability to enhance the IgE-mediated secretion of histamine, IL-4, and IL-13 from most basophils (particularly those expressing IgE−) makes it somewhat like IL-3 in its ability to prime basophils (Schroeder et al., 1997a). Since it has been identified in the late-phase response lavage fluids taken from allergic lesions, HRF is thought to represent a novel cytokine having an important role in allergic inflammation.

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At this time, there is no cytokine or factor known that has a more profound effect on the basophil response than does IL-3. This cytokine is capable of acting both as a complete secretogogue and as an enhancer of IgE-dependent and -independent stimulation. At relatively high concentrations (>100 ng/mL), IL-3 has been shown to induce histamine release from the cells of selected allergic donors, particularly those also reacting to HRF (MacDonald et al., 1989). Most significantly, IL-3 has been shown to be a potent stimulus of IL-13, inducing the secretion of this cytokine from most donor cells even in the absence of histamine and IL-4 release (Ochensberger et al., 1996; Redrup et al., 1998). This latter activity of IL-3 is of biological importance because it implies that basophils need not express IgE in order to make IL-13. Thus, IL-13 secreted from basophils exposed to IL-3 may have a role in inflammatory processes not directly linked to immediate hypersensitivity. Finally, it is well known that IL-3, like HRF, will enhance IgE-mediated responses, resulting in the increased secretion of histamine, LTC4, IL-4, and IL-13 induced by a wide range of anti-IgE or antigen concentrations. However, unlike HRF, IL-3 will also prime basophils for increased histamine and LTC4 release when activated with IgE-independent stimuli, such as C5a and FMLP (Kurimoto et al., 1989; MacGlashan and Warner, 1991). This priming effect occurs within minutes of IL-3 exposure, and we are just now beginning to understand some of the intracellular mechanisms involved (see below). While IL-3 and HRF are the predominant cytokines modulating basophil secretion, there is evidence that other cytokines may have similar capabilities. Both IL-1 and NGF are reported to enhance IgE-mediated secretion of histamine and LTC4 (Bischoff and Dahinden, 1992; Massey et al., 1989). For NGF, this is apparently mediated through the trk A rather than trk B, trk C, or the low-affinity NGF receptors, suggesting that other neurotrophins lack priming capabilities (Burgi et al., 1996). It is not presently known whether these cytokines have similar effects on basophil cytokine production. VI. Signal Transduction and Pharmacological Control of Secretion

A great deal of information concerning the intracellular signals regulating FcεRI-mediated signaling has come from studies investigating various rodent cell lines, including transformed rat basophilic leukemia cells and murine (IL-3– dependent) mast cell lines (Okazaki and Siraganian, 1999). It remains uncertain, however, whether the signal transduction mechanisms described in these models have relevance to human cells. Both the HMC-1 and KU812 cell lines (Butterfield et al., 1988; Fukuda et al., 1987), which resemble immature human mast cells and basophils, respectively, lack the expression of functional FcεRI receptors, therefore making it difficult to perform studies investigating IgEmediated signaling in these cells. Additional complications exist in that most subclones of these human cell lines are generally poor producers of IL-4 and

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IL-13 and, in many instances, fail to secrete these cytokines. As noted earlier, the same seems true for tissue-derived human mast cells, not to mention that these cells are extremely difficult to purify from tissue. In contrast, human basophils secrete large quantities of mediators and cytokines, making it possible to detect these products from relatively few cells. Furthermore, it has become possible to routinely purify basophils from blood to >99% purity and in numbers exceeding tens of millions of cells, which has led to marked progress in further delineating the signal transduction processes that account for the production and release of histamine, LTC4, IL-4, and IL-13. As a result, this section focuses primarily on the developments pertaining to the signals regulating the release of these three classes of mediators from human basophils, with particular emphasis on cytokine generation. The aggregation of FcεRI on human basophils results in the recruitment and activation of receptor-associated tyrosine kinases, namely, p53/56lyn (a member of the src family of kinases) and p72syk (Kepley et al., 1998b; Lavens-Phillips and MacGlashan, 2000). The expression and phosphorylation of these kinases in basophil lysates can be detected within minutes following IgE-mediated activation. Inhibitors of tyrosine kinases (e.g., PP1 and PP2) that appear selective for src kinases have been shown to reduce the phosphorylation of lyn and the subsequent activation of several proteins, including syk (Lavens-Phillips and MacGlashan, 2000). Most significantly, this inhibition causes a marked reduction in the secretion of all classes of mediators, suggesting that lyn and syk activation constitute early signals regulating IgE-mediated secretion. Studies suggest, in fact, that the so-called “nonreleaser”phenotype, which is characterized by basophils completely unresponsive to cross-linking stimuli, is the result of a deficiency in syk expression (Kepley et al., 1999). Furthermore, nonreleaser basophils have been shown to convert into “releaser” basophils following 4 days’ incubation in IL-3 (Yamaguchi et al., 1996), which implies that IL-3 signaling likely modulates syk expression. It is not known whether other components in the signaling cascade are also deficient. It is important to note that tyrosine kinase inhibitors fail to inhibit basophil histamine and LTC4 rapidly released through GTP-binding protein–coupled receptors, such as that occurring with FMLP activation. Maximal degranulation with this univalent stimulus is extremely rapid (∼1 min), or some 5- to 10-fold faster than that occurring with IgE-mediated stimulation, and is sensitive to pertussis toxin, which causes ADP ribosylation of GTP-binding proteins (Saito et al., 1987; Warner et al., 1987). Taking into account that FMLP also does not readily induce IL-4 and IL-13 secretion, the phosphorylation of lyn and syk associated with FcεRI activation appears to play a critical role in regulating downstream signals important for cytokine generation in basophils. While pharmacological studies indicate a commonality in the early events (i.e., lyn and syk activation) regulating the three classes of mediators generated in response to cross-linking, there is the belief that their intracellular

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mechanisms diverge from one another in downstream events (Schroeder and MacGlashan, 1997). The approach taken in identifying such divergent pathways in human basophils has been to measure specific intracellular events and their kinetics of activation and to determine whether specific inhibitors of these events also prevent the secretion of one or all of the classes of mediators. Using this approach, we have recently gained an understanding of the intracellular signaling involved in LTC4 formation. As noted above, the generation of this lipid mediator is very much dependent on cytosolic phospholipase A2 (cPLA2) activity. The so-called extracellular signal–regulated kinases (ERKs, such as ERK1 and ERK2), which are downstream of p21ras, are thought to target cPLA2 for activation. In recent studies, the sequential phosphorylation of the ERKs and cPLA2 correlated with the IgE-dependent (and -independent) release of LTC4 from basophils. By reducing the phosphorylation of ERK1 and ERK2 with the kinase inhibitor, PD098059, the subsequent phosphorylation of cPLA2 was also interrupted and the release of LTC4 was inhibited (Gibbs and Grabbe, 1999; Miura et al., 1999). Most interestingly, this inhibitor had little effect, if any, on the secretion of histamine and IL-4, suggesting that the intracellular events regulating the release of these mediators are upstream of the ERK1/ERK2 pathway. One potential pathway involved in both cytokine generation and mediator release may involve phosphatidylinositol 3-kinase, which is downstream of lyn and syk. This enzyme initiates a variety of signaling pathways that are important for ribosomal activity in the translation of some proteins. Both wortmannin and LY294002 inhibit phosphatidylinositol 3-kinase, and both compounds also prevent the secretion of all three classes of mediators released in response to the IgE-mediated activation (Gibbs and Grabbe, 1999). There is a large amount of data, derived mostly from studies using rodent cell models, that cytosolic calcium and protein kinase C (PKC) play pivitol roles in the secretory responses of basophils and mast cells. The basic features for their generation are as follows: both univalent (e.g., FMLP and C5a) and FcεRI cross-linking stimuli are thought to activate a phospholipase C enzyme via a GTP-binding protein or via a tyrosine kinase (i.e., syk), respectively. One of the consequences of both pathways is the metabolism of phospholipids resulting in the formation of two important messengers that relay the initiating signal further along. Diacylglycerol constitutes one of these messengers and functions as an important regulator of PKC activity. The other is triphosphates (derived from phosphoinositol metabolism), which have a role in regulating free calcium levels by causing the release of this ion from intracellular stores into the cytosol. Whereas the activation of PKC is well documented in the pathways leading to mediator release in rat basophilic leukemia cells, its role in the prodegranulatory events in human basophils following IgE-mediated activation has been challenged (Miura et al., 1998). Several PKC isozymes have been identified

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in basophils, including ␤1, ␤2, and ␦. However, the activation of these isozymes, as assessed by their translocation to the membrane fraction, has been observed only after stimulation either with phorbol myristate acetate (PMA)—a direct activator of PKC activity— or with FMLP. In contrast, activation with anti-IgE has not produced evidence of PKC translocation. In fact, inhibitors of PKC activity, such as the bisindolylmaleimides (BIS I and II), have actually been found to enhance anti-IgE–induced histamine release, suggesting that PKC activation plays more of an inhibitory role during IgE-mediated degranulation. Furthermore, the BIS compounds have no effect on histamine released by FMLP. It is important to note that staurosporine, which was used in early studies as an inhibitor of PKC, does prevent IgE-mediated but not FMLP-mediated histamine release (Warner and MacGlashan, 1990). However, the results with staurosporine are less clear in light of more recent information that this compound also inhibits the early and late tyrosine kinase activity associated with FcεRI activation. PKC activation plays an even more complex role in the generation of cytokines in basophils by possessing both anti- and pro-secretory activity. Studies show that PMA is a potent activator of basophils, causing nearly 100% histamine release at concentrations beginning at 1 ng/mL (Schleimer et al., 1981a). However, little, if any, IL-4 protein is secreted by cells cultured with this amount, or higher concentrations, of PMA even after 48 hr of incubation. In fact, the large quantities of IL-4 produced by cells cultured with calcium ionophores (e.g., ionomycin) are, remarkably, down-regulated some 70% with the simultaneous addition of PMA. This inhibitory effect of PMA is reversed with BIS II, suggesting that PKC activity negatively affects IL-4 generation in basophils (Schroeder et al., 1998a). In sharp contrast, PMA exerts quite an opposite effect on the secretion of IL-13 by basophils. In this instance, the phorbol ester will directly stimulate the secretion of this cytokine, and this response, as expected, is prevented by PKC inhibitors (Redrup et al., 1998). Thus, PKC activation appears to play a dual role in the production of cytokines by basophils by negatively regulating IL-4 while promoting the secretion of IL-13. Cytosolic calcium responses play a critical role in the pro-secretory events occurring in basophils, and both IgE-mediated and univalent stimuli are capable of inducing a calcium response resulting in degranulation. Although several studies have noted a good correlation between elevations in free cytosolic calcium and histamine release from basophils (Knol et al., 1992; Warner and MacGlashan, 1990), causal testing of this linkage indicates that the relationship is more complex than was first anticipated (MacGlashan and Botana, 1993). In particular, the linkage is better for secretion of LTC4 and IL-4 than for histamine release. The tight linkage for LTC4 secretion comes about because of the absolute requirement for cytosolic calcium elevations and the activity of cPLA2 and 5-lipoxygenase. Based on the current understanding of signaling elements needed for cytokine generation, secretion of IL-4 may have similar absolute

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requirements. First, early studies showed that IgE-mediated secretion of IL-4 is very much dependent on adequate elevations in cytosolic free calcium (Schroeder et al., 1994b). Second, during low-level cross-linking of FcεRI, which, as noted above, is optimal for the secretion of IL-4 (and IL-13), the cytosolic calcium responses are sustained compared to the short-lived responses seen during activation with the 10-fold greater concentrations of cross-linking stimuli that are optimal for histamine release (MacGlashan and Botana, 1993). Finally, the lack of IL-4 and IL-13 secreted in response to FMLP and C5a may reflect the fact that these univalent stimuli induce cytosolic calcium responses in basophils that are also short-lived, despite being sufficient for histamine and LTC4 release (MacGlashan and Warner, 1991; Warner and MacGlashan, 1990). At this time, there is reason to believe that a calcium/calcineurin pathway has a role in the transcription of cytokine induced with FcεRI activation (Schroeder and MacGlashan, 1997). This belief is founded, in part, by several observations that are relevant to the importance of free cytosolic calcium in this response. First, the addition of a chelator of calcium (e.g., EGTA) will immediately halt cytokine secretion induced by anti-IgE by preventing the accumulation of mRNA for this cytokine. Second, calcium ionophores, which sustain intracellular calcium levels, are the most potent activators of IL-4 and IL-13, inducing the secretion of up to 1000 pg/106 basophils for either cytokine. Most significant is the fact that FK-506 and cyclosporine A are the most potent inhibitors of IL-4 and IL-13 secreted in response to IgE-mediated activation (Redrup et al., 1998; Schroeder et al., 1999). Well known as selective inhibitors of calcineurin phosphatase activity, these immunophilins prevent IL-4 secretion in basophils at subpicomolar concentrations and are some 50- to 100-fold more effective at preventing the secretion of this cytokine than they are at inhibiting histamine release (De Paulis et al., 1991). It is important to note that while these drugs similarly inhibit IL-13 induced by anti-IgE, they have little to no effect on the secretion of this cytokine induced by PMA or by IL-3, both of which do not directly induce cytosolic calcium changes in basophils. Finally, the translocation of specific members of the nuclear factor of activated T cell (NFAT) family of transcription factors from the cytoplasm to the nucleus of activated T cells has been associated with the generation of cytokines, including IL-4 (Rao et al., 1997). Calcineurin initiates this subcellular localization to the nucleus by removing phosphates on inactive cytosolic NFAT. Thus, FK-506 and cyclosporine A are well-known selective inhibitors of this nuclear translocation. In data not yet published, we have evidence that antibodies specific for the NFAT2 and -4 isoforms, but not for NFAT1, detect cytosolic proteins in basophils that translocate to the nucleus within a time (1 hr) that is consistent with the generation of IL-4 (J. T. Schroeder, 2000). At this time, it is not known whether these antibodies detect both isoforms or if they cross-react with a unique NFAT isoform found in basophils.

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Whereas similarities exist between the generation of IL-4 and IL-13, particularly following IgE-mediated activation, there are clear differences in the stimuli and mechanisms regulating their generation in basophils. As suggested above, IL-13 is somewhat unique in that its release is not dependent chiefly on IgE– FcεRI interactions. In other words, its release is linked to neither histamine nor IL-4. It is also the case that a variety of IgE-independent stimuli can induce IL-13, a major difference from IL-4, suggesting that this cytokine has a more significant role in the pathogenesis of chronic allergic disease—a concept that has recently gained considerable attention in murine models of asthma (Grunig et al., 1998; Wills-Karp et al., 1998). There is pharmacological evidence to support the existence of at least three distinct pathways resulting in the generation of IL-13, all of which are dependent on the specific stimuli that induce its secretion (Schroeder et al., 1999). As noted, the production of cytokines such as IL-4 and IL-13 in response to stimuli that induce a sustained calcium response (e.g., calcium ionophores or anti-IgE) is inhibited by FK-506 and cyclosporine A. However, the generation of IL-13 mRNA or protein that occurs following induction by IL-3 is completely resistant to these drugs, and only marginal inhibition is observed for the IL-13 induced by PMA. As predicted, inhibitors of PKC prevent the secretion of IL-13 made in response to PMA, but not to IL-3. Glucocorticoids, which have proven efficacy in the treatment of allergic disease, have been shown to inhibit both IL-4 and IL-13 (Schroeder et al., 1997b, 1998c; Shimitzu et al., 1998) induced by all the stimuli tested thus far. In fact, these drugs require little exposure time (<1 hr) to inhibit cytokine secretion compared to the 8–20 hr necessary to prevent histamine released by activated basophils (Schleimer et al., 1981b). However, steroids clearly support the existence of multiple pathways for IL-13 secretion by showing very different potencies in their ability to inhibit this cytokine. For example, dexamethasone inhibits the IL-13 made in response to IL-3 at subnanomolar concentrations, which are some 30-fold less than those necessary to prevent this cytokine induced by anti-IgE. This finding is surprising in light of the profound developmental and priming effects that IL-3 is known to have on basophil function, and that this cytokine often lessens the inhibitory effects steroids mediate on histamine and LTC4 released by these cells (Schleimer et al., 1989). There are several lines of evidence suggesting that the mechanisms involved in the direct stimulation of IL-13 by IL-3 (which is a relatively late effect requiring several hours’ incubation) are very different from the rapid priming effect IL-3 typically has on basophil mediator release. First, studies show that IL-3 priming of basophils for 15 min does lead to the transient phosphorylation of cPLA2 and that this is associated with increased LTC4 release upon activation with IgE-dependent and -independent stimuli (Miura et al., 1998). Phosphorylation of cPLA2 is not seen after 2 hr of exposure nor is there increased LTC4 released upon activation. However, cPLA2 is once again phosphorylated after

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18 hr of incubation in IL-3, and this correlates with the ability of secretagogues to produce a calcium response. This later event is prevented by an inhibitor of protein synthesis, such as cycloheximide (Miura and MacGlashan, 2000). Likewise, cycloheximide prevents the ability of IL-3 to induce IL-13 mRNA expression (J. T. Schroeder, 2000). These findings suggest that IL-3 induces the synthesis of a protein that is important in regulating the release of both IL-13 and LTC4. The exact nature of this unidentified protein is not yet known, but its induction by IL-3 is relatively late (i.e., hours), and likely has little to do with acute priming, which occurs within minutes and is insensitive to inhibitors of protein synthesis. VII. Basophils and Allergic Disease

As noted in Section I, studies performed nearly 30 years ago in our laboratory showed that the in vitro release of histamine from basophils challenged with various allergens predicted the severity of the respiratory symptoms experienced by the donor when exposed to that allergen (Lichtenstein et al., 1968). Numerous studies have since expanded on these findings to further show that clinical correlates exist between the presence of diseases such as asthma and urticaria and the numbers of circulating basophils, their progenitors, and their releasability to various stimuli (summarized in Schroeder et al., 1995a). One study found, in fact, that the basophil was the only cell type whose presence correlated with bronchial hypersensitivity, as measured by mecholyl challenge. To further support a role for basophils in allergic disease, it has long been acknowledged, but not fully appreciated, that these cells are also found in tissue sites along with eosinophils and lymphocytes following exposure to allergen. Most recently, Koshino and colleagues (1995), using biopsied specimens from the lungs of individuals dying from severe asthma, concluded that the percentage of basophils found in this tissue equaled the percentage of mast cells. In contrast, only mast cells were found in the lungs of individuals suffering nonasthmatic deaths. Although findings such as this imply that basophils participate in allergic lesions, many questions remain concerning the exact role they provide in disease. With the recent knowledge that they secrete IL-4 and IL-13, in addition to histamine and LTC4, it seems certain that a greater amount of emphasis will be directed in the future toward detecting these cells in allergic lesions to determine their potential as cytokine-producing cells. In fact, the development of basophilspecific monoclonal antibodies suitable for immunohistochemical staining will likely facilitate such studies. While there is evidence for basophil participation in natural disease, much of what is known regarding the involvement of this cell in allergic lesions has come from studies investigating a clinical model, the late-phase response (LPR) to allergen challenge (Lichtenstein and Bochner, 1991; Solley et al., 1976). This

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response occurs several hours after the early or immediate symptoms to allergen exposure have subsided and is accompanied not only by a late episode of symptoms, but also by a cellular infiltrate consisting of eosinophils, lymphocytes, and basophils into the reaction site. Since the basophils in these lesions can be many times greater (30- to 50-fold) than their frequency in blood, their presence is thought to result from a selective recruitment into the extravascular tissue. Models of the LPR exist for both the upper (nose) and lower (lung) airways as well as for skin; the relative ease with which consistent data can be obtained from each has meant that many therapeutic experimental maneuvers can be tested (Bascom et al., 1988a,b; Charlesworth et al., 1989, 1991; Guo et al., 1993; Iliopoulos et al., 1992). Early studies of the LPR relied on the presence of histamine and the absence of prostaglandin D2 and tryptase (mast cell markers) in reaction site lavage fluids for implicating the involvement of basophils, since staining techniques identified both mast cells and basophils. In later studies, a combination of ultramorphological, functional, and phenotypic analyses were used in identifying basophils in the LPR, since in vitro studies using isolated mast cells and basophils had detected a number of differences between the two cell types. Definitive evidence confirming the presence of basophils in the LPR has recently been obtained by immunohistochemical staining using basophil-specific monoclonal antibodies (Irani et al., 1998; Macfarlane et al., 1999). These antibodies, in fact, identified a remarkable infiltration of basophils in skin biopsies taken 6–20 hr after allergen challenge, suggesting that previous studies identifying these cells using histochemical stains may have underestimated the extent of their involvement in these lesions. While the data at this time clearly show basophils as the primary producers of IL-4 and IL-13 of the leukocytes circulating in blood, their contribution to the production of these cytokines in late reactions and chronic allergic disease has not been fully addressed. IL-4 and IL-13, along with IL-5, are recognized as important players in the pathogenesis of disease, and all three cytokines have been detected in lavage fluids taken from late reactions in the lung (Huang et al., 1995; Walker et al., 1992). Interestingly, the time course for the generation of IL-4 by activated basophils (i.e., 1–4 hr), is consistent with the onset of the LPR, whereas lymphocyte IL-4 generation is generally thought to require longer duration. It has been shown, in preliminary studies, that basophils infiltrating the lungs 20 hr following segmental allergen challenge do have the potential for IL-4 secretion, and there is reason to believe that IL-13 is also generated by these cells (Schroeder et al., 2001). However, it now seems pertinent to determine whether basophils contribute to the early production of IL-4 and IL-13 or whether this response is mediated by other cell types, such as lymphocytes and eosinophils. In light of the biological properties of IL-4 and IL-13, the cells producing these cytokines at the onset of the late reaction likely play a role in amplifying the overall response by promoting the influx of eosinophils, lymphocytes, and basophils

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and by modulating the microenvironment to favor the development of lymphocytes producing Th2-like cytokines, all of which characterizes chronic allergic disease. ACKNOWLEDGMENT This work was supported by National Institute of Health Grants AI-42221, AI-07290, and AI-27906.

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ADVANCES IN IMMUNOLOGY, VOL. 77

Btk and BLNK in B Cell Development SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan

I. Introduction

Development of B-lineage cells is tightly regulated by sequential expressions of immunoreceptors whereupon growth factors such as cytokines or other receptor ligands promote developmental progression. In particular, studies have demonstrated that expression of the B cell antigen receptor (BCR) and of the pre–B cell receptor (pre-BCR) at appropriate differentiation stages is mandatory for B cell development. Not only the signals resulting from ligand binding to the receptors, but also continuous signals from the BCR (Lam et al., 1997) and pre-BCR, are thought to be important for the proliferation and survival of cells. Therefore, one of the keys to understanding the whole framework of B cell differentiation is identification of the precise molecular mechanism by which the BCR and pre-BCR transmit their signals. Although the pre-BCR signal pathway is still far from being well understood, it has been shown that BCR signaling is mediated by a distinct set of cytoplasmic protein tyrosine kinases (PTKs) (Fig. 1). Src family PTKs such as Lyn are located immediately downstream of the BCR and initiate the PTK cascade by phosphorylating immunoreceptor tyrosine–based activation motifs (ITAMs) in the cytoplasmic regions of immunoglobulin ␣ (Ig␣) (CD79a) and Ig␤ (CD79b), which are the signal-transducing units of the BCR. Tyrosine phosphorylation of ITAMs recruits another PTK, Syk, via its two tandem Src homology 2 (SH2) domains, which results in the activation of Syk by an Src family PTK or its autoactivation mechanism (Kurosaki, 1999; Tamir and Cambier, 1998). The activated Syk subsequently phosphorylates adapter molecules such as BLNK, which integrate the upstream PTKs with downstream multiple effectors. Bruton’s tyrosine kinase (Btk) represents the third PTK family involved in the BCR (and presumably pre-BCR) signaling pathway. Btk is a member of Btk/Tec family PTKs and was originally identified as a PTK deficient in human X-linked agammaglobulinemia (XLA) (Tsukada et al., 1993; Vetrie et al., 1993). The Btk/Tec family is an expanding family of cytoplasmic PTKs that includes Btk, Tec, Itk, Rlk (Txk), and Bmx (Desiderio, 1997; Rawlings, 1999; Tsukada et al., 1994a). An ancestral form of this PTK family is also found in Drosophila (Tec29; alternatively, Dsrc29A) (K. Baba et al., 1999). Structurally, Btk/Tec family PTKs share the conserved arrangement of the SH3, SH2, and kinase (SH1) domains also found in Src family PTKs (Fig. 1) but are distinguished 123 C 2001 by Academic Press. Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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FIG. 1. Structural overview of key protein tyrosine kinases (PTKs) in B cell antigen receptor signaling. Bruton’s tyrosine kinase (Btk) has four protein- or lipid-interacting domains in addition to the catalytic domain. The pleckstrin homology (PH) domain exhibits a high affinity for binding to phosphatidylinositol-3,4,5-trisphosphate, the product of the action of phosphatidylinositol-3 kinase. This binding activity seems to enhance the translocation of Btk to the cell membrane and promote the activation of Btk by membrane-localized PTKs such as Lyn. The Src homology 2 (SH2) domain binds to BLNK phosphorylated by Syk, while the SH3 domain of Btk seems to negatively regulate its kinase activity. The physiological ligand of the Tec homology (TH) domain is unclear. The Src family PTK transphosphorylates the tyrosine 551 in the Btk catalytic domain and enhances the Btk kinase activity, which is followed by the autophosphorylation of the tyrosine 223 in the Btk SH3 domain. The tyrosine 551 in Btk corresponds to the consensus autophosphorylation site in the catalytic domain of Lyn or Syk. Btk is also distinguished from Src family PTKs by the absence of N-terminal myristylation (Myr), the absence of C-terminal regulatory tyrosine.

by the absence of N-terminal myristylation essential for the membrane localization of Src family PTKs, the absence of C-terminal regulatory tyrosine, and the presence of a pleckstrin homology (PH) domain (except Rlk) and a Tec homology (TH) domain (Rawlings et al., 1993; Tsukada et al., 1993). The functional significance of each domain in B cell development has been recently elucidated by the identification of interacting molecules and their genetic dissections. The purpose of this chapter is to summarize the major advances in the study of Btk, especially identification of the link between Btk and BLNK in the context of B cell development, with an attempt to incorporate these studies into a complete picture of the molecular framework of the Btk signaling pathway. II. Btk and B Cell Development

The genetic defect of Btk causes human XLA (Tsukada et al., 1993; Vetrie et al., 1993). XLA is characterized by the early onset of bacterial infections, very low serum Ig levels of all isotypes, and severely reduced numbers of peripheral B lymphocytes (Conley et al., 1994; Ochs and Smith, 1996). More than 400 XLA-associated Btk mutations have been characterized to date (Conley et al., 1998; Vihinen et al., 1998, 1999), demonstrating that mutations in every domain

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of Btk (except the SH3 domain) can cause XLA. Although significant variance in the disease phenotypes has been recognized among XLA patients, very few genotype–phenotype correlations have been identified. Flow-cytometric analyses of Btk protein expression have demonstrated that most of the Btk mutations in XLA resulted in deficient expression of the Btk protein, probably because of a reduction in Btk mRNA or an instability of the produced protein (Futatani et al., 1998). In addition to typical XLA cases, the rapid progress in diagnostic methods led to the extension of Btk protein evaluation to other hypogammaglobulinemic cases and to the identification of several atypical XLA patients who exhibited mild, if any, clinical symptoms prior to adulthood (Hashimoto et al., 1999a; Kanegane et al., 2000). While the exact characterization of B cell defects in XLA is important for clarifying the precise role of Btk in B cell development, the question of how genetic defects in XLA affect early B cell development has been rather controversial. This is probably because of imprecise characterization of the human B cell developmental stages and heterogeneity of patients’ phenotypes, as well as limited availability of patients’ bone marrow (BM) samples. An early study by Pearl and co-workers (1978) reported that XLA patients had a normal frequency of cytoplasmic ␮+ pre-B cells in BM and suggested that the transition from pre-B cells to B cells might be impaired in XLA. In contrast, later studies demonstrated a greater heterogeneity in the number of pre-B cells in XLA BM (Campana et al., 1990; Landreth et al., 1985). Although pre-B cells are more or less detectable in XLA BM, it has been shown that substantial numbers of pro-B cells are present in BM from a majority of XLA cases, resulting in an increased ratio of pro-B cells to pre-B cells. A dramatic reduction in the number of cytoplasmic ␮+ pre-B cells entering the S phase has been observed (Campana et al., 1990), which also suggested that the progression of pre–B cell differentiation is retarded in XLA BM. Conley (1985) proposed that the defect in XLA might interfere with multiple stages of B cell differentiation because the small numbers of peripheral B cells (leaky B cells) seen in XLA patients also exhibited an immature phenotype and appeared to have been arrested at an early stage of differentiation. In contrast to earlier studies, which used relatively classical surface markers for distinguishing the developmental stages, a study by Nomura and associates (2000) adopted flow-cytometric analysis of the surrogate light (SL) chain expression in XLA and normal BM B-lineage cells. Their analysis of normal BM identified five discrete types of human B cells, namely, cytoplasmic ␮−SL++ (pro-B), ␮lowSL++ (pre-B1a), ␮lowSL+ (pre-B1b), ␮lowSL− (pre-B2), and ␮highSL− (B) cells (Fig. 2). The larger cells, presumably in cycling states, were enriched in the pre-B1a stage. A large numeric expansion occurs from pre-B1a through the pre-B1b and pre-B2 stages which is especially active in normal young age. This scheme, particularly the expression pattern of the SL chain, seems consistent with that of mice, in which it was previously demonstrated that the production

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FIG. 2. Schematic diagrams of human and murine B cell developments. In X-linked agammaglobulinemia patients, Bruton’s tyrosine kinase (Btk) mutations impede the transition from pro-B cells to pre-B cells. In mice, some reports have documented that Btk mutation results in the failure to expand to small pre-B cells [Btk (1)], whereas Hendriks et al. (1996) reported that Btk mutation impedes the transition from small pre-B to immature B cells as well as the late maturation step [Btk (2)]. SL, Surrogate light.

of the SL chain is high at the earliest stage of pre-B cells (large pre-BII in terms of murine B cells), which are actively cycling and exhibit dramatic proliferative expansion in normal murine BM (Karasuyama et al., 1994, 1996; Melehers et al., 1994). Nomura and colleagues (2000) reported that the pre-B cells seen in XLA BM were largely composed of pre-B1a cells, the earliest pre–B cell population, and that the later stages of pre-B cells (pre-B1b and pre-B2) were dramatically reduced in XLA BM. These pre-B1a cells in XLA BM were also fewer than those in normal BM and consisted of small cells, suggesting that they might not be in cycling states. This observation indicated that, in humans, Btk mutations impede the evolution of pro-B cells to the earlier pre-B cells. The earliest bottleneck in XLA B cell development is thus at the pre-BCR checkpoint (Karasuyama et al., 1996). It is therefore noteworthy that the maturation block in patients with mutations in pre-BCR components (␮-chain, ␭5, and Ig␣) is reportedly also at the transition from pro-B cells to pre-B cells, which is indistinguishable from that in XLA (reviewed in Minegishi et al., 1999b; see also Minegishi et al., 1998, 1999a;

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Yel et al., 1996). These observations raise the possibility that Btk may be involved in the signaling downstream of the pre-BCR, as discussed later. In mice, B cell abnormality caused by Btk mutation has been observed in the CBA/N strain, which carries a missense mutation (arginine-to-cysteine substitution at position 28 [R28C]) in the PH domain of Btk (Rawlings et al., 1993; Thomas et al., 1993). There are remarkable differences in the severity of the B cell defects in XID mice and human XLA. XID mice have relatively normal serum concentrations of IgG1, IgG2a, and IgG2b but markedly reduced IgM and IgG3. Although unable to mount antibody responses to type 2 T cell–independent antigens, XID mice respond well to challenges with T cell–dependent antigens. These mice have 30–50% of the normal levels of conventional B cells and no B1 cells (Scher, 1982; Wicker and Scher, 1986). The B cell phenotype in these mice is immature (IgMhighIgDlow profile) and shows defects in response to lipopolysaccharide (Scher, 1982; Wicker and Scher, 1986), interleukin 5 (IL-5) (Hitoshi et al., 1993; Koike et al., 1995), IL-10 (Go et al., 1990), stimulations of CD38 (Santos-Argumedo et al., 1995) or RP105 (Miyake et al., 1995), and cross-linking of the BCR (Scher, 1982; Wicker and Scher, 1986). The phenotypic difference in XID mice and human XLA cannot be explained by the specific nature of the XID mutation (R28C) because the phenotype of Btk null mice generated by gene targeting is very similar to that of XID mice, but not that of human XLA (Kerner et al., 1995; Khan et al., 1995). This contradicts the hypothesis that the R28C mutation might only partially impair the Btk function and that the less severe phenotype of XID mice might thus be attributable to the residual Btk function. Another observation to support the idea of a species-specific difference in Btk utility was that XLA patients with missense mutations at the same residue (R28) as XID mice exhibited an almost complete block of B cell development. Several amino acid substitutions for arginine at position 28, including histidine (de Weers et al., 1994), proline (Hashimoto et al., 1996), and cysteine (Conley et al., 1998), have been found in typical XLA patients. (It should be noted, however, that various amino acid substitutions for R28 of Btk may have different effects on the expression of Btk protein in human hematopoietic cells. Our unpublished observations [H. Kanagane and T. Miyawaki, 1999] demonstrated that R28C Btk is stably expressed but the R28P mutation greatly reduces the expression of Btk protein. No evaluation of the expression of R28H Btk has been reported.) The exact stage in murine B cell differentiation at which defects in Btk become apparent is still controversial (Fig. 2). The most important point that should be clarified is whether, as in humans, Btk deficiency affects the early developmental stage of B lineage. Early studies have shown that XID mice have nearly normal numbers of pro-B and pre-B cells in their BM (Kincade et al., 1982). Similar observations were reported for Btk knockout mice (Kerner et al., 1995; Khan et al., 1995), suggesting a less stringent requirement for Btk in early murine B

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cell development. Additionally, in XID/+ animals, selection against XID cells is evident for surface IgM+ B cells but not for pre-B cells (Forrester et al., 1987). However, an in vivo competition assay, in which Btk-deficient (null) embryonic stem cells were injected into wild-type blastocysts, demonstrated a severe failure of Btk-deficient cells to expand to the small pre–B cell stage (Kerner et al., 1995). This suggests that Btk-deficient B-lineage cells in mice manifest an abnormality in the precursor cell expansion that is similar to that in humans and also suggests that the phenotypic difference between XID mice and human XLA is only quantitative. This observation also indicated that the XID mutation is not completely null, because no selection against XID pre-B cells has been observed. Apparently contradicting this observation, the experiment by Hendriks and collaborators (1996) demonstrated that Btk is critical only after the pre–B cell stage in murine B cell development. These authors used homologous recombination to produce Btk-deficient mice in which the Btk gene was replaced with the in-frame ␤-galactosidase (lacZ) gene. The X chromosome inactivation process in Btk+/− (lacZ+) heterozygous female mice enabled them to evaluate selective disadvantages of the B-lineage cells that used the Btk−/lacZ+ allele by monitoring lacZ expression. Based on this experiment, Hendriks’ group (1996) concluded that the disadvantage of the Btk-deficient cells occurs only at relatively later stages of B cell differentiation (at the transition from small pre-B cells to immature B cells and at the maturation step from IgMhighIgDlow to IgMlowIgDhigh cells in the periphery). Thus, the earlier question—whether Btk is also necessary for the differentiation or expansion of pre-B cells in mice—appears to remain unanswered. It was also suggested (Hendriks et al., 1996) that one reason for this confusion seems to be that the severity of the B cell defect accompanying the XID mutation depends on the genetic background. It is known that, relative to the CBA/N background, XID mutation or Btk deficiency confers a more severe B cell defect with the C3H/HeN background (Bona et al., 1980) and, in contrast, a less severe defect with the C57BL/6 background (Khan et al., 1995). Many more studies using different experimental systems thus seem to be needed to clarify the exact role of Btk in murine B cell development. Although there is still no satisfying explanation based on experiments regarding the phenotypic difference between human XLA and XID mice, it is tempting to hypothesize that both Btk-dependent and Btk-independent B cell differentiation pathways may contribute to the generation of peripheral B cell pools and that the former may contribute much more to the B cell pool of humans than to that of mice. The latter, Btk-independent, pathway may be T cell–dependent, since XID/nu (Karagogeos et al., 1986) or XID/CD40-deficient (Khan et al., 1997; Oka et al., 1996) mice exhibit a much more profound block in their B cell differentiation. In humans, too, the peripheral leaky B cells are capable of undergoing differentiation into the cells producing all of the Ig subclasses in a CD40-dependent manner (Nonoyama et al., 1998). The former, Btk-dependent, pathway may

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generate a B cell pool in a pre-BCR–dependent manner, in which Btk may play a role in the downstream of pre-BCR. In this connection, it is intriguing that the defect of ␭5, a component of pre-BCR, has a more deleterious effect on human B cell development (Minegishi et al., 1998) than in the case of ␭5 knockout mice (Kitamura et al., 1992). However, at present, we do not know which cell population with any specific markers corresponds to the putative Btk-dependent and -independent B cell pools, and the possibility cannot also be excluded that the contribution of Btk/Tec family PTKs other than Btk may simply be more significant in murine B cell development than in the development of human B cells. III. Activation of Btk

A. ACTIVATION AND TYROSINE PHOSPHORYLATION OF Btk The engagement of immunoreceptors on hematopoietic cells induces activation of distinct families of PTKs (Justement, 2000; Kurosaki, 1999; Wienands, 2000). The enhancement of this PTK activity is generally associated with its tyrosine phosphorylation in the activation loop of the catalytic domain (Hanks and Hunter, 1995). Since the identification of Btk, several immunoreceptor signals have been shown to activate and tyrosine phosphorylate Btk. The first observation of this was in mast cells, where Btk is tyrosine phosphorylated and activated following the cross-linking of the high-affinity IgE receptor (FcεRI) (Kawakami et al., 1994). During this activation, a fraction of the cytoplasmic pool of Btk is translocated to the cell membrane, although no physical association between Btk and the receptor has been observed. While Btk lacks the N-terminal lipid modification required for membrane localization, this observation was the first indication that the activation of Btk accompanies its membrane localization. Subsequent reports have demonstrated that many other signals also activate Btk, including BCR engagement (de Weer et al., 1994; Saouaf et al., 1994); IL-5 (Koike et al., 1995; Sato et al., 1994), CD38 (Kikuchi et al., 1995; SantosArgumedo et al., 1995), or gp130 (Matsuda et al., 1995) stimulation in B cells; and the engagement of collagen receptor (Oda et al., 2000; Quek et al., 1998), thrombin receptor (Laffargue et al., 1999), or CD32 (Oda et al., 2000) in platelets. In BCR engagement, the activation of Src family PTKs (e.g., Lyn) leads that of Btk (Saouaf et al., 1994), which appears to place the Src family PTKs upstream of Btk. The in vitro association of Src family PTKs with the proline-rich region of the TH domain of Btk (Cheng et al., 1994) also suggests a possible link between the activation of Btk and that of Src family PTKs. A coexpression experiment of Btk and Lyn in Epstein–Barr virus–transformed B cell lines and fibroblast cell lines suggested the existence of one mechanism controlling Btk activity through the sequential phosphorylation of specific regulatory tyrosine residues

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(Y 551 and Y223) (Park et al., 1996; Rawlings et al., 1996). In this coexpression system, the phosphorylation of Y551, which is located in the activation loop of the catalytic domain and at a site homologous to the Src family PTK consensus autophosphorylation site, requires Lyn activation, indicating that Y551 is a transphosphorylation site. This phosphorylation dramatically enhances Btk catalytic activity (Rawlings et al., 1996). Btk is then further phosphorylated at Y223 in the SH3 domain, which is dependent on the Btk catalytic activity (Park et al., 1996). The validity of this activation model of Btk by Src family PTKs has been supported by several observations. It was demonstrated that a coexpression of Csk with Src family PTKs, which down-regulates their kinase activities by phosphorylating the C-terminal regulatory tyrosine residue of Src family PTKs, suppresses the phosphorylation of Btk (Afar et al., 1996). In contrast, Btk activity was found to have been reduced in J558L␮mM3 plasmacytoma cells lacking CD45, which should up-regulate the activities of Src family PTKs by dephosphorylating the C-terminal regulatory tyrosine residue (Pao et al., 1997). Impairment of Btk activations by BCR engagement or CD38 stimulation in B cells from Lyn−/− and Lyn−/−Fyn−/− mice (Yasue et al., 1997) also supports the role of Src family PTKs in Btk activation. Involvement of an Src family PTK in the activation of a Btk homologue (Tec29; alternatively, Dsrc29A) during Drosophila development was demonstrated by genetic analyses (Guarnieri et al., 1998; Roulier et al., 1998), indicating that the activation link between Src family PTKs and Btk has been conserved during evolution. Studies using phosphopeptide-specific antibodies that recognize two distinct phosphotyrosines actually demonstrated that the phosphorylation of these residues occurs sequentially (Nisitani et al., 1999; Wahl et al., 1997). It was found that Y551 was maximally phosphorylated within 30 sec following BCR cross-linking, whereas the phosphorylation of Y223 was maximal at 5 min. This observation further supports the concept of a mechanism that controls Btk activity through the sequential phosphorylation of Y551 and Y223. Compared with the situation in other PTK families, it is unusual that Y551, which is located in the activation loop of the catalytic domain and corresponds to other PTKs’ consensus autophosphorylation site, is the transphosphorylation site, and Y223, located in the SH3 domain, is the autophosphorylation site in Btk. However, the flanking sequence of Y551 (LDDEYTSS) exhibits an excellent match to the substrate specificity for Src family PTKs, which has been demonstrated with the aid of a chemical peptide library (Zhou et al., 1995) or a phage display approach (Schmitz et al., 1996). Although the substrate specificity for Btk has been poorly studied because a very limited number of molecules are able to serve as the Btk substrate, we previously reported that Btk phosphorylates Wiskott–Aldrich syndrome protein (WASP) on its Y291 in vivo (Y. Baba et al., 1999). The sequence surrounding Y291 of WASP (SKLIYDFI) exhibited a significant similarity to that surrounding Y223 of Btk (VVALYDYM). A comparison of these flanking

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sequences yields the following common characteristics: −4 to −2 (nonacidic), −1 (hydrophobic), +1 (acidic), +2 (aromatic), and +3 (hydrophobic). This common sequence appears to represent the substrate specificity of Btk. Although it has been clearly demonstrated that the phosphorylation of Y551 enhances Btk catalytic activity as described, the significance of the phosphorylation of Y223 is still unclear. The mutation of Y223 to phenylalanine (Y223F) does not significantly change Btk activity (Kurosaki and Kurosaki, 1997; Part et al., 1996). The finding that Y223F Btk could normally restore the BCRinduced phospholipase C␥ 2 (PLC␥ 2) activation and calcium mobilization when introduced into a Btk-deficient DT40 chicken B cell line suggests that Y223 is dispensable for this signaling context (Kurosaki and Kurosaki, 1997). Park and associates (1996) demonstrated that the Y223F mutation enhances the trans∗ forming ability of the activated version of Btk (E41K mutation; Btk [Li et al., 1995]) in a manner similar to that of block deletion of the SH3 domain. Given that Y223 is located in a highly conserved region of the proline-rich peptide binding groove (Erpel et al., 1995; Noble et al., 1993), this observation raises the possibility that the autophosphorylation of Y223 might mediate the down-regulation of Btk activity by modulating the binding of cellular regulatory molecules or intramolecular conformational changes. In the case of the SH3 domains of other signaling molecules, phosphorylation of the Y138 residue of the c-Src SH3 domain (although not equivalent to Y223 of Btk), which can be mediated by the platelet-derived growth factor receptor, significantly reduced proline-rich ligand binding, although the functional significance of this finding remains elusive (Broome and Hunter, 1997). Another study has shown that the association of WASP with the cytoskeletonassociated protein PSTPIP is disrupted by phosphorylation of a tyrosine residue within the SH3 domain of PSTPIP, which may play a role in control of the cytoskeleton (Wu et al., 1998). Using a fusion protein [glutathione S-transferase (GST)–SH3] experiment, Morrogh and co-workers (1999) reported that the in vitro binding of WASP to the SH3 domain of Btk was drastically reduced when Y223 was phosphorylated. In contrast to this observation, however, our data indicated that the in vivo binding of WASP (Y. Baba et al., 1999) or Sab (Matsushita et al., 1998) to the Btk SH3 domain was unaltered by the phosphorylation of Btk. There still seem to be no definitive data supporting the concept that the phosphorylation of Y223 may modulate the binding affinity of SH3 domain binding ligands. It can also be speculated that the SH3 domain of Btk interacts with the upstream proline-rich region in the TH domain (Andreotti et al., 1997) and that the phosphorylation of Y223 blocks this intramolecular interaction, resulting in an altered accessibility for ligands. However, no experimental evidence for this speculation is currently available. Another possible significance of the tyrosine phosphorylation of Y223 is that it may create a potential binding site for SH2 domain–containing molecules. A

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pull-down experiment by Morrogh and associates (1999) detected Syk as a binding molecule of GST–SH3(Btk) in a manner dependent on the phosphorylation of Y223. However, this binding was mediated by the catalytic, not SH2, domain, of Syk and depended on the phosphorylation of Syk. It is possible that the observed binding of Syk to the phosphorylated Btk SH3 domain may be indirect and may be mediated by other SH2 domain–containing molecules. Although the flanking sequence of Y223 (YDYM) conforms to the consensus binding sequence of the SH2 domain of the phosphatidylinositol 3-kinase (PI3K) p85 subunit (Songyang et al., 1993), no association of PI3K p85 with the phosphorylated Btk SH3 domain has been detected. Besides the role of Src family PTKs, recent findings indicate that Syk is also involved in Btk activation (Kurosaki and Kurosaki, 1997). Genetic dissection experiments on the DT40 B cell line demonstrated that the phosphorylation of Btk is partially reduced in Lyn- or Syk-deficient cells, indicating that both Lyn and Syk contribute to Btk phosphorylation. These single deficient cells exhibited a complementary time course of Btk phosphorylation. In Lyn-deficient cells, early-phase Btk phosphorylation (1–3 min after BCR cross-linking) was significantly reduced, whereas late-phase (10-min) phosphorylation reached almost the same level as that in wild-type cells. In Syk-deficient cells, only early-phase Btk phosphorylation was observed. Furthermore, Lyn/Syk double-deficient DT40 cells failed to exhibit any Btk phosphorylation following BCR cross-linking. These observations suggest that Syk contributes to the Btk phosphorylation of a phase different from that contributed by Lyn. In addition, some reports (Morrogh et al., 1999; Wan et al., 1997) have suggested the physical association of Btk and Syk in B cells. However, in COS cells or 293 cells, coexpression of Btk and Syk did not affect the tyrosine phosphorylation or catalytic activity of Btk (Mahajan et al., 1995; our unpublished observations). This discrepancy suggests the requirement of another B cell–specific molecule for Btk phosphorylation by Syk. B. ACTIVATION AND MEMBRANE TARGETING OF Btk Several observations have suggested that the membrane association of Btk is a critical step in its activation. As first demonstrated in the FcεRI receptor system of mast cells, activation of Btk by receptor cross-linking correlates with the translocation of Btk to the cell membrane (Kawakami et al., 1994). When Btk was coexpressed with Lyn in fibroblasts, most tyrosine-phosphorylated Btk was found in the membrane fraction (T. Li et al., 1997). In addition, activation ∗ of Btk by a point mutation (E41K) within the PH domain (Btk ), which acquires the ability to transform fibroblasts, resulted in increased membrane localization of Btk (T. Li et al., 1995). Constitutively, membrane-associated Btk chimeras exhibited an enhanced ability to transform in the presence of Src family PTKs,

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which also suggests the importance of membrane association for Btk activation (T. Li et al., 1997). At least in some experimental systems, the transient localization of Btk to the membrane seems to be mainly mediated by its PH domain. The Btk PH domain has been reported to interact with membrane-associated molecules, including subunits of heterotrimeric G proteins and phosphoinositides. With the aid of an in vitro binding assay and an in vivo competition assay, the Btk PH domain was shown to bind the ␤␥ dimer of heterotrimeric G proteins (Tsukada et al., 1994b). In this binding, the C-terminal half of the PH domain plays the major role and the presence of W124 in subdomain 6 of the PH domain, which is highly conserved through almost all PH domains, is critical. Because the ␥ -subunit of heterotrimeric G protein is localized in the cell membrane as a result of its lipid modification (Simon et al., 1991), the observed interaction of Btk and the ␤␥ dimer has been implicated to transiently localize Btk to the membrane. However, the actual contribution of this interaction to the membrane localization of Btk has not yet been assessed. In the past few years, mounting evidence has demonstrated that the Nterminal region of certain PH domains can bind phosphoinositides (Lemmon and Ferquson, 1998; Shaw, 1996). Although the results of affinity studies of PH domains and phosphoinositides are often contradictory because of different assay systems, the results have suggested that the Btk PH domain binds at least two inositol compounds, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) (Rameh et al., 1997; Salim et al., 1996) and inositol-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) (Fukuda et al., 1996; Kojima et al., 1997), with a KD of <1 ␮M. This interaction is mediated by the positively charged surface of the PH domain that binds the negatively charged phosphate groups of inositol compounds. XID mutation (R28C) was found to dramatically reduce the affinity of the PH domain for both PtdIns-3,4,5-P3 and Ins-1,3,4,5-P4 (Fukuda et al., 1996; Salim et al., 1996). In addition, a crystal structure study suggested that binding to inositol compounds on a membrane surface stabilizes Btk in the dimeric state, which is predicted to enhance the transphosphorylation and activation of Btk (Baraldi et al., 1999). Because PtdIns-3,4,5-P3 is generated by the action of PI3K, the interaction between the Btk PH domain and PtdIns3,4,5-P3 puts Btk downstream of PI3K. The extrapolated molecular scenario is that, after receptor stimulation, PtdIns-3,4,5-P3 generated by the activated PI3K enhances the translocation of Btk to the cell membrane, which in turn promotes the activation of Btk by membrane-anchoring PTKs such as Lyn. It has been demonstrated that the coexpression of PI3K with Btk in Rat2 cells harboring Srcfamily PTKs enhances the tyrosine phosphorylation of Btk, which in turn is reduced by treatment with the PI3K inhibitor wortmannin (Z. Li et al., 1997). It was also demonstrated that in a Rat2 soft agar transformation assay, a coexpression of Btk and an activated form of the p110 subunit of PI3K

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results in a great enhancement of colony formation (Scharenberg et al., 1998). In B cells, too, the BCR-induced tyrosine phosphorylation of Btk was blocked by wortmannin or LY294002 (Nore et al., 2000). In NIH–3T3 cells coexpressing a Btk PH–GFP (green fluorescent protein) fusion protein and the epidermal growth factor receptor, the membrane translocation of the Btk PH–GFP by epidermal growth factor was completely inhibited by the treatment with wortmannin or LY294002 (Varnai et al., 1999). Moreover, a membrane- targeted form of PI3K led to membrane localization of the Btk PH domain. In another study, membrane translocation of the Btk–GFP chimeric protein by surface receptor stimulations upstream of PI3K was observed (Nore et al., 2000). Furthermore, it was demonstrated that the genetic dissection of SH2-containing inositol 5′ -phosphatase, which hydrolyzes PtdIns-3,4,5-P3 to phosphatidylinositol-3,4bisphosphate (PtdIns-3,4-P2) and Ins-1,3,4,5-P4 to inositol-1,3,4-trisphosphate (Ins-1,3,4-P3), leads to significant membrane association of Btk in DT40 B cells (Bolland et al., 1998). These observations suggest that the intracellular concentration of PtdIns-3,4,5-P3 (and Ins-1,3,4,5-P4) is one of the major determinants leading to the membrane localization and subsequent activation of Btk. It should be noted, however, that in several surface receptor systems the actual contribution of the interaction of the Btk PH domain and phosphoinositides to the activation of Btk remains to be determined. One example showed that, although XID mutation dramatically reduces the affinity of the Btk PH domain for phosphoinositides, the BCR-induced enhancement of Btk activity remains unaltered compared to that in the wild-type cells and that the events downstream of Btk, such as calcium mobilization following BCR cross-linking, are only partially impaired in XID cells (Rigley et al., 1989). It is possible that additional interactions mediated by other domains of Btk may affect the overall localization of the whole molecule. As mentioned later in detail, Btk is inducibly associated with BLNK through its SH2 domain (Hashimoto et al., 1999b). Because BLNK translocates to the membrane fraction following BCR cross-linking (Ishiai et al., 1999a), this interaction may also allow for the translocation of Btk. It also remains to be determined whether the association of PtdIns-3,4,5-P3 and Btk contributes to the activation of Btk in a manner different from its tyrosine phosphorylation by Src family PTKs. It has been recognized that the interaction of certain phosphoinositides and the PH domain directly allow for the activation of the enzyme harboring the PH domain. It is known that the activity of Akt/protein kinase B, a serine–threonine kinase with a PH domain, is partly regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain through facilitating the dimerization of Akt (Franke et al., 1997). Also in the case of Btk, a crystal structure study suggested the dimeric formation of Btk with phosphoinositides (Baraldi et al., 1999), so that it is possible that part of the Btk activity is also directly regulated by the interaction of PtdIns3,4,5-P3 or Ins-1,3,4,5-P4.

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C. REGULATION OF Btk ACTIVITY The catalytic activities of PTKs generally appear to be strictly controlled, which contributes to the homeostatic regulation of cytoplasmic signal transductions. In addition to tyrosine phosphorylation and intracellular translocation, this regulatory process includes serine–threonine phosphorylation and protein interactions with other molecules called trans-activators or trans-inhibitors (Fig. 3). Btk is constitutively phosphorylated on serine and threonine residues both in mast cells and in B cells (Yao et al., 1994). As one of the kinases involved in this phosphorylation, protein kinase C␤ (PKC␤) is reported to constitutively associate with Btk (Yao et al., 1994). This binding is mediated by the N-terminal region (residues 28–45) of the PH domain of Btk (Yao et al., 1997) and is diminished by introduction of the XID mutation (R28C) (Yao et al., 1994). It was also observed that the phosphorylation mediated by PKC down-regulated the catalytic activity of Btk, leading to the hypothesis that the phosphorylation by PKC might keep the basal and peak tyrosine phosphorylation levels of Btk in check (Kawakami et al., 1999; Yao et al., 1994). While the importance of the Btk PH and SH2 domains in the Btk-related signaling pathway has been emphasized by several experimental systems, the functional significance of the Btk SH3 domain remains elusive, as mentioned previously. The Btk SH3 domain is thought to be dispensable for the calcium

FIG. 3. Bruton’s tyrosine kinase (Btk)–related signaling pathways in B cells. Protein kinase C␤ (PKC␤) and Sab seem to suppress the Btk activity. BCR, B cell antigen receptor; DAG, diacylglycerol; IP3, inositol-1,3,4-trisphosphate; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol3,4-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PLC␥ 2, phospholipase C␥ 2; SHIP, Src homology 2–containing inosotol 5′ -phosphatase; WASP, Wiskott–Aldrich syndrome protein.

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signal or apoptotic process induced by BCR cross-linking. The deletion of the Btk SH3 domain rather enhanced the transformational ability of the activated version of Btk (E41K) (Li et al., 1995). Seemingly in agreement with these observations, no missense mutation has been found in the Btk SH3 domain of XLA patients (Vihinen et al., 1999). This suggests that the mutations in the SH3 domain do not result in the loss of function of Btk, and that instead this domain probably plays a regulatory role in the Btk signaling pathway. We previously reported the identification of a 70-kDa Btk SH3 domain–binding protein, termed Sab (SH3BP5), by using a Far Western cloning method (Matsushita et al., 1998). Sab exhibited a higher selectivity for binding to the SH3 domain of Btk than to those of other PTKs (Lyn, Fyn, Lck, and Src) or other cytoplamic molecules (PLC␥ 2, PI3K, Grb2, and Crk). Although Sab can associate with Btk in vivo, tyrosine phosphorylation of Sab by Btk has not been observed, suggesting that Sab is not a substrate of Btk but rather participates in the regulation of the Btk activity. Subsequent experiments have actually demonstrated that Sab inhibits the auto- and transphosphorylation activity of Btk. The possibility that Sab is the regulator of the Btk activity was further supported by observations that the forced expression of Sab in the DT40 B cell line suppressed the BCR-induced tyrosine phosphorylation of Btk as well as BCR-coupled early signaling events such as calcium mobilization and inositol-1,4,5-trisphosphate (IP3) generation, in which Btk activity has previously been shown to be involved (Yamadori et al., 1999). The interaction of the ␤␥ dimer of the heterotrimeric G protein and the PH domain of Btk was initially proposed as the mechanism by which Btk is translocated to the membrane fraction (Tsukada et al., 1994b). A subsequent report using a cotransfection assay and an in vitro reconstitution assay (LanghansRajasekaran et al., 1995) demonstrated that certain ␤␥ dimers enhance the kinase activity of Btk (and also of Itk). However, in our unpublished experiment (S. Tsukada et al.), we could not reproduce this result, and at any rate, the extent of the Btk activation by the ␤␥ dimer seems to be relatively weak. Recently, the same group (Bence et al., 1997) reported that the ␣-subunit of the heterotrimeric G protein directly stimulates Btk and that the purified ␣-subunit of the Gq, but not of the Gi1, G0, or G2 class, increased the activity of purified Btk protein. They also demonstrated that an agonist stimulation of the Gq-coupled receptor in DT40 cells enhanced Btk activity. A subsequent report (Y. Jiang et al., 1998; Ma and Huang, 1998) mapped the binding site of ␣-subunit to the region composed of the TH domain and the SH3 domain, prompting them to hypothesize that the binding of the ␣-subunit to this region disrupts the intramolecular interaction of the SH3 domain and the proline-rich region in the TH domain, thereby activating Btk. Although these proposed regulatory mechanisms are likely to be of biological significance, the data presently available are still fragmentary. Further studies

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are absolutely required for the integration of these observations into the entire molecular framework of the Btk signaling pathway.

IV. Downstream of Btk

A. CALCIUM SIGNALING The role of Btk in cytoplasmic signal transduction has been most extensively studied in the BCR-coupled signaling pathway. The availability of Btk-deficient B cell lines, such as those generated by genetic dissection in DT40 chicken B cells (Kurosaki, 1999) and established from XLA patients, has greatly contributed to this progress. In these studies, one of the most striking findings has been that Btk is the PTK linking the BCR to calcium signaling. BCR engagement on B cells activates a remarkably diverse set of intracellular signaling cascades (Justement, 2000; Kurosaki, 1999, 2000; Tamir and Cambier, 1998; Wienands, 2000). The earliest known event is the tyrosine phosphorylation of multiple cellular proteins. Since the BCR itself lacks any intrinsic tyrosine kinase activity, several BCR-associated PTKs mediate downstream signaling events. In the BCR system, the overall induction of tyrosine phosphorylation in multiple cellular proteins seems to be mediated mainly by Lyn and Syk. As demonstrated by genetic dissection experiments in DT40 cells, the global tyrosine phosphorylation of cellular molecules is greatly reduced by dissection of Lyn or Syk (Takata et al., 1994) and is almost completely eliminated in Lyn/Syk double-deficient cells (Takata and Kurosaki, 1996). In contrast, the global tyrosine phosphorylation in Btk-deficient DT40 cells is essentially the same as that in wild-type DT40 cells (Takata and Kurosaki, 1996). Seemingly consistent with this observation in DT40 cells, the BCR-induced tyrosine phosphorylation patterns in XLA-derived and wild-type human B cell lines are not very different (our unpublished observations). These observations indicate that Btk does not participate in triggering of the PTK cascade which induces the tyrosine phosphorylation of multiple signaling molecules and also indicate that only a limited number of cellular molecules are able to serve as the substrate of Btk. Another striking BCR-induced event with rapid kinetics is the release of ionized calcium from intracellular stores, which in turn induces the entry of extracellular calcium. In B cells, as in many other cell types, calcium signals are triggered by the second messenger IP3, the product of the action of PLC on phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2).The generation of IP3 is followed by the release of internal calcium storage through IP3 receptors on the endoplasmic reticulum. Once these endoplasmic reticulum calcium stores are emptied, this triggers extracellular calcium entry through store-operated calcium channels in the plasma membrane, which in turn regulates the selectivity of

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transcriptional factors (Berridge et al., 1998, 2000). In contrast to the minor contribution of Btk to the initiation of the BCR-coupled PTK cascade, accumulating data have indicated that Btk is a critical component in BCR-coupled calcium signaling. The first evidence came from an experiment with genetic dissection of the Btk gene in DT40 cells, which exhibited a complete loss of BCR-coupled IP3 production and calcium mobilization (Takata and Kurosaki, et al., 1996). It was also shown that the function of both the PH and SH2 domains as well as Btk kinase activity is required for calcium signaling. This observation was followed by experiments using XLA B cell lines in which BCR-coupled calcium signaling is markedly blunted (Fluckiger et al., 1998). An overexpression of Btk in the human B cell line enhanced the sustained phase (extracellular calcium entry) of the BCR-induced calcium response, which was further potentiated by expressing the activated version of Btk (Li et al., 1995) or the constitutively membrane-associated Btk chimeric construct. These observations indicate that the intracellular dosage of Btk is one of the major determinants of BCR-coupled calcium signaling. Similarly, the important role of Btk/Tecfamily PTKs in calcium signaling has also been demonstrated in T cells. A noticeable impairment of the TCR-induced calcium signal was observed in T cells lacking Itk (Liu et al., 1998), which became even more profound in the case of the double knockout of Itk and Rlk (Schaeffer et al., 1999). Activation of the PLC enzyme is the crucial step for initiation of the calcium signal. Of several PLC isoforms in existence, the PLC␥ isoform plays a dominant role in antigen receptor–induced calcium signals. T cells express mainly the PLC␥ 1 isoform, while PLC␥ 2 is predominantly expressed in B cells (Coggeshall et al., 1992; Hempel et al., 1992). Because biochemical studies have clearly demonstrated that PLC␥ activation occurs mainly as a result of its tyrosine phosphorylation (Weiss et al., 1991), the observation that the dissection of Btk abolishes BCR-coupled IP3 production and calcium mobilization, which reflect PLC␥ activation, indicates the possibility that Btk is the PTK responsible for the tyrosine phosphorylation of PLC␥ 2 in B cells. Actually, a significantly reduced level of PLC␥ 2 phosphorylation upon BCR cross-linking was observed in Btk-deficient DT40 cells (Takata and Kurosaki, et al., 1996). Genetic dissection experiments also demonstrated the important role of another PTK family, the Syk family PTKs (Syk in B cells and Zap70 in T cells) in antigen receptor–coupled calcium signaling. Both IP3 production and calcium mobilization were almost completely eliminated in Syk-deficient DT40 chicken B cells (Takata et al., 1994) or Zap70-deficient Jurkat T cells (Williams et al., 1999). In Syk-deficient DT40 cells, moreover, the BCR-induced tyrosine phosphorylation of PLC␥ 2 is abolished (Takata et al., 1994). This makes it clear that Btk and Syk are the PTKs responsible for PLC␥ 2 activation in B cells. The molecular framework used by Btk to activate PLC␥ 2 is discussed extensively in later sections.

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B. APOPTOSIS AND THE c-jun N-TERMINAL KINASE (JNK) PATHWAY Several observations have supported the notion that Btk regulates the apoptotic processes of B cells and mast cells. In DT40 cells, BCR-induced apoptosis is blocked in both Btk- and Syk-deficient cells, whereas Lyn-deficient cells undergo apoptosis normally (Takata and Kurosaki, 1996; Takata et al., 1994). The block of apoptosis is also observed in PLC␥ 2-deficient cells (Takata et al., 1995), which indicates the importance of PLC␥ 2 activation, which is the downstream of Btk and Syk, for this apoptosis of DT40 cells. Furthermore, BCR-induced apoptosis is significantly suppressed also in triple IP3 receptor–deficient DT40 cells (Sugawara et al., 1997), indicating that calcium mobilization plays a role in apoptosis. However, in contrast to the almost complete block of apoptosis in PLC␥ 2-deficient cells, residual apoptosis remains in IP3 receptor–deficient cells, suggesting that calcium mobilization and the PKC pathway, both of which are downstream of PLC␥ 2, are required for this apoptosis. The regulatory role of Btk in apoptosis can also be observed in mast cells deprived of cytokine. XID and Btk null murine BM mast cells died by apoptosis at a significantly slower rate than did wild-type cells upon IL-3 deprivation. This suppression of apoptosis was undone by introduction of wild-type Btk but not of kinase-negative or XID (R28C) Btk (Kawakami et al., 1997). It has been suggested that activation of mitogen-activated kinase (MAPK), especially that of c-jun N-terminal kinase (JNK), contributes to the induction of these apoptotic processes, because the activation patterns of JNK in the above cells correlate nicely with the induction of apoptosis. Although wild-type and Lyn-deficient DT40 cells show significant activation of JNK1 upon BCR cross-linking, Btk- and Syk-deficient cells do not (A. Jiang et al., 1998). PLC␥ 2- and IP3 receptor–deficient cells also exhibit an impaired JNK1 response (Hashimoto et al., 1998). Furthermore, JNK1 and JNK2 activation induced by IL-3 deprivation as well as by FcεRI cross-linking was drastically reduced in Btk null mast cells (Kawakami et al., 1997). Although the exact causal relationship among these findings remains to be determined by further experiments, they suggest the involvement of JNK activation in the apoptosis of these cells. In contrast to the close correlation between JNK activation and apoptosis, the activation of other MAPKs, (extracellular signal–regulated kinase (ERK)) and p38 MAPK, does not correlate with apoptosis, nor does the lack of Btk lead to any significant loss of ERK or p38 activation (A. Jiang et al., 1998; Kawakami et al., 1997). Experiments based on both biochemical and genetic dissection methods indicate that BCR-induced JNK activation requires both the Rac1 and PLC␥ 2 pathways (Hashimoto et al., 1998; A. Jiang et al., 1998). Rac1, a Rhofamily GTPase, appears to activate JNK through the phosphorylation cascade Rac1—PAK65—MEKK1—SEK1 (or MKK7)—JNK (Bagrodia et al., 1995; Kawakami et al., 1998; Zhang et al., 1995), JNK activation also requires PLC␥ 2 activation because both calcium signaling and PKC activation are necessary for

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FIG. 4. Bruton’s tyrosine kinase (Btk) signaling pathways that lead to transcriptional controls of several genes and apoptosis through calcium mobilization or c-jun N-terminal kinase (JNK) activation. PKC, Protein kinase C; PLC␥ 2, phospholipase C␥ 2.

the full activation of JNK (A. Jiang et al. 1998; Sutherland et al., 1996). Because both Btk and Syk are required for PLC␥ 2 activation, as discussed previously, it stands to reason that a deficiency of Btk or Syk leads to the loss of JNK activation and apoptosis. It has been suggested that Btk is also required for Rac1 activation, because PAK65 activity, which occurs immediately downstream of Rac1, was significantly higher in wild-type mast cells upon FcεRI cross-linking than in Btk null mast cells (Kawakami et al., 1997) (Fig. 4). However, the exact molecular link between Btk and Rac1 is currently unknown. It has been noted that the regulation of the JNK pathway by Btk controls not only apoptosis but also the transcriptions of several genes, including those of cytokines such as IL-2 and tumor necrosis factor ␣ (Hata et al., 1998). It was also found that splenic B cells from XID mice exhibit a rather high rate of spontaneous apoptosis in in vitro culture compared to those from normal mice (Anderson et al., 1996; Brorson et al., 1997), suggesting that the biological outcomes of Btk deficiency depend on the differentiation stage of B cells. Reportedly, this high spontaneous apoptosis results from the inability of XID splenic B cells to induce bcl-XL, which is also calcium dependent (Anderson et al., 1996). Finally, some studies have demonstrated the involvement of Btk in radiation-induced (Uckun et al., 1996) or Fas-induced apoptosis (Uckun, 1998; Vassilev et al., 1999). C. OTHER DOWNSTREAM PATHWAYS OF Btk In addition to PLC␥ 2, biochemical analyses have identified several Btk substrates and suggested the presence of other downstream pathways.

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WASP is the gene product responsible for Wiskott–Aldrich syndrome, which is an X-linked recessive disorder associated with severe thrombocytopenia, eczema, and immunodeficiency (Derry et al., 1994). It has been demonstrated that WASP is involved in organizing the cytoskeleton through the Arp2/3 complex (actinnucleating assembly), which regulates the structure and dynamics of the actin filament network (Machesky and Gould, 1999). The observations that WASP is transiently tyrosine phosphorylated following BCR cross linking in B cells (Y. Baba et al., 1999), IgE receptor cross-linking in mast cells (Guinamard et al., 1998), and collagen stimulation of platelets (Oda et al., 1998) suggest the presence of a link between WASP and PTK pathways. In reconstituted cells, Btk associates with WASP via the SH3 domain and phosphorylates the Y291 residue of WASP (Baba et al., 1999). Consistent with this indication that WASP is the Btk substrate in vivo, the collagen-induced tyrosine phosphorylation of WASP in platelets from XLA patients was shown to be reduced when compared to that in normal platelets (Gross et al., 1999). Although the functional significance of the tyrosine phosphorylation of WASP is not yet known, it may modulate the conformational change or function of WASP. It was further reported that WASP is autoinhibited by intramolecular binding of the GTPase binding domain with the C-terminal region. Association of WASP with Cdc42 via the GTPase binding domain causes a conformational change in WASP, resulting in disruption of the autoinhibition fold, which allows WASP to interact with other molecules (Kim et al., 2000). Because the Y291 residue of WASP, the site of phosphorylation by Btk, is located in an important region for forming the autoinhibition fold, it can be assumed that the phosphorylation of WASP may destabilize the intramolecular binding and cause conformational change, leading to actin polymerization in a GTPase-independent manner. Alternatively, the tyrosine phophorylation of WASP may create a docking site for SH2 or phosphotyrosine binding domain–containing molecules, which would allow for intermolecular interaction. Although WAS patients’ hematopoietic cells, including B cells, were reported to have some abnormalities in their differentiation and function (Lau et al., 1992; Morio et al., 1989; Ochs et al., 1980; Simon et al., 1992; Wengler et al., 1995), the defect in WAS B cells has been poorly defined in contrast to the clearly identified defect in XLA B cells. In WASP knockout mice, B cell differentiation and function are reported to be almost normal (Snapper et al., 1998; J. Zhang et al., 1999). The functional significance of the link between Btk and WASP in B cell development remains to be investigated. TFII-I is a ubiquitously expressed multifunctional transcription factor that was initially characterized as a binding protein to the initiator sites of various promoters (Roy et al., 1993) and has also been demonstrated to enhance the activation of the c-fos promoter through interaction with its upstream elements. TFII-I is also encoded by the gene that is altered in Williams–Beuren syndrome (Perez-Jurado et al., 1998). It was found that TFII-I is identical to

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BAP-135, which was cloned as the molecule that associates with the PH domain of Btk and is tyrosine phosphorylated in vitro by Btk (Yang and Desiderio, 1997). Novina and colleagues (1999) have reported that TFII-I constitutively associates with wild-type and kinase-deficient Btk but not with XID mutant (R28C) Btk. Following BCR cross-linking, this association is disrupted and the dissociated TFII-I translocates to the nucleus. These observations imply that Btk plays a role in the retention of TFII-I in the cytoplasm of resting cells and that the BCR-induced tyrosine phosphorylation of TFII-I reduces its association with Btk. Furthermore, wild-type but not XID mutant nor kinase-deficient Btk was found to enhance TFII-I–dependent transcriptional activation of genes such as V␤5.2 (Novina et al., 1999), suggesting that both kinase activity and a direct association with TFII-I are necessary for Btk to function as a positive regulator of TFII-I. Since the transcripton of many genes important for B cell development, including ␭5, Vpre-B, TdT, RAG, CD5, bcl-2, and bcl-XL, could be regulated by TFII-I (Novina et al., 1999), the functional link of Btk and TFII-I can be assumed to be involved in normal B cell development. V. BLNK Connects Btk Activity to Downstream Effectors

A. BLNK INTEGRATES Btk AND Syk ACTIVITIES For the precise identification of the Btk signaling pathway, the next important issue to be solved was the determination of the molecular mechanism that connects Btk activity and the multiple downstream pathways, the latter then being biologically integrated and ultimately deciding the fate of cells. It was also an important question how the activities of Btk and another PTK such as Syk are integrated and transmitted downstream. As discussed previously, both Btk and Syk are necessary for the full activation of PLC␥ 2. Why are two different classes of kinase, Syk and Btk, necessary, and how are these kinases integrated to activate PLC␥ 2 in B cells? The key to solving these questions came from the identification of hematopoietic cell–specific adapter molecules that integrate PTKs with downstream effectors. SLP76 (Jackman et al., 1995), which is primarily expressed in T cells and phosphorylated by Zap70 after T cell receptor (TCR) engagement, is a prototype of such adapter molecules. The important role of SLP76 in TCR signals is illustrated by the fact that SLP76-deficient T cells manifest a severe defect in the TCR-induced calcium signaling even though the activations of TCR-coupled PTKs such as the Src family and Zap70 were normal (Yablonski et al., 1998). This finding indicates that the loss of SLP76 uncouples the activation of upstream PTKs and the downstream calcium signal in T cells. Although B cells do not express SLP76, an adapter molecule, BLNK (Fu et al., 1998)—alternatively SLP65 (Wienands et al., 1998) or BASH (Goitsuka et al., 1998), with a similar structure to that of SLP76—was identified in B cells (Fig. 5). Common structural features of this adapter family (SLP76-like adapter)

FIG. 5. Structure of BLNK and a model for B cell antigen receptor (BCR)−induced calcium mobilization. BLNK contains a number of domains that dictate interactions with other proteins, including an Sre homology 2 (SH2) domain, a proline-rich region that presumably interacts with SH3 domain− containing molecules, and several tyrosine residues in the N-terminal region (P−Y) that are phosphorylated after BCR engagement and allow for the interactions with several SH2 domain−containing molecules, such as Btk and phospholipase C␥ 2 (PLC␥ 2). IP3, Inositol-1,3,4-trisphosphate; PH, plecstrin homology; PIP2, phosphatidylinositol-3,4 bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; TH, Tec homology.

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are the presence of N-terminal multiple tyrosine phosphorylation sites, a central proline-rich region, and a C-terminal SH2 domain. BLNK was initially identified as a molecule that is tyrosine phosphorylated following BCR engagement (Fu et al., 1998) or pervanadate stimulation (Wienands et al., 1998), and also as a molecule preferentially expressed in bursal B cells (Goitsuka et al., 1998). It was demonstrated that Syk is the tyrosine kinase responsible for the phosphorylation of BLNK, which, after being phosphorylated, binds to the SH2 domain of PLC␥ (Fu et al., 1998b). Because the SH2 domain of PLC␥ 2 was found to be essential for the BCR-induced calcium signaling (Ishiai et al., 1999b; Takata et al., 1995), this observation raised the possibility that BLNK may serve as a linker protein connecting Syk with PLC␥ 2 for efficient PLC␥ 2 activation by Syk. This scenario was clearly validated by a subsequent experiment using BLNK-deficient DT40 cells in which BCR-induced PLC␥ 2 phosphorylation, IP3 production, and calcium mobilization were eliminated (Ishiai et al., 1999a). While these results established that the linking of Syk, BLNK, and PLC␥ 2 is essential for the generation of the BCR-induced calcium signaling, it remained to be determined how Btk, another PTK responsible for PLC␥ 2 activation, is integrated into this molecular framework. Because Btk seemed not to phosphorylate BLNK (Fu et al., 1998), Btk must utilize a mechanism for PLC␥ 2 activation different from that used by Syk. The clue to the nature of this mechanism came from the characterization of the SH2 domain–binding protein of Btk (Hashimoto et al., 1999b; Su et al., 1999). In vitro binding and protein purification experiments demonstrated that one of the major Btk SH2 domain–binding proteins in B cells is BLNK. In B cells, Btk was shown to inducibly associate with BLNK after BCR engagement via its SH2 domain. Furthermore, reconstitution experiments demonstrated that the presence of BLNK phosphorylated by Syk enhanced the tyrosine phosphorylation of PLC␥ 2 by Btk (Hashimoto et al., 1999b). These findings explained the essential role of the Btk SH2 domain in the BCR-induced calcium signal, which had been previously demonstrated by mutational analysis of Btk (Takata and Kurosaki, 1996) and led to the emergence of a new molecular connection of Btk, Syk, BLNK, and PLC␥ 2 in calcium signals in B cells (Fig. 5). After BCR engagement, Syk is activated and phosphorylates BLNK, which allows for the association of Btk with BLNK, as well as for the association of PLC␥ 2 with BLNK via their respective SH2 domains. As Syk also colocalizes on phosphorylated BLNK (Fu et al., 1998; Goitsuka et al., 1998), BLNK might nucleate an activation complex including Syk, Btk, and PLC␥ 2, for which PLC␥ 2 is fully phosphorylated and subsequently activated. The calcium defects observed in cells deficient in Syk (Takata et al., 1994), Btk (Takata and Kurosaki, 1996), PLC␥ 2 (Takata et al., 1995), and BLNK (Ishiai et al., 1999a) seem to be entirely consistent with this molecular scenario. The exact biochemical basis of the PLC␥ activation, however, remains elusive. In the case of PLC␥ 1, it has been reported that receptor-type PTKs such as

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the epidermal growth factor and platelet-derived growth factor receptors phosphorylate PLC␥ 1 on its tyrosine residues 771, 783, and 1254 (Kim et al., 1991; Wahl et al., 1990). The Y783F mutation completely blocked PLC␥ 1 activation, whereas the Y1254F mutation partially inhibited and the Y771F mutation enhanced the PLC␥ 1 activity by platelet-derived growth factor in NIH–3T3 cells (Kim et al., 1991), indicating that multiple tyrosine residues are phosphorylated and have different impacts on PLC␥ 1 activation. Although the exact phosphorylation sites in PLC␥ 2 have not been reported, it has been shown that, in Sykdeficient DT40 B cells, both PLC␥ 2 phosphorylation and IP3 production are almost abolished (Takata et al., 1994). In contrast, the reduction of PLC␥ 2 phosphorylation is relatively small in Btk-deficient DT40 B cells (Takata and Kurosaki, 1996) or XLA-derived B cell lines (Fluckiger et al., 1998; our unpublished observations). Given that multiple tyrosine residues in PLC␥ 1 are phosphorylated upon stimulation of receptor-type PTKs, one possible explanation of the difference in inhibition of PLC␥ 2 phosphorylation by the loss of either Syk or Btk is that these PTKs mediate the phosphorylation of different tyrosine residues. According to the aforementioned molecular scenario connecting Btk, Syk, and PLC␥ 2, the deletion of Syk can be expected to eliminate both Btk-dependent and Syk-dependent (if they exist) PLC␥ 2 phosphorylations because Syk is also required for Btk-dependent PLC␥ 2 phosphorylation. In contrast, the residual phosphorylation of PLC␥ 2 in Btk-deficient cells may be caused by the activity of Syk, as Btk is not necessarily required for Syk-dependent PLC␥ 2 phosphorylation. However, it is at present unclear whether Syk can directly phosphorylate PLC␥ 2 in cells. Because Btk is capable of phosphorylating PLC␥ 2 in heterogeneous transfection systems (Fluckiger et al., 1998; Hashimoto et al., 1999b) and also in in vitro kinase assays (S. Hashimoto and S. Tsukada, 1999, unpublished observations), it is almost certain that PLC␥ 2 is the direct substrate of Btk and is phosphorylated on one or more tyrosine residues critical for its activation. On the other hand, although PLC␥ 1 is reported to be phosphorylated by Syk in vitro and by constitutively membrane-associated CD8–Syk chimeras in COS cells (Law et al., 1996), no significant tyrosine phosphorylation of PLC␥ 2 by Syk was observed in a heterogeneous transfection system (Hashimoto et al., 1999b). In addition, our unpublished observations indicated that PLC␥ 2 was not phosphorylated by Syk in an in vitro kinase assay, casting doubt on the assumption that PLC␥ 2 is the direct substrate of Syk. This observation raises the possibility that Syk may directly participate only in the phosphorylation of BLNK to provide docking sites for Btk and PLC␥ 2. If this is true, the residual PLC␥ 2 phosphorylation observed in Btk-deficient cells, which is not sufficient for the full activation of PLC␥ 2, might be attributable to other unidentified PTKs located downstream of Syk. Other mechanisms, independent of PLC␥ tyrosine phosphorylation, are also reported to play roles in the control of PLC␥ activity. These include PH

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domain– and PtdIns-3,4,5-P3–dependent activation (Falasca et al., 1998) and the autoregulation of PLC␥ activity by its intrinsic inhibitory region (Homma and Takenawa, 1992). The relatively small decrease in PLC␥ phosphorylation in Btkdeficient cells, compared to the drastic reduction in IP3 production, may suggest that Btk utilizes another mechanism for PLC␥ activation in addition to phosphorylation of PLC␥ . Alternatively, the presence of tyrosine phosphorylation– dependent and –independent activation mechanisms of PLC␥ may mean that multiple pathways must operate together to allow for the IP3-gated internal calcium store to be released. Admittedly, certain reported observations remain difficult to explain in terms of the simple scenario discussed earlier, which connects Btk, Syk, and PLC␥ 2 through BLNK. While the defective calcium signal in the Btk-deficient DT40 cells could barely be restored by PH-mutated (R28C) Btk (Takata and Kurosaki, 1996), B cells from XID mice were reported to exhibit only a limited (40–50%) reduction of BCR-induced IP3 production and calcium flux (Rigley et al., 1989). This discrepancy may reflect species differences in the use of PLC␥ isoforms or Tec family PTKs. While DT40 B cells express exclusively the PLC␥ 2 isoform (Takata et al., 1995), both PLC␥ 1 and PLC␥ 2 are expressed in murine B cells (Coggeshall et al., 1992; Hempel et al., 1992). The contribution of PLC␥ 1 to the calcium signaling in murine B cells has not been assessed, and in addition, there is at present no evidence that PLC␥ 1 and PLC␥ 2 use the same activation mechanism. Fluckiger and collaborators (1998) reported that overexpression of Btk in B cells enhanced the sustained phase (extracellular calcium entry) of BCRinduced calcium response but had a minimal detectable effect on the initial phase (intracellular calcium release). Overexpression of Syk, in contrast, resulted in no enhancement in the sustained phase, and the dominant negative form of Syk specifically blocked the initial phase. This observation led Fluckiger’s group to conclude that Btk and Syk regulate distinct phases in calcium signaling. However, this conclusion is difficult to explain from the molecular connection of Btk, Syk, and BLNK, which simply predicts that Btk and Syk on BLNK act in concert in PLC␥ 2 activation. It is possible that the process is more complicated than the simple scenario discussed above so that the BCR-induced calcium signaling is appropriately regulated. BLNK thus represents a typical example of the adapter molecules that integrate the activities of two distinct PTKs (Btk and Syk) to the downstream pathway. However, it remains to be resolved whether BLNK also couples Btk activity with other downstream pathways in addition to PLC␥ 2. The BLNK-deficient DT40 cells also display a severe defect in BCR-coupled Rac1 activation, in which Btk activity seems to be involved (Ishiai et al., 1999a). Furthermore, the mechanism needs to be clarified how BLNK can selectively activate the downstream effectors like a switchboard. Ishiai and co-workers (1999a) assumed that this selection might be determined by phosphorylation of distinct tyrosine residues

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within BLNK. If this is true, how can Syk and Btk, which are at present the only two PTKs proven to associate with BLNK, regulate the phosphorylation sites in BLNK? Another question that must be clarified is whether B cells utilize other adapter molecules in addition to BLNK. Although BLNK is at present the only adapter molecule proven to be involved in the BCR signaling pathway, T cells appear to utilize at least two adapter molecules (SLP76 and linker for activation of T cells [LAT]) for the TCR signaling pathway (Clements et al., 1999; van Leeuwen and Samelson, 1999). LAT is an integral membrane protein and contains multiple tyrosine residues that are phosphorylated by Zap70 after TCR engagement (Weber et al., 1997; Zhang et al., 1998). The phosphorylated LAT then interacts with several SH2 domain–containing molecules, including PLC␥ 1. Dissection of LAT eliminates the TCR-induced calcium mobilization as well as the tyrosine phosphorylation of SLP76 and PLC␥ 1 (Finco et al., 1998; W. Zhang et al., 1999). In contrast, the phosphorylation of LAT and the recruitment of PLC␥ 1 to LAT remain unaffected in SLP76-deficient T cells even though the PLC␥ 1 phosphorylation and calcium mobilization are eliminated (Yablonski et al., 1998). These findings demonstrate that the concerted action of SLP76 and LAT leads to the activation of PLC␥ 1 and, finally, to calcium mobilization. The linking of two adapter molecules in T cells raises the possibility that, in B cells, too, an additional adapter molecule may participate in the BLNK-related signaling pathway. Another possibility, as initially proposed by Chan and colleagues (Fu et al., 1998), is that BLNK may function as the unique adapter molecule in the BCR signaling pathway. While BLNK and SLP76 share structural and functional similarities, BLNK possesses a greater number of phosphorylation sites, to interact with a greater number of downstream molecules, than does SLP76. While BLNK is capable of interacting with PLC␥ , Grb2, Vav, and Nck (Fu et al., 1998), SLP76 interacts only with Vav and Nck (Onodera et al., 1996; Tuosto et al., 1996; Wu et al., 1996). Complementarily with the binding of molecules to SLP76, LAT interacts with PLC␥ and Grb2 (Weber et al., 1997; Zhang et al., 1998). It is therefore reasonable to assume that BLNK serves as the unique central adapter molecule that integrates the functions of both SLP76 and LAT. At present, however, there is no clear evidence to substantiate these possibilities. Furthermore, growing evidence is beginning to indicate that, in addition to the SLP76-like adapters, Grb2-like adapter molecules (which have SH3–SH2–SH3 structure) may also be involved in the molecular framework of the antigen receptor signaling pathway. Although Grb2 is ubiquitously expressed, recent studies have identified several Grb2-like adapter molecules whose expressions are restricted to hematopoietic cells. One of these molecules, Gads (Liu et al., 1999), also known as GrpL (Law et al., 1999) or Grf40 (Asada et al., 1999), is primarily expressed in T cells but also in B cells. Coimmunoprecipitation experiments have shown that Gads interacts with SLP76 via its SH3 domain (Asada et al., 1999;

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Law et al., 1999; Liu et al., 1999) and probably also with LAT via its SH2 domain (Asada et al., 1999; Liu et al., 1999), which suggests the possibility that Gads links these two adapter molecules in T cells. It has also been demonstrated that Gads synergizes with SLP76 to augment nuclear factor activation in T cells, which presumably reflects the enhancement of PLC␥ 1-mediated calcium mobilization (Asada et al., 1999; Law et al., 1999; Liu et al., 1999). One of the hypothesized molecular scenarios in TCR-coupled calcium signaling is that, after being phosphorylated by Zap70, LAT and SLP76 bind PLC␥ 1 (Weber et al., 1997; Zhang et al., 1998) and Itk (Bunnell et al., 2000), respectively. Grb2-like adapters such as Gads bridge LAT and SLP76 (Asada et al., 1999; Liu et al., 1999), which allows for PLC␥ 1 activation by Itk (Kurosaki and Tsukada, 2000). Given the similarity of BCR- and TCR-coupled signalings, these findings in the T cell system also suggest the presence of other players besides Syk, Btk, and BLNK in B cells, thus accounting for the proper regulation of the BCR-induced calcium signaling. While BLNK is predominantly expressed in B cells, the recently identified third member of the SLP76 family, CLNK/MIST, is mainly expressed in cytokine-dependent cell lines (Cao et al., 1999) and mast cells (Goitsuka et al., 2000). In contrast to BLNK, which is phosphorylated by Syk, CLNK/MIST is phosphorylated by Lyn and subsequently associates with PLC␥ , Vav, Grb2, and LAT (Goitsuka et al., 2000). Considering that Btk is strongly expressed in mast cells and plays an important role in FcεRI signaling (Kawakami et al., 1999), it is possible that the functional link of BLNK and Btk is conserved also in the case of CLNK/MIST and Btk, playing a role in mast cell activation. B. BLNK AND B CELL DEVELOPMENT The results discussed above indicate that the activities of Btk and Syk strongly associate with BLNK, at least in a certain signaling context in B cells. This conclusion suggests that, if the function of BLNK is nonredundant in B cell development, mutations in BLNK might also result in the arrest of B cell maturation, presumably at the same stage, as in cases with defects of Btk and Syk. Actually, Minegishi and colleagues (1999c) reported an immunodeficiency patient with mutations in BLNK. The disease phenotype of the patient was indistinguishable from that of the typical XLA patient (no detectable serum Ig and <1% B cells in the peripheral circulation). BM analysis of the patient showed that the block in B cell differentiation occurred at the transition from pro-B cells to pre–B cells, thus demonstrating the essential role of BLNK in human early B cell development and also that there is no alternative molecule that can replace the function of BLNK, at least in humans. Moreover, the maturation arrest at the early stage of pre-B cells in the BLNK-deficient patient suggested that BLNK is also required at the pre–B cell stage, presumably for pre-BCR signaling. The phenotype of BLNK knockout mice was also reported by at least four groups (Hayashi et al., 2000; Jumaa et al., 1999; Pappu et al., 1999; Xu et al., 2000).

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Overall, the phenotype of BLNK-deficient mice bears a striking resemblance to that of XID mice. They exhibit lower IgM and IgG3 serum concentrations, unresponsiveness to type 2 T cell–independent antigens, and absence of B1 cells. The B cell development is blocked at the pre-B and immature B cell stages in BM, and the maturation of peripheral B cells is also impaired. Their B cells show reduced BCR-coupled calcium mobilization and reduced proliferative responses to B cell mitogens. The phenotypic difference between BLNK-deficient humans and mice is very similar to the discrepancy observed in the phenotypes of human and murine Btk deficiencies. This probably indicates that the functions of Btk and BLNK are highly interdependent and that the B cell deficiency in BLNK-deficient humans and mice mainly originates from the disruption in Btk–BLNK interaction. In contrast, the apparently more severe phenotype of Syk-deficient mice, which shows an almost complete block at the transition from pro-B cells to pre-B cells and no leaky peripheral B cells (Cheng et al., 1995; Turner et al., 1995), may imply that additional Syk substrates besides BLNK also play critical roles in B cell development. C. Btk AND BLNK IN EARLY B CELL DEVELOPMENT The expression of pre-BCR, which is composed of heavy (␮) chain and SL chain (consisting of Vpre-B and ␭5) with signal-transducing units Ig␣ and Ig␤, is a mandatory step for the progression of B cell development. It was believed that the continuous signal produced through functional pre-BCR results in the dramatic expansion of early pre-B cells and is also essential for allelic exclusion at the heavy-chain locus (Karasuyama et al., 1996; Melchers et al., 1994). Pre-B cells also express Btk and BLNK, as well as other PTKs, such as Lyn and Syk (Minegishi et al., 1999c; Tsukada et al., 1993). Considering that the earliest bottleneck in the B cell development of both XLA- and BLNK-deficient patients is at the early pre–B cell stage and given the structural similarity of BCR and pre-BCR, it is a reasonable assumption that Btk and BLNK might also be involved in pre-BCR signaling. However, the current understanding of pre-BCR signaling is very limited. Although studies have revealed the important roles of Btk and BLNK in the signals generated upon BCR cross-linking which mimic the signals via BCR engagement by foreign antigens that lead to B cell activation and antibody production, other studies have suggested that the signal-transducing units Ig␣ and Ig␤ also play important roles in sustaining B cell development and survival by means of continuous signals in which the participation of ligand binding is unclear (Lam et al., 1997). In the case of pre-BCR, no ligand has been identified despite intensive searches, which probably means that pre-BCR may also transmit some ligand-independent/continuous signals (Karasuyama et al., 1996). In this context, it may be a reasonable observation that in a murine pre–B cell line NFS70.15, several proteins, including Btk, are constitutively tyrosine phosphorylated independently of pre-BCR cross-linking on the surface (Aoki

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et al., 1994). We have also observed that in a human pre–B cell line, NALM6, Btk as well as its substrate, WASP, is constitutively tyrosine phosphorylated (Y. Baba et al., 1999). We also found that the tyrosine phosphorylation of Btk is significantly higher in an Abelson virus–transformed pre–B cell line than in the pro–B cell line from which the pre–B cell line was cloned and established (Y. Baba and S. Tsukada, 1999, unpublished observations). While these data seem to suggest the involvement of Btk in the constitutive signaling via pre-BCR, it is currently unclear whether they can be extrapolated to pre-B cells under physiological conditions. At present, there are no available data that suggest the role of BLNK in the early stage of B lineage cells. Although early studies reported that in some pre-B cell lines, pre-BCR cross-linking induced a certain amount of intracellular calcium mobilization (Misener et al., 1991; Nakamura et al., 1993; Nomura et al., 1991; Takemori et al., 1990), others reported that no significant calcium flux was detected upon cross-linking of Ig␤ on murine BM pro-B cells, in contrast to the prominent calcium flux in splenic B cells treated with the anti-Ig␤ antibody (Nagata et al., 1997). This appears to suggest that pre-BCR and BCR activate distinct signaling modules besides common ones, even though both receptors utilize the Ig␣/Ig␤ heterodimer as a common signaling unit. In addition, no induced tyrosine phosphorylation of Btk or BLNK following Ig␤ cross-linking on pre-B or pro-B cells has been reported. While both Btk and BLNK seem to play their roles at the transition from pro-B cells to pre–B cells, the major difficulty in investigating B cell development at this stage was the lack of an appropriate in vivo system in which the requirements of specific molecules can be tested. Karasuyama and colleagues reported a unique differentiation system utilizing recombination activating gene 2 (RAG-2) knockout mice (Nagata et al., 1997). Cross-linking of Ig␤, by injecting an anti-Ig␤ antibody into RAG-2 knockout mice, induces differentiation of the RAG-2–deficient pro-B cells to small pre-B cells in vivo. This system seems to be particularly useful for investigating the functions of Btk and BLNK, by assessing the roles of these molecules in this stage of B cell development in the case of the double knockout of Btk or BLNK with RAG-2. VI. Conclusion

One of the most significant advances in the study of Btk signaling is the identification of the functional connection of Btk and BLNK. BLNK integrates two distinct PTKs, Btk and Syk, into the downstream effectors such as PLC␥ 2, which allows for the intracellular calcium store to be released upon BCR cross-linking. It is assumed that several other Btk downstreams may also be regulated by BLNK or by other, as yet unidentified adapters. The question remains, however, how BLNK can appropriately select the multiple downstream effectors for activation like a switchboard and integrate specific effectors with Btk and Syk. Is

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it regulated by its tyrosine phosphorylation or by other factors? Further studies are clearly needed to completely understand the functional link of Btk and BLNK. Although details of the Btk signaling through the BCR are beginning to emerge, the role of Btk at the pre–B cell stage, at which XLA mutation impedes B cell differentiation, is still far from being understood. In particular, the significance of the “continuous signals” from the pre-BCR and BCR necessary for cell proliferation and survival as well as the role of Btk in this context remain completely unidentified. Clarification of these Btk-dependent signals is essential for understanding the whole molecular framework that allows B cells to develop. ACKNOWLEDGMENTS We thank Toshio Miyawaki and Hirokazu Kanegane for unpublished data, Ryo Goitsuka for preprint, and Tomohiro Kurosaki and Hajime Karasuyama for critical comments. Our work has been supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Ministry of Health and Welfare of Japan.

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ADVANCES IN IMMUNOLOGY, VOL. 77

Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2s MAKOTO MURAKAMI AND ICHIRO KUDO Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

I. Introduction

Phospholipase A2 (PLA2) has emerged as a growing superfamily of enzymes that catalyze the hydrolysis of membrane glycerophospholipids at the sn-2 position, liberating free fatty acids and lysophospholipids. PLA2 provides precursors for the biosynthesis of eicosanoids, such as prostaglandins (PGs) and leukotrienes, when the liberated fatty acid is arachidonic acid (AA), as well as lysophospholipid-derived mediators, such as platelet-activating factor (PAF) and lysophosphatidic acid. As overproduction of these lipid mediators causes various diseases and tissue disorders, it is important to understand the mechanisms that regulate the functions of PLA2. Recent advances in molecular and cellular biology have led to the identification of a number of mammalian PLA2 enzymes, which are subdivided into several groups based on their structures, enzymatic characteristics, subcellular distributions, and cellular functions. There are four major families of PLA2: secretory PLA2s (sPLA2s), cytosolic PLA2s (cPLA2s), Ca2+ -independent PLA2s (iPLA2s), and PAF acetylhydrolases (PAF-AH). Among them, cPLA2 (group IV) has received much attention as a key regulator of stimulus-initiated eicosanoid and PAF biosynthesis, because it selectively releases AA, shows submicromolar Ca2+ sensitivity, and is activated by mitogen-activated protein kinase–directed phosphorylation (Clark et al., 1991; Lin et al., 1993). cPLA2 undergoes Ca2+-dependent translocation from the cytosol to perinuclear and endoplasmic reticular membranes, where several downstream eicosanoid-generating enzymes, including cyclooxygenase (COX) and lipoxygenase (LOX), are localized (Schievella et al., 1995). Studies of cPLA2-deficient mice have confirmed its critical role in lipid mediator generation during the acute allergic response, parturition, and postischemic brain injury (Bonventre et al., 1997; Uozumi et al., 1997). Cytosolic iPLA2 (group VI), which occurs as several splicing variants (Larsson et al., 1998; Tang et al., 1997), plays a pivotal role in the phospholipid remodeling reaction (Balsinde et al., 1997). PAF-AHs (groups VII and VIII) are a group of unique PLA2 subtypes that degrade PAF and related oxidized phospholipids, thereby contributing to sequestering inflammatory responses (Hattori et al., 1994, 1996; Nakajima et al., 1997; Stafforini et al., 1997). 163 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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sPLA2 comprises Ca2+-dependent interfacial enzymes that share a low molecular mass (∼14–16 kDa). Numerous sPLA2s have been found in venom from vertebrate and invertebrate animals as well as in plants. To date, at least ten structurally related isozymes (groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals: these enzymes display distinct yet partially overlapping tissue distributions (Cupillard et al., 1997; Gelb et al., 2000; Ishizaki et al., 1999; Kramer et al., 1989; Seilhamer et al., 1989a; Suzuki et al., 2000, Tischfield, 1997; Valentin et al., 1999a,b, 2000). Continued awareness of the diversity of sPLA2s has cast doubt on earlier studies reporting the molecular identity of sPLA2 species expressed in various cells. Understanding the physiological functions of various sPLA2s is now a complex and challenging area of research in the eicosanoid field. Although the biological roles of each of these enzymes has not yet been clearly defined, they have been implicated in various physiological and pathological functions, including lipid digestion, lipid mediator generation, cell proliferation, exocytosis, antibacterial defense, cancer, and inflammatory diseases. In this chapter, we introduce the recent advances in the diversity, enzymatic properties, and functions of the sPLA2 family. In particular, the roles of each sPLA2 in eicosanoid generation in the context of functional coupling between other eicosanoid-biosynthetic enzymes in different phases of cell activation are described. II. Structures and Enzymatic Properties of sPLA2s

A. STRUCTURES The structural features of mammalian sPLA2s are summarized in Fig. 1 sPLA2s (except sPLA2-III; see below) contain highly conserved amino acid residues and sequences that are characteristic of most sPLA2s sequenced to date: (i) an ␣-helical N-terminal segment containing lipophilic residues at positions 2, 5, and 9; (ii) a Ca2+-binding loop with a typical glycine-rich sequence at Tyr25–Pro37 and at the residue Asp49; (iii) an active site residue His48 as well as residues Tyr52, Tyr73 and Asp99; and (iv) 12–16 cysteine residues, most of which are in the same positions. sPLA2-IB (and snake venom group I sPLA2s) possesses 14 cysteines, of which residues 11 and 77 form a characteristic disulfide bond, an N-terminal propeptide, which is removed during the secretion/activation process, and an extra segment called the pancreatic loop (residues 54–56) (Murakami et al., 1997; Verheij et al., 1981). The gene for sPLA2-IB has been mapped to human chromosome 12 and mouse chromosome 5 (Seilhamer et al., 1989b). The group II subfamily of sPLA2s includes six isozymes (IIA, IIC, IID, IIE, IIF, and V), which possess similar structural characteristics that are not found in sPLA2-IB, and their genes are tightly clustered in the same chromosomal locus (human chromosome 1 and mouse chromosome 4) (Tischfield, 1997; Valentin et al., 1999a,b).

FIG. 1. The structures of mammalian secretory phospholipase A2s (sPLA2s). The genes for sPLA2-IIA, -IIC, -IID, -IIE, -IIF, and -V (the group II subfamily of sPLA2s) map to the same chromosomal locus.

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FIG. 2. The phylogenetic tree of mammalian secretory phospholipase A2s.

sPLA2s-IIA, -IID, and -IIE display all of the specific features of group II sPLA2s (including snake venom group II enzymes), possessing a cysteine at position 50 and a cysteine that terminates the group II–specific C-terminal extension composed of seven residues (Ishizaki et al., 1999; Kramer et al., 1989; Seilhamer et al., 1989a; Suzuki et al., 2000; Valentin et al., 1999a,b). The refined threedimensional crystal structure of human sPLA2-IIA indicates that its catalytic mechanism is essentially identical to those inferred from the crystal structures of other venom and pancreatic group I and venom II sPLA2s (Scott et al., 1991; Wery et al., 1991). sPLA2-IIC is unique in that it has 16 cysteines, of which residues Cys86 and Cys92 are postulated to form an extra disulfide bridge (Chen et al., 1994b). This isozyme is expressed as an active enzyme in rodents, but is present in the form of a pseudogene in humans. sPLA2-IIF has a long C-terminal extension of 23 amino acids containing an extra cysteine (Valentin et al., 1999b). sPLA2-V has only 12 cysteines and lacks a group II–specific Cys50 and C-terminal extension (Chen et al., 1994a), yet its overall properties, revealed by the phylogenetic tree (Fig. 2), are more similar to those of the other group II subfamily of sPLA2s than to those of sPLA2-IB and -X. sPLA2-X, which has 16 cysteines, exhibits several structural features that are found in both groups I and II, including a group I–specific N-terminal propeptide, group I– and group II–specific disulfide bridges, and a group II– specific C-terminal extension (Cupillard et al., 1997). sPLA2-X is the only isozyme that undergoes N-glycosylation, although this sugar chain is not essential for catalytic function (Hanasaki et al., 1999). The gene for human sPLA2-X has been mapped to chromosome 16 (Cupillard et al., 1997). The amino acid sequence homology among these sPLA2s of the same species is ∼30–50%, and the

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exon–intron structures of their genes resemble one another, indicating that they are evolutionarily conserved. Besides these sPLA2s, onconin 90, a major protein component of otoconia, contains two domains homologous to sPLA2 (Wang et al., 1998). Alignment of the sPLA2-like domains in onconin 90 with the known sPLA2s reveals a highly conserved region close to the N terminus, which includes the Ca2+-binding loop and catalytic site. However, some of the residues considered essential for Ca2+binding and catalytic activity are replaced in both the sPLA2-like domains, suggesting that onconin 90 has no enzymatic function. The phylogenetic tree places the sPLA2-like domains in onconin 90, sPLA2-IB and sPLA2-X, on the same branch ancestral to other contemporarily expressed mammalian sPLA2s (Fig. 2). Very recently, another novel class of sPLA2, group XII, has been identified (Gelb et al., 2000). This isozyme displays homology to other sPLA2s only over a short stretch of amino acids in the active site region. The activity is Ca2+dependent (albeit weak) despite the fact that it has an unusual Ca2+-binding loop. B. ENZYMATIC PROPERTIES Mechanistic studies have shown that the sPLA2s do not form a classical acyl enzyme intermediate characteristic of serine esterases, including cPLA2s, iPLA2s, and PAF-AHs. Instead, they utilize the catalytic site His48, assisted by Asp49, to polarize a bound water, which then attacks the carbonyl group: the essential Ca2+ ion, bound in the conserved Ca2+-binding loop, stabilizes the transition state (Dennis, 1994). In vitro PLA2 assays provide somewhat varied results according to the protocol used. Generally, sPLA2s do not discriminate between fatty acid moieties at the sn-2 position, but have rather more specificity for the polarized head groups (Kudo et al., 1993; Murakami et al., 1997). The optimal enzyme reaction occurs under neutral to mildly alkaline conditions (pH ∼ 7–9) in the presence of millimolar Ca2+. Using pure phospholipid vesicles as a substrate, the group II subfamily of sPLA2s prefers anionic phospholipids such as phosphatidylglycerol, phosphatidylethanolamine (PE), and phosphatidylserine (PS) to charge-neutral phosphatidylcholine (PC); sPLA2-X does not discriminate between the head group moieties; and sPLA2-IB exhibits an intermediate pattern. On the basis of the strict lipid–water interfacial studies, sPLA2-IB binds much more tightly to anionic phospholipids than to PC, although the binding to PC is significant (Snitko et al., 1999). Hydrolysis of PC is greatly accelerated, to a level comparable with that of PE, in the presence of a low concentration of detergents such as deoxycholate (Hanasaki et al., 1999; Kudo et al., 1993). sPLA2-IIA binds very weakly to PC vesicles even at millimolar concentrations, and interfacial binding is enhanced by >106-fold when anionic phospholipids are mixed (Kinkaid and Wilton, 1995). sPLA2-IIC, -IID, -IIE, and -IIF exhibit substrate specificity similar to that of sPLA2-IIA (Ishizaki et al., 1999; Murakami et al., 1998; Suzuki et al.,

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2000; Valentin et al., 1999a,b). sPLA2-V binds at least 100-fold more tightly to anionic phospholipid vesicles than to PC vesicles, but binds to the latter with much higher affinity than do sPLA2-IB and -IIA (Han et al., 1999). Paradoxically, AA-containing phospholipids are rather poorer substrates for sPLA2-V than are linoleate-containing phospholipids (Chen and Dennis, 1998; Hanasaki et al., 1999), even though this isozyme has the potent ability to mobilize AA metabolism in mammalian cells (see below). sPLA2-X hydrolyzes PE and PC vesicles in an almost equal ratio (Hanasaki et al., 1999; Murakami et al., 1999c). Sphingomyelin (SM) inhibits the enzymatic activity of sPLA2-IB and -IIA in vitro (Koumanov et al., 1997). The high packing density of lipid bilayers enriched in SM hinders the penetration of sPLA2s into the membrane. Cholesterol counteracts the effect of SM-based inhibition of sPLA2s (Koumanov et al., 1998). Several hydrophobic residues near the ␣-helical N terminus of sPLA2s contribute to interfacial binding to phospholipid vesicles (Murakami et al., 1997). Tryptophan residues located on the putative interfacial binding surfaces are postulated to be critical for penetration of sPLA2 into PC vesicles. Indeed, Trp31 and Trp67 are crucial for the binding of sPLA2-V and sPLA2-X, respectively, to PC vesicles (Bezzine et al., 2000; Han et al., 1999). sPLA2-IIA, which binds very poorly to PC vesicles, does not have such a tryptophan, yet replacement of Val3 by Trp increases the affinity for PC dramatically (Baker et al., 1998). sPLA2IIA, -IID and -V contain a number of basic amino acid residues throughout the molecules, and at least in the case of sPLA2-IIA, some of them contribute to binding to anionic phospholipid vesicles (Koduri et al., 1998). C. HEPARANOID BINDING sPLA2-IIA, -IID, and -V are highly cationic isozymes and bind tightly to heparin–Sepharose, whereas the other sPLA2 isozymes with acidic to neutral pI show no, or very low, affinity (Ishizaki et al., 1999; Murakami et al., 1996, 1998, 1999b,c; Valentin et al., 1999a). An initial site-directed mutagenesis study demonstrated that the cluster of basic amino acids near the C terminus of sPLA2IIA and V plays a crucial role in heparanoid binding (Murakami et al., 1996). Later, it was shown that the affinity of sPLA2-IIA for heparanoids is modulated not only by a highly localized site of basic residues but also by diffuse sites that partially overlap with the interfacial binding site (Koduri et al., 1998). As described below, this heparin-binding property affects the cellular functions of these sPLA2s under various conditions. Heparin and chondroitin sulfate, but not heparan sulfate, increase severalfold the activity of sPLA2-IIA toward lowdensity lipoprotein (Sartipy et al., 1996). On the other hand, heparan sulfate on mast cells plays a negative regulatory role for sPLA2-IIA, in which the enzyme is bound to heparan sulfate on cell surfaces, internalized, and then degraded rapidly (Enomoto et al., 2000b).

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III. Expression and Functions of sPLA2s

A. SPLA2-IB The sPLA2 present in pancreatic juice is classified as sPLA2-IB, and its main role is the digestion of phospholipids in food (Verheij et al., 1981). sPLA2-IB is synthesized in the pancreatic acinar cells, liberated into the pancreatic juice, and secreted into the duodenum. sPLA2-IB in pancreatic tissue exists exclusively as an inactive proenzyme, and removal of an N-terminal heptapeptide by trypsin yields an active enzyme. sPLA2-IB is also expressed in trace amounts in several tissues such as lung, kidney, and spleen (Hara et al., 1995; Tojo et al., 1988), where it is assumed to play nondigestive roles. Plasma plasmin is a possible candidate to promote the conversion of inactive zymogen to mature enzyme in these nondigestive organs (Nakano et al., 1994). Although only limited information about transcriptional regulation of sPLA2-IB is available, expression of sPLA2-IB mRNA in the pancreas is reported to be decreased following excess intake of glucose (Metz et al., 1991). Intact cellular membrane is usually rather resistant to sPLA2-IB–directed hydrolysis (Bezzine et al., 2000; Murakami et al., 1998). This is probably because this isozyme shows poor interfacial binding to PC-rich plasma membrane surfaces and has virtually no affinity for heparan sulfate proteoglycan, which acts as a cell surface adapter for the heparin-binding group II subfamily of sPLA2s (see below). However, as described later in detail, sPLA2-IB binds to the M-type sPLA2 receptor with high affinity, through which it stimulates various cellular responses, including AA release (Lambeau and Lazdunski, 1999). sPLA2-IB has been implicated in the pathogenesis and pathophysiology of acute pancreatitis. The initial enthusiasm concerning pancreatic sPLA2-IB as an enzyme responsible for pancreatic necrosis and systemic manifestations of acute pancreatitis has gradually waned, as the mechanisms of the pathogenesis and pathophysiology of acute pancreatitis have been revealed. The overactive systemic inflammatory response seen in severe acute pancreatitis, associated with the activation of different cascade systems and increased levels of inflammatory mediators, closely resembles that associated with other severe inflammatory diseases (e.g., septic shock). Although activation of sPLA2-IB in the pancreas may cause tissue damage, the elevated serum PLA2 levels in patients with acute pancreatitis are due to sPLA2-IIA, production of which is generally induced during the inflammatory response (see below), rather than to sPLA2-IB that has leaked from the damaged pancreas (Nevalainen et al., 1999). B. SPLA2-IIA 1. Expression Discovery of sPLA2-IIA dates back a decade, when several inflammation researchers succeeded in purification of an sPLA2 distinct from pancreatic sPLA2

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from inflammatory exudates and platelets (Chang et al., 1987; Horigome et al., 1987; Kramer et al., 1989; Seilhamer et al., 1989a). sPLA2-IIA is synthesized as a precursor form containing a signal sequence and is then processed to a mature enzyme during translocation from the cytosolic to the luminal side of the endoplasmic reticulum. Constitutive expression of sPLA2-IIA has been detected in several tissues, such as the spleen, thymus, intestine, tonsil, liver, and bone marrow (Murakami et al., 1997), and also in inflammatory effector cells, such as platelets, neutrophils, macrophages, and mast cells, in which sPLA2-IIA is stored in secretory granules and released into the extracellular fluids immediately after cell activation (Horigome et al., 1987; Murakami et al., 1992). In the intestine, sPLA2-IIA is localized in Paneth cells, where it plays a role in antimicrobial defense (Senegas-Balas et al., 1984); in the liver, it is distributed preferentially in the Kupffer cells (Inada et al., 1991). Large amounts of sPLA2-IIA have been detected at various inflamed sites and in the plasma of patients with rheumatoid arthritis and septic shock, as well as in experimental animal models of inflammation (Kudo et al., 1993; Murakami et al., 1997; Pruzanski and Vadas, 1988; Vadas and Pruzanski, 1986). These findings strongly argue that sPLA2-IIA is involved in inflammatory responses and host defense. Notably, sPLA2-IIA is an inducible isozyme in response to various stimuli (Couturier et al., 1999; Crowl et al., 1991; Kuwata et al., 1998; Murakami et al., 1993a; Nakazato et al., 1991; Oka and Arita, 1991; Pfeilschifter et al., 1993;. Suga et al., 1993). The major inducers of sPLA2-IIA expression include bacterial lipopolysaccharide (LPS); the proinflammatory cytokines, such as interleukin 1 (IL-1), tumor necrosis factor, and IL-6; and cAMP-elevating agents. Cytokineinduced sPLA2-IIA expression occurs in various types of cells, such as chondrocytes, smooth muscle cells, hepatocytes, astrocytes, renal mesangial cells, endothelial cells, mast cells, macrophages, and fibroblasts. The time-dependent induction of sPLA2-IIA expression generally occurs after an initial lag period of several hours and then continues to increase throughout the culture period, accompanied by sustained PG generation. Injection of LPS into rats markedly induces sPLA2-IIA expression in many tissues (Nakano and Arita, 1990; Sawada et al., 1999). The promoter region of the sPLA2-IIA gene contains TATA and CAAT boxes and several elements homologous with consensus sequences for binding of transcription factors such as activator protein-2, nuclear factor–IL-6, nuclear factor-␬B, and peroxisome proliferator-activated receptor (PPAR) ␥ (Couturier et al., 1999; Crowl et al., 1991). Anti-inflammatory glucocorticoids are potent suppressors of the induction of sPLA2-IIA expression (Kuwata et al., 1998; Nakano and Arita, 1990; Nakano et al., 1990). Transforming growth factor ␤, an anti-inflammatory cytokine, as well as platelet-derived growth factor and insulinlike growth factors also reduce sPLA2-IIA induction, with a concordant decrease in PG generation (Muhl ¨ et al., 1991; Schalkwijk et al., 1992).

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2. Eicosanoid Biosynthesis Arachidonic acid metabolism is subdivided into two phases according to the time courses of the production of particular kinds of eicosanoids, which in turn depend on the type of cell and stimulus involved (Bingham et al., 1996; Murakami et al., 1994, 1997, 1998, 1999a; Reddy and Herschman, 1997; Reddy et al., 1997). Many cells respond to a number of ligands coupled to G proteins or tyrosine kinases, which cause transient intracellular Ca2+ mobilization and elicit “immediate” eicosanoid generation that lasts only for several minutes. Thromboxane A2 generation by platelets and PGD2 and leukotriene C4 generation by mast cells activated with immunoglobulin E and antigen are typical examples of immediate eicosanoid generation utilizing constitutively expressed enzymes for eicosanoid biosynthesis. Another aspect of eicosanoid generation is the “delayed” production of prostanoids following stimulation with cytokines, growth factors, and mitogens that lasts for several hours and is accompanied by de novo synthesis of regulatory proteins. The existence of two kinetically distinct PG-biosynthetic responses, the immediate and delayed phases, implies the recruitment of different sets of biosynthetic enzymes to this pathway. A rapidly expanding body of evidence suggests that the two COX isozymes, the constitutive COX-1 and the inducible COX-2, play distinct roles in regulating AA metabolism (Smith et al., 1996). Generally, utilization of COX-1 is observed during the immediate phase of PG biosynthesis, whereas COX-2–dependent PG generation proceeds over several hours in parallel with the induction of COX-2 expression. Segregated utilization of COX-1 and COX-2 in different phases of the PG-biosynthetic responses depends, at least in part, on the amounts of AA released by PLA2s at the moment when PG generation takes place. At the cellular level, COX-1 requires higher concentrations of AA for its function than does COX-2 (Murakami et al., 1999a; Shitashige et al., 1998). Thus, during the immediate phase, when a burst of AA is released in a short time, the local concentration of AA reaches a level high enough to activate constitutive COX-1, whereas a limited amount of AA is supplied gradually in the delayed phase, during which only inducible COX-2 is active. The fact that the induction of sPLA2-IIA expression is often associated with concomitant changes in COX-2 expression and PG generation implies that this isozyme contributes to supplying AA to COX-2 to promote delayed PG biosynthesis. Support for this speculation was provided by earlier studies using antibodies, chemical inhibitors, and antisense oligonucleotides that were assumed to be specific for sPLA2-IIA (Barbour and Dennis, 1993; Murakami et al., 1993a; Pfeilschifter et al., 1993; Suga et al.,1993). However, the identification of many novel sPLA2 isozymes has forced a reassessment of a significant fraction of the large literature describing the functions of sPLA2-IIA (Tischfield, 1997). Indeed, some of the functions that were ascribed to sPLA2-IIA

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have turned out to be attributable to sPLA2-V, as described below. Nevertheless, recent more careful evaluation of the correlation between expression and function of sPLA2-IIA (Kuwata et al., 1998; Naraba et al., 1998; Tada et al., 1998) has provided unequivocal evidence that this isozyme functions as an amplifier of the delayed PG-biosynthetic response. Cotransfection of sPLA2-IIA and COX-2 in human embryonic kidney 293 cells led to marked augmentation of IL-1–induced delayed AA release and PGE2 generation (Murakami et al., 1998, 1999a–c). Furthermore, coculture of sPLA2-IIA and COX-2 transfectants revealed that extracellular sPLA2-IIA augments PGE2 generation by neighboring COXexpressing cells, implying that it plays a particular role as a mediator of transcellular PG biosynthesis from one cell to another (Murakami et al., 1999a; Tada et al., 1998). Importantly, continuous production and supply of sPLA2-IIA are crucial for it to function appropriately in the delayed PG-biosynthetic response. It is noteworthy that AA release by sPLA2-IIA occurs only when cells are activated by proinflammatory stimuli, whereas quiescent cells are fairly refractory to sPLA2-IIA (Kudo et al., 1993; Murakami et al., 1998). This indicates the existence of regulatory mechanisms that render the membranes susceptible to the action of sPLA2-IIA only after cell activation. The following regulatory mechanisms for the cellular actions of sPLA2-IIA have been proposed. a. Heparan Sulfate Proteoglycan. In many (but not all) cases, the regulatory functions of sPLA2-IIA (as well as other heparin-binding sPLA2s) in the delayed PG-biosynthetic response depend on heparan sulfate proteoglycans, which act as a functional adapter on cell surfaces (Kuwata et al., 1998; Murakami et al., 1993a, 1996, 1998, 1999b; Suga et al., 1993). A considerable portion of de novo– synthesized sPLA2-IIA exists as a cell surface–associated form, which is washed out by extracellular addition of heparin or heparinase. This solubilization process is accompanied by reduction of sPLA2-IIA–mediated PG biosynthesis. Replacement of the C-terminal basic amino acid cluster with acidic amino acids abolishes the ability of sPLA2-IIA to bind heparan sulfate without affecting in vitro enzyme activity, and this particular mutant is incapable of promoting in vivo AA release when transfected into cells (Murakami et al., 1996, 1998). The cell surface heparan sulfate proteoglycans fall into two families of molecules that differ in their core protein domain structures (David, 1993). The syndecans have core proteins with a transmembrane and a cytoplasmic domain, and they possess heparan and/or chondroitin sulfate chains near the N terminus distal to the plasma membrane (Bernfield et al., 1992). The glypicans, by contrast, lack a membrane-spanning domain, are anchored to the external surface of the plasma membrane via glycosylphosphatidylinositol (GPI), and have three heparan sulfate chains near the C terminus, which are close to the plasma membrane (David et al., 1990). Consistent with a GPI-anchored moiety, glypicans are mobile in the cell membrane and exhibit both apical and basolateral distributions, whereas syndecans are distributed basolaterally to be attached to

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extracellular matrix proteins (Mertens et al., 1996). A significant portion of glypican translocates to the nucleus in cells undergoing cell division and activation (Liang et al., 1997). Several lines of evidence have suggested that endogenously expressed sPLA2IIA preferentially associates with the GPI-anchored form of heparan sulfate proteoglycan, glypican (Murakami et al., 1999b). With the aid of glypican, the nanograms per milliliter amount of endogenous sPLA2-IIA that is continuously produced is sufficient to promote AA release. GPI-anchored proteins generally occur in microdomains of the cell membrane called caveolae or the caveolae-related domain (Friedrichson and Kurzchalia, 1998; Hoessli and Robinson, 1998; Smart et al., 1996). Dynamic changes occur in the subcellular distribution of glypican, which moves to the nucleus and punctate caveolaelike domains, depending on cell activation states (Liang et al., 1997). Glypican appears to play a specific role in the sorting of sPLA2-IIA into caveolae-like compartments in activated cells (Murakami et al., 1999b). Caveolae form a unique endocytic and exocytic compartment at the surface of most cells, capable of importing molecules, delivering them to specific locations within the cell, and compartmentalizing a variety of signaling activities (Anderson, 1998). By means of the caveolae-mediated endocytotic event called potocytosis, sPLA2-IIA is capable of being translocated to the perinuclear compartments, in proximity to COX-2 (Murakami et al., 1999b). Since there is considerable evidence to indicate that caveolae are a site of Ca2+ storage and entry into the cell (Anderson, 1998), it is likely that sPLA2-IIA, a Ca2+-dependent enzyme, present inside caveolae signalsomes retains its enzyme activity even after internalization and translocation to the perinuclear domain. The caveolae membrane is enriched in SM, which inhibits the enzymatic activity of sPLA2-IIA in vitro (Koumanov et al., 1997). A decrease in the cellular SM content caused by sphingomyelinase in response to cytokines (Adam-Klages et al., 1996) may allow the otherwise silent sPLA2-IIA to become active toward the caveolae membrane. Cholesterol, which is also abundant in caveolae (Anderson, 1998), counteracts the effect of SMbased inhibition of sPLA2-IIA (Koumanov et al., 1998) and may contribute to the temporal and spatial regulation of this enzyme during cell activation. Studies using a sPLA2-IIA mutant with altered interfacial binding further supported the idea that sPLA2-IIA does not simply act on the PC-rich outer plasma membrane but on a membrane compartment rich in acidic phospholipids in the glypicandependent pathway (Murakami et al., 2001). b. Membrane Asymmetry. That sPLA2-IIA hardly acts on resting cell membranes is likely to be a reflection of its poor interfacial binding capacity to PC, which is enriched in the external surface of the plasma membrane (Baker et al., 1998; Bezzine et al., 2000; Koduri et al., 1998). In order for exogenous sPLA2-IIA to enhance agonist-stimulated AA release, high concentrations of the enzyme (on the order of micrograms per milliliter) are often required (Murakami et al.,

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1997). Mutational analyses have shown that the action of exogenous sPLA2-IIA on cells is transient and depends essentially on its interfacial binding to PC, but not on heparan sulfate binding (Koduri et al., 1998). This notion is further supported by the observations that exogenous sPLA2-X (Bezzine et al., 2000; Hanasaki et al., 1999) and sPLA2-V (Han et al., 1999), which exhibit much higher PC-hydrolytic activity than sPLA2-IIA, elicit AA release at lower concentrations than sPLA2-IIA, even in unstimulated cells. It has been proposed that the transbilayer movement of anionic phospholipids, the preferred substrates for sPLA2-IIA, to the external surface of the plasma membrane is one of the mechanisms underlying increased cellular sensitivity to the enzyme (Kudo et al., 1993; Murakami et al., 1997). Whether perturbed membrane asymmetry indeed influences the cellular actions of sPLA2-IIA has been verified by transfection experiments with phospholipid scramblase, which facilitates the transbilayer movement of anionic phospholipids (Zhao et al., 1998; Zhou et al., 1997). Thus, overexpression of phospholipid scramblase led to exposure of PS on the external surface of the plasma membrane, accompanied by increased cellular susceptibility to sPLA2-IIA, even without cell activation (Murakami et al., 1999c). However, it still remains uncertain whether the phospholipid scramblase–dependent process occurs under physiological conditions. Damaged or apoptotic cell membranes (Atsumi et al., 1997) and microvesicles shed from activated cells (Fourcade et al., 1995), in which PS is exposed to the external surface (Martin et al., 1996), are potential targets for exogenous sPLA2-IIA. c. Oxidation of cPLA2-Derived Products. Several lines of evidence have suggested that cPLA2 is required in order for sPLA2-IIA (and sPLA2-V; see below) to act properly (Kambe et al., 1999; Kuwata et al., 1998; Murakami et al., 1998). Supporting this idea are the observations that sPLA2-IIA-dependent AA release was blocked by cPLA2 inhibitors and restored by supplementation with exogenous AA, and that cotransfection of cPLA2 and sPLA2-IIA augmented AA release in a synergistic manner. cPLA2 is also required in several cell types for the induction of sPLA2-IIA at the transcriptional level by cytokines (Couturier et al., 1999; Kuwata et al., 1998). In the search for a regulatory molecule that links cPLA2 and sPLA2-IIA, 12/15LOX, a LOX isozyme that oxygenates free AA as well as esterified polyunsaturated fatty acids in the cellular membranes (Brash, 1999), has been found to play a pivotal role in the regulation of sPLA2-IIA (Kuwata et al., 2000). 12/15-LOX regulation of sPLA2-IIA occurs in two ways. First, 12/15-LOX–oxidized lipids sensitize the cellular membranes to be more susceptible to sPLA2-mediated AA release. This is compatible with the observations that sPLA2-IIA–mediated hydrolysis is accelerated by chemical oxidization of membranes (Akiba et al., 1997). Second, 12/15-LOX products up-regulate the induction of sPLA2-IIA expression. As the ligands for the nuclear receptor PPAR␥ are fatty acids and their

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oxidized derivatives, including 12/15-LOX metabolites (Huang et al., 1999), the putative PPAR␥ -binding site in the sPLA2-IIA gene promoter region has been suggested to be involved in 12/15-LOX–mediated regulation of sPLA2-IIA expression (Couturier et al., 1999). Thus, cPLA2 activation immediately after cell activation leads to production of 12/15-LOX metabolites, which in turn trigger sensitization of the membranes to be susceptible to sPLA2-IIA–mediated AA release. The AA thus released is further oxidized by 12/15-LOX and contributes to amplification of sPLA2IIA gene transcription and the membrane rearrangement process, leading to sustained activation of sPLA2-IIA and the attendant delayed generation of PG. This scenario has revealed a functional array of enzymes in separate arms of the AA cascade, the LOX and COX pathways, and the biological importance of LOX-directed lipid oxidation signaling in regulating the expression and function of particular lipid-metabolizing enzymes. 3. Antimicrobial Activity Phagocytosis of gram-negative Escherichia coli by neutrophils, which is an essential first-line defense against invading bacteria, triggers bacterial envelope phospholipid degradation; the extent of intracellular destruction of these ingested bacteria is closely linked to the magnitude of PLA2 action (Elsbach and Weiss, 1988). sPLA2-IIA participates in intracellular bacterial digestion by associating with the surfaces of bacteria and neutrophils before phagocytosis and acting after co-internalization with ingested bacteria (Wright et al., 1990). Although sPLA2-IIA alone cannot manifest antimicrobial activity directly by degrading the phospholipids in intact bacterial membranes, in combination with a neutrophilderived protein called bactericidal permeability–increasing protein (BPI) it reduces the viability of microorganisms (Wright et al., 1990). In the presence of BPI, phospholipids in intact bacteria are hydrolyzed by sPLA2-IIA both extracellularly and intracellularly. sPLA2-IB is unresponsive to BPI, although both isozymes show virtually the same activity toward E. coli phospholipids after the bacterial structure has been altered by autoclaving or after extraction. The clusters of basic residues (Arg7, Lys10, and Lys15) in the N-terminal region of human sPLA2-IIA may account, in part, for its ability to act on BPI-treated bacterial membranes (Weiss et al., 1991). The close correlation between the effects of these mutations on BPI-dependent PLA2 binding and hydrolytic activity toward E. coli suggests that a major role of these basic residues is to mediate electrostatic interactions with acidic bacterial envelope sites that become available after BPI treatment, as well as hydrogen bond–mediated interactions. The bactericidal activity of intestinal sPLA2-IIA, which is produced by Paneth cells, may play a crucial role in protecting the small intestinal crypts from microbial invasion (Harwig et al., 1995).

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A biological role for sPLA2-IIA in contributing to the antimicrobial arsenal mobilized by the host in response to invading microorganisms has been established by several studies using sPLA2-IIA transgenic mice. These mice exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia (Grass et al., 1996). The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. After the administration of Staphylococcus aureus or E. coli, the transgenic mice showed reduced mortality, improved resistance, and increased bacterial killing (Laine et al., 1999, 2000). 4. Anticoagulation The anticoagulant activities of the sPLA2 family depend on the presence of basic residues within a specific variable surface region (residues 54–77) distinct from both the conserved catalytic machinery and the surface sites mediating the antimicrobial action of these enzymes (Kini and Evans, 1987). Substitution of Lys56 by Gln in human sPLA2-IIA reduced, whereas that of Asp59 by Arg and Ser60 by Gly in porcine sPLA2-IB increased, the anti-prothrombinase activity of the enzyme (Inada et al., 1994), providing the first direct evidence for a role of basic residues within this region in the effects of sPLA2 against reactions that promote coagulation. The anticoagulation effect can occur independently of the presence of phospholipids but requires factor Va, leading to the hypothesis that sPLA2-IIA inhibits this factor (Mounier et al., 1996). In addition to the noncatalytic and phospholipid-independent component, the degradation of PS, an essential component of the prothrombinase complex, also plays an important role in the inhibition of prothrombinase by sPLA2IIA. The role of sPLA2-IIA released from activated platelets is more likely to regulate the clotting reaction negatively by degrading PS, thereby suppressing prothrombinase complex formation on platelet membranes and microvesicles (Fourcade et al., 1995; Yokoyama et al., 1995), rather than by contributing to thromboxane A2 biosynthesis, leading to acceleration of coagulation (Mounier et al., 1993). sPLA2-IIA also potentiates PGI2 generation by vascular endothelial cells (Murakami et al., 1993a), further implying its potential role as a negative regulator of blood coagulation. 5. Degranulation Several pharmacological and immunochemical studies have suggested the participation of sPLA2 in exocytosis of several endocrine cells, including mast cells and chromaffin cells (Matsuzawa et al., 1996; Murakami et al., 1992). sPLA2-IIA added exogenously at high concentrations directly elicited degranulation of rat peritoneal mast cells (Murakami et al., 1993b). Direct evidence for the involvement of sPLA2-IIA in mast cell degranulation has been provided

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by transfection studies, where immunoglobulin E–dependent degranulation of rat mastocytoma RBL-2H3 cells was markedly augmented by overexpression of sPLA2-IIA (Enomoto et al., 2000a). sPLA2-IIA, which is stored in secretory granules in unstimulated cells, accumulates on the membranous sites where the fusion between the plasma membrane and granule membranes occurs in activated cells. As the lysophospholipid perturbs the structure of bilayer membranes (Karli et al., 1990), its production around the opening granular membranes by the enzymatic action of sPLA2-IIA may facilitate the ongoing membranefusion. 6. Pathology a. Inflammation. Detection of high levels of sPLA2-IIA at various inflamed sites suggests that it is involved in pathogenesis of the inflammatory responses (Kudo et al., 1993). The pathological roles of sPLA2-IIA in local inflammatory processes have been confirmed by injecting purified or recombinant sPLA2-IIA into different sites of experimental animals (Bomalaski et al., 1991; Murakami et al., 1990) or by administering particular inhibitors or antibodies that are fairly specific for sPLA2-IIA into various inflamed sites (Fleisch et al., 1996; GarciaPastor et al., 1999; Kakutani et al., 1994; Marshall et al., 1995; Snyder et al., 1999; Tanaka et al.,1994). It should be noted, however, that the latter observations may be due to inhibition of sPLA2 isozymes other than sPLA2-IIA, particularly in studies using mice as an experimental model, where the expression of sPLA2-IIA is limited to the intestine, whereas that of sPLA2-V is ubiquitous and inducible (Sawada et al., 1999). b. Ischemia. PLA2-induced changes in phospholipid integrity and the toxic actions of free fatty acids and lysophospholipids may be critical for the altered plasma membrane and mitochondrial permeability properties and bioenergetic capacity associated with ischemia and perfusion. Activation of sPLA2-IIA is associated with ischemia and related tissue injury (Hatch et al., 1993; KikuchiYanoshita et al., 1993; Koike et al., 1995; Lauritzen et al., 1994). Antibodies against sPLA2-IIA prevented renal injury due to ischemia and reperfusion in rats (Takasaki et al., 1998). c. Atherosclerosis. Studies using sPLA2-IIA transgenic mice have revealed its unexplored role in the development of atherosclerosis (Ivandic et al., 1999; Leitinger et al., 1999; Tietge et al., 2000). The transgenic mice exhibited increased atherosclerotic lesions, around which sPLA2-IIA accumulated. sPLA2IIA may promote atherogenesis, in part, through decreasing high-density lipoprotein levels. Furthermore, the levels of biologically active oxidized phospholipids are increased in sPLA2-IIA transgenic mice. The correlation between the expression of sPLA2-IIA and the degree of atherosclerosis underlines its possible importance in atherogenesis (Schiering et al., 1999).

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d. Cancer. The sPLA2-IIA gene is naturally disrupted by a frameshift mutation in exon 3 in some inbred mouse strains (Kennedy et al., 1995; MacPhee et al., 1995). The lack of observable differences in the physiology and pathology of sPLA2-IIA–deficient and normal mouse strains suggests that sPLA2s are redundant in mammals and that other isozymes can compensate for loss of sPLA2-IIA. Mutations in the APC gene are responsible for various familial and sporadic colorectal cancers. Min mice carrying a dominant mutation in the homologue of the APC gene develop multiple adenomas throughout their small and large intestines. Of particular importance, the gene for mouse sPLA2-IIA maps to the Mom1 locus in the distal region of chromosome 4 that dramatically modifies Min-induced tumor number (MacPhee et al., 1995). Thus, sPLA2-IIA is a candidate for modifier of polyp numbers by altering the cellular microenvironment within the intestinal crypts. The most likely explanation for this is that the presence of wild-type sPLA2-IIA activity confers resistance to multiple adenoma formation, whereas the truncated product has no effect on tumor formation. In marked contrast to the situation in the mouse model, however, sPLA2-IIA gene mutations do not appear to play a major role in the development of colorectal cancer in humans (Riggins et al., 1995). Rather, the high level of sPLA2-IIA expression often detected in human familial adenomatous polyposis is more likely to contribute to the elevated levels of AA found in colorectal cancer and, in conjunction with the elevated expression of COX-2, which is crucially involved in tumorigenesis (Oshima et al., 1996), could be another factor in tumor formation (Kennedy et al., 1998). In relation to this, overexpression of sPLA2-IIA prevents apoptosis in certain cell lines (Zhang et al., 1999). Although the reason that sPLA2-IIA has an antitumorigenic potential in mice is still unclear, mechanisms other than prostanoid generation may be involved. The lack of bactericidal sPLA2-IIA in the intestine may enable proliferation of certain types of bacteria that produce carcinogenic products facilitating the formation of polyps and malignant transformation. Alternatively, loss of asymmetry in colon cancer cells leads to increased accessibility to cell membrane phospholipids, providing a suitable target for sPLA2-IIA membrane hydrolysis. In support of this idea, cotransfection of sPLA2-IIA and phospholipid scramblase, which disturbs membrane asymmetry, into 293 cells led to marked reduction in cell growth, accompanied by increased membrane hydrolysis (Murakami et al., 1999c). C. SPLA2-V 1. Expression sPLA2-V appears to be a primary sPLA2 isozyme in mice, where its mRNA is detected in a wide variety of tissues and cells, whereas its expression is rather restricted to the heart and, to a lesser extent, to the lung in humans and rats (Chen et al., 1994a; Sawada et al., 1999). Conversely, sPLA2-IIA is widely distributed in humans and rats, whereas it is detected only in the intestine of mice. Injection of LPS increases sPLA2-V expression in various organs in mice and in the heart in

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rats (Sawada et al., 1999). Stimulation of mouse macrophage-like P388D1 cells with LPS (Shinohara et al., 1999) and mouse cultured mast cells with particular combinations of cytokines (Sawada et al., 1999) results in marked up-regulation of sPLA2-V expression. Therefore, it is likely that sPLA2-V in mice takes the place of sPLA2-IIA in rats and humans under various conditions. 2. Functions sPLA2-V is constitutively expressed in mouse macrophage-like P388D1 cells (Balboa et al., 1996; Balsinde and Dennis, 1996; Balsinde et al., 1998) and mouse mast cells (Reddy and Herschman, 1997; Reddy et al., 1997; Sawada et al., 1999), in which it plays a role in augmentation of immediate PGE2 production induced by PAF following LPS priming and immunoglobulin E–dependent immediate PGD2 production, respectively. Inducible sPLA2-V promotes LPS-induced delayed PGE2 generation in P388D1 cells (Shinohara et al., 1999). Transfection of sPLA2-V into 293 cells and Chinese hamster ovary (CHO) cells led to increases in both the immediate and delayed phases of AA release elicited by appropriate stimuli (Murakami et al., 1998). AA released by sPLA2-V is converted to PGs via both COX-1 and COX-2 in the immediate response and predominantly by COX-2 in the delayed response (Balsinde et al., 1998; Murakami et al., 1999a; Reddy and Herchman, 1997; Reddy et al., 1997; Sawada et al., 1999; Shinohara et al., 1999). sPLA2-V is also capable of promoting transcellular PG biosynthesis (Murakami et al., 1999a; Reddy and Herschman, 1996). Prior activation of cPLA2 is necessary for sPLA2-V to act (Balsinde et al., 1998; Shinohara et al., 1999). All of these functional features of sPLA2-V are very similar to those of sPLA2-IIA (see above). Moreover, both sPLA2-IIA and -V have the ability to increase COX-2 expression, which further contributes to augmentation of delayed PG generation (Balsinde et al., 1999; Murakami et al., 1999c; Tada et al., 1998). Consistent with these close similarities, sPLA2-V, like sPLA2-IIA, utilizes the glypican-dependent route for the promotion of delayed PG biosynthesis (Murakami et al., 1999a, 2001). Collectively, these findings imply that both isozymes are functionally compensatory. However, several studies have suggested that the functions of sPLA2-IIA and -V are not perfectly identical. When sPLA2-V was overexpressed in RBL2H3 cells, it markedly augmented immunoglobulin E–dependent immediate PGD2 and leukotriene C4 generation and degranulation, whereas sPLA2-IIA augmented degranulation (see above) without affecting eicosanoid biosynthesis (Enomoto et al., 2000a; Murakami et al., 2001; Sawada et al., 1999). Exogenously added sPLA2-V, but not -IIA, directly promoted leukotriene B4 generation in human neutrophils (Han et al., 1999). In mouse P388D1 macrophages, exogenous sPLA2-V was capable of eliciting AA release even without additional stimuli (Balsinde et al., 1999), whereas exogenous sPLA2-IIA required appropriate costimulators to do so (Balsinde et al., 1998). The more potent eicosanoidbiosynthetic action of sPLA2-V than that of sPLA2-IIA in these settings is most

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probably because the former enzyme has much higher PC-hydrolytic activity than the latter (Han et al., 1999). Thus, when acting on the outer leaflet of the plasma membrane independently of glypican, the cellular action of sPLA2-V depends essentially on its interfacial binding ability to PC, on which sPLA2-IIA is unable to act. In this regard, sPLA2-V behaves like sPLA2-X (see below). In mouse mast cells, sPLA2-IIA is stored in secretory granules, whereas sPLA2-V is distributed mainly in intracellular compartments such as the Golgi apparatus and perinuclear membranes (Bingham et al., 1999). Such a different subcellular localization in the same cell also suggests their functional segregation under certain conditions. D. OTHER MEMBERS OF THE GROUP II SUBFAMILY OF SPLA2S sPLA2-IIC is expressed in rat and mouse testis, but is a pseudogene in humans (Chen et al., 1994b; Tischfield, 1997). In mouse testis, sPLA2-IIC is expressed in cells undergoing meiosis, including pachytene spermatocytes, secondary spermatocytes, and round spermatids (Chen et al., 1997). sPLA2-IIC has little affinity for heparanoids and is unable to promote AA release when overexpressed in 293 cells (Murakami et al., 1998). sPLA2-IID, -IIE, and -IIF were identified during the searches of DNA databases for expressed sequence tags representing parts of genes for sPLA2 homologues. sPLA2-IID shows high homology (48%) with sPLA2-IIA, and its expression is detected in several tissues, including spleen, thymus, skin, lung, ovary, and colon (Ishizaki et al., 1999; Valentin et al., 1999a). The expression of sPLA2-IID is elevated in the thymus after treatment with LPS (Ishizaki et al., 1999). As in the case of sPLA2-IIA and -V, overexpression of sPLA2-IID in 293 cells augments agonist-induced immediate and delayed AA release and attendant PGE2 generation, which occurs through the glypican shuttling mechanism (Murakami et al., 2001). sPLA2-IIE shows the highest homology (51%) with sPLA2-IIA (Suzuki et al., 2000; Valentin et al., 1999b). The enzymatic properties of human sPLA2-IIE are almost identical to those of sPLA2-IIA and -IID, whereas the mouse orthologue exhibits very low enzymatic activity. In contrast to the broad expression profiles of sPLA2-IIA and -IID, the expression of sPLA2-IIE is restricted to the brain, heart, placenta, and uterus and is markedly enhanced in the lung and intestine following LPS challenge. sPLA2-IIE is expressed in alveolar macrophages in the lungs of LPS-treated mice. The genomic organizations of sPLA2-IIA, -IID and -IIE are almost identical, revealing their evolutionary conservation (Suzuki et al., 2000). sPLA2-IIF, which has a unique long C-terminal extension, is strongly expressed during embryogenesis and in adult testis of mice (Valentin et al., 1999b). Like the other members of the group II subfamily of sPLA2s, sPLA2-IIF hydrolyzes anionic phospholipids in preference to PC in vitro. The functions of sPLA2-IIE and -IIF have yet to be elucidated.

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E. SPLA2-X 1. Expression sPLA2-X is produced as a zymogen with weak catalytic activity, and a propeptide composed of 11 amino acids is removed during the secretion process to produce an active mature enzyme (Hanasaki et al., 1999). Human sPLA2-X is expressed in several organs and tissues related to the inflammatory response, such as spleen, thymus, and peripheral blood leukocytes (Cupillard et al., 1997). In human lung, sPLA2-X is expressed in alveolar epithelial cells (Hanasaki et al., 1999). In contrast, its expression in mice is restricted to the testis and the stomach (Valentin et al., 1999b). 2. Functions sPLA2-X is able to hydrolyze PC much more efficiently than any other mammalian sPLA2 isozymes identified to date (Bezzine et al., 2000; Hanasaki et al., 1999; Murakami et al., 1999c). Although sPLA2-X does not bind heparan sulfate appreciably and is therefore secreted into the supernatant without being retained on cell surfaces when overexpressed in 293 cells, it is capable of promoting fatty acid release even in the absence of stimulus (Murakami et al., 1999c). Exogenous sPLA2-X is also strongly active on mammalian cells, releasing fatty acids even from cells that are refractory to sPLA2-IB, -IIA, -IID and even -V (Bezzine et al., 2000; Hanasaki et al., 1999). The cellular action of sPLA2-X apparently occurs through randomly hydrolyzing PC in the outer leaflet of the plasma membrane. The AA released by sPLA2-X is converted to PGE2 mainly via COX-2 (Bezzine et al., 2000; Murakami et al., 1999c). F. SPLA2-III So far, structurally related groups I and II sPLA2s have been found in vertebrates such as mammals and snakes, whereas group III sPLA2s have mainly been found in venom from invertebrates such as bees and scorpions. cDNA coding for a novel human sPLA2 that displays 31% homology with bee venom group III enzyme has been identified (Valentin et al., 2000). The full-length human sPLA2-III cDNA codes for a signal peptide of 19 residues followed by a protein of 490 amino acids made up of a central sPLA2 domain (141 residues) flanked by large N- and C-terminal regions (130 and 219 residues, respectively). The sPLA2 domain displays all of the features of group III sPLA2s, including 10 cysteines. The sPLA2-III gene maps to chromosome 22. The sPLA2-III transcript is found in kidney, heart, liver, and skeletal muscle. sPLA2-III shows an 11-fold preference for phosphatidylglycerol over PC and optimal activity at pH 8. The function of sPLA2-III remains to be elucidated.

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IV. sPLA2 Receptors

Venom sPLA2s display different types of toxicity, including neurotoxicity, myotoxicity, and anticoagulant and proinflammatory effects, and these varying effects are linked to the existence of a variety of high-affinity receptors for these toxic enzymes. The N-type sPLA2 receptor, initially identified in brain and then in other tissues, recognizes several neurotoxic sPLA2s for which it shows high affinity (Lambeau et al., 1989). The M-type receptor represents another class of sPLA2 receptor, initially identified in skeletal muscle, for myotoxic sPLA2s (Lambeau et al., 1990). The M-type sPLA2 receptor is a 180-kDa type I transmembrane protein with an NH2-terminal cysteine-rich domain, a fibronectin type II domain, eight repeats of a carbohydrate recognition domain (CRD), and transmembrane and cytoplasmic domains, and its entire structure is related to the macrophage mannose receptor (Ishizaki et al., 1994; Lambeau et al., 1994). These characteristics are the first definitive demonstration that sPLA2 has a function unrelated to its phospholipid-hydrolyzing activity. The rabbit sPLA2 receptor binds both human sPLA2-IB and -IIA, and the mouse receptor binds both mouse sPLA2-IB and IIA with high affinity; this contrasts with the receptors of other animal species, including humans, rats, and cows, which are fairly specific for sPLA2-IB (Cupillard et al., 1999). sPLA2-X reportedly acts as a ligand for the receptor (Morioka et al., 2000) whereas sPLA2-IID does not (Valentin et al., 1999a). However, whether other sPLA2 isozymes bind to this receptor is still largely obscure. Alternative splicing of the receptor transcript results in production of a soluble sPLA2 receptor that lacks a transmembrane domain, but still binds to sPLA2-IB with high affinity (Ancian et al., 1995). The human sPLA2 receptor gene maps in the q23–q24 bands of chromosome 2 (Ancian et al., 1995) and has a similar exon–intron structure to the mannose receptor gene (Ishizaki et al., 1994; Lambeau et al., 1994). sPLA2 receptor mRNA is expressed in several tissues, including pancreas, liver, lung, kidney, and spleen, where sPLA2-IB is detectable, although the tissue distributions of the receptor and ligand differ considerably among animal species (Higashino et al., 1994). The N-terminal region of the M-type receptor, including the CRD, is responsible for binding of sPLA2-IB (Kd ∼ 1–10 nM). The domains surrounding CRD4 to CRD6, particularly CRD5, are essential for sPLA2 binding to its receptor (Nicolas et al., 1995). Residues within or close to the Ca2+-binding loop (Gly30, Leu31, and Asp49) of sPLA2-IB are crucial for the binding step, although the presence of Ca2+, which is essential for enzymatic activity, is not required for binding to the receptor (Lambeau et al., 1995). The N-terminal ␣-helices and the pancreatic loop are not essential for binding of sPLA2-IB to the receptor. The sPLA2 receptor undergoes rapid internalization, which is mediated by a clathrin-coated pit and independent of ligand binding (Zvaritch et al., 1996).

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The NSYY motif in the cytoplasmic domain encodes the major endocytic signal, with the distal tyrosine residue playing the key role. Although the importance of sPLA2 receptor internalization remains obscure, it has been proposed that it terminates the signals produced by sPLA2 on target cells, that it serves as a system delivering sPLA2 to specific intracellular components where the ligand can manifest its enzymatic activity, and that it serves a clearance function by selectively removing sPLA2 from the extracellular fluid. M-type sPLA2 receptor knockout mice are viable, fertile, and without evident histopathological abnormalities (Hanasaki et al., 1997). There is no difference in clearance of circulating sPLA2-IB. After challenge with LPS, these mice exhibit longer survival than wild-type mice. The increase in tumor necrosis factor and IL-1 in plasma after LPS treatment is significantly attenuated in the mutant mice. These findings suggest a potential role of sPLA2 receptor in the progression of endotoxin shock. Cross-talk of sPLA2-IB to other PG-biosynthetic enzymes via the M-type sPLA2 receptor, leading to enhanced PG biosynthesis, has been demonstrated (Kishino et al., 1994; Tohkin et al., 1993). sPLA2-IB elicited PGE2 generation by inducing COX-2 in osteoblastic cells (Tohkin et al., 1993) and sPLA2-IIA in renal mesangial cells (Kishino et al., 1994). Activation of cPLA2 by sPLA2s via the sPLA2 receptor–mediated process has been also suggested (Fonteh et al., 1998; Hernandez et al., 1998). Therefore, sPLA2-IB–induced PG biosynthesis depends on de novo protein synthesis and transmembrane signaling through its receptor, rather than direct hydrolysis of membrane phospholipids by its own enzymatic activity. The molecular nature of the N-type sPLA2 receptor is unclear. Photoaffinity labeling and chemical cross-linking techniques have identified a few binding proteins for some snake venom neurotoxic sPLA2s, and one subunit of the binding proteins for several venom sPLA2s is a 45-kDa polypeptide distributed preferentially in neuronal membranes (Yen and Tzeng, 1991). Tyr22 of neurotoxic sPLA2s is essential for the enzymes to bind to this 45-kDa protein, to which sPLA2-IB cannot bind, but substitution of Phe22 by Tyr resulted in sPLA2-IB’s acquiring the capacity to do so (Tzeng et al., 1995). More recently, a novel sPLA2 receptor immunochemically distinct from the M-type receptor has been identified and partially purified from porcine cerebral cortex (Copic et al., 1999). V. Conclusion

With the cloning of ten sPLA2s, it is obvious that a diversity of sPLA2s exists in mammals. These sPLA2s exhibit different tissue distributions and inducibility by stimuli, which also differ according to animal species. The expression levels of these sPLA2s are not the same; to date, numerous expressed sequences tags have been identified for sPLA2-IIA and -IB (Valentin et al., 1999b), indicating

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that these two isozymes are widespread and overwhelming in amount compared with the other recently identified isozymes in rats and humans. The functions of sPLA2s are to regulate the release of lipid mediators in different tissues and cells, acting on various phospholipid substrates, extracellularly or within different cellular compartments, and under physiological and pathological conditions. In regulating AA release from live cells, sPLA2s can utilize both the heparan sulfate–dependent and lipid interface–dependent pathways according to the type and activation state of the cell, as well as the molecular properties, subcellular locations, and dynamics of each isozyme. Moreover, sPLA2s can function not only as enzymes but also as ligands. Further work is clearly needed to understand the biological functions of the different members of the growing family of sPLA2s. REFERENCES Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus, W., Schneider-Mergener, J., and Kronke, M. (1996). FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86, 937–947. Akiba, S., Nagatomo, R., Hayama, M., and Sato, T. (1997). Lipid peroxide overcomes the inability of platelet secretory phospholipase A2 to hydrolyze membrane phospholipids in rabbit platelets. J. Biochem. (Tokyo) 122, 859–864. Ancian, P., Lambeau, G., Mattei, M. G., and Lazdunski, M. (1995). The human 180–kDa receptor for secretory phospholipases A2: Molecular cloning, identification of a secreted soluble form, expression, and chromosomal localization. J. Biol. Chem. 270, 8963–8970. Anderson, R. G. W. (1998). The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225. Atsumi, G., Murakami, M., Tajima, M., Shimbara, S., Hara, N., and Kudo, I. (1997). The perturbed membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A2 to liberate arachidonic acid. Biochim. Biophys. Acta 1349, 43–54. Baker, S. F., Othman, R., and Wilton, D. C. (1998). Tryptophan-containing mutant of human (group IIa) secreted phospholipase A2 has a dramatically increased ability to hydrolyze phosphatidylcholine vesicles and cell membranes. Biochemistry 37, 13203–13211. Balboa, M. A., Balsinde, J., Winstead, M. V., Tischfield, J. A., and Dennis, E. A. (1996). Novel group V phospholipase A2 involved in arachidonic acid mobilization in murine P388D1 macrophages. J. Biol. Chem. 271, 32381–32384. Balsinde, J., and Dennis, E. A. (1996). Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. J. Biol. Chem. 271, 6758–6765. Balsinde, J., Balboa, M. A., and Dennis, E. A. (1997). Antisense inhibition of group VI Ca2+independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages. J. Biol. Chem. 272, 29317–29321. Balsinde, J., Balboa, M. A., and Dennis, E. A. (1998). Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2. Proc. Natl. Acad. Sci. USA 95, 7951–7956. Balsinde, J., Shinohara, H., Lefkowitz, L. J., Johnson, C. A., Balboa, M. A., and Dennis, E. A. (1999). Group V phospholipase A2–dependent induction of cyclooxygenase-2 in macrophages. J. Biol. Chem. 274, 25967–25970. Barbour, S. E., and Dennis, E. A. (1993). Antisense inhibition of group II phospholipase A2 expression blocks the production of prostaglandin E2 by P388D1 cells. J. Biol. Chem. 268, 21875– 21882.

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ADVANCES IN IMMUNOLOGY, VOL. 77

The Antiviral Activity of Antibodies in Vitro and in Vivo PAUL W. H. I. PARREN AND DENNIS R. BURTON Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

I. Introduction

Antibodies can display antiviral activities in vivo and in vitro. Since experimentation is far easier in vitro, researchers have long sought to develop in vitro assays that will predict activity in vivo. Furthermore, successful prediction implies that one is studying the mechanisms that are operative in vivo, and therefore one can hope to manipulate antibody activity. This could be important in both vaccine design and in passive antibody administration. However, despite a large body of work, there are still major controversies in the area. Many of these spring from fundamentally different views of how antibodies neutralize viruses. The proposed mechanisms of in vitro neutralization range from those requiring binding of a single antibody molecule to virus to those requiring substantially complete antibody coating of virus. The relationship between binding to virus and neutralization has attracted controversy, with some researchers arguing for binding but nonneutralizing antibodies, and others dissenting. The relationship between neutralization in vitro and protection in vivo has attracted much attention, but still one hears debate about whether neutralization in vitro is an absolute requirement for efficacy in vivo. Of course, all of these considerations can vary from one set of circumstances (virus, antibody, cells used in vitro, animals used in vivo, etc.) to another, so one can question whether any general rules can be established. We believe that some general rules can indeed be established. We begin with the viewpoint that antibodies are not molecules like enzymes that have evolved over long time periods for narrow, highly specialized functions. Rather, they are the products of mutation and selection that are generated afresh in each individual animal as a result of antigen challenge. Each antibody, even directed to the same epitope, has a different sequence and a different detailed mode of binding to antigen. Such molecules are not, for example, likely to trigger common conformational changes in the antigen. They can cross-link antigen molecules, but otherwise their modus operandi is likely to be through their bulk and through the ability of the Fc region, in an array, to trigger effector systems. In other words, in the first instance, we look for antibodies to function antivirally by binding to virus or infected cells and blocking steps in the infection process by nonspecific steric interference or by associating on the virusinfected cell surface and triggering effector functions such as complement and 195 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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antibody-dependent cellular cytotoxicity (ADCC). If “straightforward” explanations for antibody activity fail, then more complex explanations can be considered. It is in this context that we present this review of the literature on the in vitro and in vivo antiviral activities of antibodies. We have included data from early studies up to the present day, as we believe that some early studies have been misinterpreted, and the misconceptions have been propagated ever since. In vivo, it is conventional to distinguish phenomenologically between two types of antibody antiviral activity. One is the ability of antibody to protect against infection when it is present before or immediately following infection. The other is the ability of antibody to interfere with an established infection. There is good reason to make this distinction because there is strong evidence that antibody is effective against many different viruses in a protective role, but there is more ambiguity about a role in ongoing infection. The distinction may have its origins, at least in part, in the activity of antibody against propagation of infection by free virus compared to its activity against propagation via cell-to-cell spread. Evidence for a number of viruses in vitro indicates that lower antibody concentrations are required to inhibit infection propagated by free virus than are required to inhibit infection propagated by cell-to-cell spread. In vitro, antiviral activity can be separated into activity against virions and activity against infected cells. The activity against virions most often considered is neutralization, which can be defined as the loss of infectivity which ensues when antibody molecule(s) bind to a virus particle, and usually occurs without the involvement of any other agency. As such this is an unusual activity of antibody paralleled only by the inhibition of toxins and enzymes Dimmock, 1995

Antibody-dependent complement-mediated virolysis is also an activity directed against free virions. Antibodies can also bind to viral products on infected cells to trigger effector functions, including activation of complement and ADCC. Antibody-mediated phagocytosis can be directed against free virions as well as some infected cells. In addition, the binding of antibody to infected cells has been proposed to inhibit intracellular viral replication in certain cases. We begin by considering the in vitro antiviral activities of antibody.

II. Mechanisms of Neutralization

A. INTRODUCTION Neutralization of viruses by antibody has been extensively studied during the last century, and many mechanisms for neutralization have been proposed (reviewed in Burnet et al., 1937; Daniels, 1975; Della-Porte and Westaway, 1977; Dimmock, 1993; Fazekas de St.Groth, 1962; Parren et al., 1999). These mechanisms include aggregation, inhibition of viral entry by inhibition of

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attachment and inhibition of fusion with the target cell, as well as post entry mechanisms, such as interference with primary and secondary uncoating of the genetic information of the virus (e.g., Dimmock, 1993). It has been suggested that some antibodies may neutralize by acting via several mechanisms simultaneously or sequentially (Dimmock, 1993). Kinetic studies of virus neutralization have been interpreted to indicate that neutralization often follows single- or few-hit kinetics in which the binding of a single or a few antibodies triggers an event leading to virion inactivation (Dimmock, 1993; Dulbecco et al., 1956; Mandel, 1976; McLain and Dimmock, 1994). To explain single-hit neutralization of viruses in cases where a number of antibody molecules are bound to a virus particle at the neutralization event [e.g., 4 antibody molecules per virion to poliovirus or 70 per virion in the case of influenza A (see below, and Icenogle et al., 1983; Taylor et al., 1987)], antibody-induced conformational changes of the viral capsid (Mandel, 1976) or the existence of antigenically identical “neutralization-relevant” and “neutralization-irrelevant” antibody binding sites on the virion surface have been proposed (Icenogle et al., 1983; Taylor et al., 1987). The myriad of proposed neutralization mechanisms, however, are somewhat difficult to reconcile with the nature of antibody generation and selection. Antibodies are superb binding molecules selected for highaffinity binding rather than for their ability to induce critical conformational changes in viral capsid proteins or envelope spikes. In this review, we propose a much more simplistic and unifying mechanism for neutralization. We suggest that neutralizing antibodies generally act by coating the surface of a pathogen, and that neutralized pathogens represent entities on which this coating has reached a critical density. This antibody coat then prevents the pathogen from interacting properly with the target cell, thereby interfering with the initiation of a productive infection. Differential effects of the antibody coat on distinct viruses, such as inhibition of attachment, viral entry, or apparent post entry effects, may be due to the epitope being recognized, but also may in part be explained by differences in virus biology rather than by the direct induction of specific events by the antibody. The mechanism suggests that so-called “neutralizing” and “nonneutralizing” epitopes do not exist as distinct entities on the viral surface, as has been suggested in the past (e.g., Dimmock, 1993). Neutralizing epitopes generally represent antibody-accessible structures on the virion surface which are present in a valence high enough that a critical antibody density, as will be calculated below, can be achieved. Low-density epitopes do not represent true nonneutralizing epitopes, as critical density could be achieved by a combination of two or more antibodies against several distinct low-density epitopes. Neo-epitopes which may exist only transiently on virions, for example during virus–target cell fusion, do not necessarily form an exception to the model discussed above. In our view, antibodies against such epitopes will only be neutralizing if the neo-epitope is accessible to antibody at some point during the virus infection process so that effective coating can occur.

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Neutralizing antibodies against such cryptic epitopes, however, appear to be relatively rare. B. ANTIBODY NEUTRALIZATION AND BINDING TO VIRUS Neutralization of a virus by antibody requires that the antibody bind to the coat of the virus. Therefore, antibody binding to virions may be a useful indicator of in vivo activity. In reality, the measurement of antibody binding to virions is often technically demanding. Furthermore, for many virus preparations, infectious particle:total particle ratios are low, introducing an element of uncertainty with regard to envelope homogeneity. Therefore, measurement of binding to envelope at the surface of infected cells has often been preferred. It should be noted, though, that some viruses express nonstructural or modified viral proteins on the infected-cell surface but not on virions. Such (non-neutralizing) antibodies are ineffective against free virus, but may be able to protect against viral infection in vivo by mediating the destruction of infected cells. This will be discussed in more detail below. For human immunodeficiency virus type 1 (HIV-1), a very good correlation has been described between neutralization and binding to envelope spikes on the surface of infected cells (Parren et al., 1998; Roben et al., 1994; Sattentau and Moore, 1995). However, problems can arise with the measurement of binding to envelope on cell surfaces. It is important to minimize these problems as much as possible by proper experimental design. First, envelope spikes such as those of HIV-1 can shed protein, providing opportunities for observation of antibodies that apparently bind well to the infected cell surface but do not bind to functional envelope spikes. Some antibodies against the HIV-1 transmembrane (TM) unit gp41 probably fall in to this category, as they may bind to inactivated envelope spikes in which gp41 is exposed, following shedding of the surface unit (SU) gp120. A second type of artifact in HIV-1 is provided by antibody binding to shed gp120 in interaction with the primary HIV-1 receptor CD4; antibodies apparently bind to the cells, but this does not reflect binding to envelope spikes. This is significant, as many nonneutralizing epitopes which are not accessible on the oligomeric HIV-1 envelope spike are exposed on monomeric gp120 (Moore et al., 1995; Parren et al., 1999). A third type of artifact may arise if envelope is overexpressed, for example, by expression of recombinant envelope glycoprotein under the control of a strong promotor. Thus it seems clear that the unprocessed HIV-1 envelope precursor glycoprotein gp160 can be expressed at cell surfaces under certain conditions (e.g., Dubay et al., 1995; Karlsson et al., 1996). Since many antibodies that do not bind to envelope spikes bind well to unprocessed gp160, there is again an opportunity for erroneous conclusions (Parren et al., 1997b). While there is general agreement that only anti-envelope antibodies that bind to the envelope spike on the virion will be neutralizing, there is considerably

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less agreement about whether all antibodies that bind to the envelope spike will neutralize virus, that is, are there antibodies that bind well to envelope spikes but do not neutralize, virus? The answer to the question has a number of important ramifications. If many antibodies that bind to envelope spikes do not neutralize, then binding is not necessarily a good indicator for likely antiviral efficacy. Further, there are implications for vaccine design. If all antibodies that bind also neutralize, then the envelope spike has the antigenic properties of an ideal vaccine candidate. On the other hand, if the envelope spike can induce nonneutralizing antibodies, it may not be an optimal antigen. In particular, the induction of nonneutralizing antibodies that can bind to envelope spikes and inhibit the binding of neutralizing antibodies would be undesirable. Our opinion on the issue of nonneutralizing but binding antibodies has been presented recently (Burton et al., 2000). We summarize here. We argue that overwhelmingly there is a good correlation between neutralization and virion binding with few exceptions. Our own experience in studies on human monoclonal antibodies (mAbs) to HIV-1, respiratory syncytial virus (RSV), and Ebola virus, has been that there is an excellent correlation between binding to envelope spikes and neutralization, which in these studies was measured as binding to infected cells. We have generally seen a close correlation between half-maximal antibody binding and antibody concentration required to give 50% neutralization, suggesting that neutralization is directly related to occupancy of sites on the virion. For HIV-1, neutralization is incremental with increasing antibody occupancy, irrespective of the epitope recognized, leading to increased inhibition of infectivity (Parren et al., 1998; Schønning et al., 1999). This issue is discussed in much greater detail below. Irrespective of our observations, there is a strong tradition in virology of “binding (i.e., to the virion) but nonneutralizing” antibodies. Early studies described a “nonneutralizable” fraction of virus which persisted even at high antibody concentrations. Addition of anti-antibody could reduce infectivity of this fraction. It seems that virus aggregation may have been partly responsible for this phenomenon (Wallis and Melnick, 1967). This will be discussed in more detail below. We also note that nonneutralizable fractions have been described for hepatitis A and hepatitis C as a result of virus association with lipids or lipoproteins (Lemon and Binn, 1985; Prince, 1994). Some descriptions of binding, nonneutralizing antibodies arose because of a failure to appreciate that antibodies that bind to isolated envelope molecules do not necessarily, and very often do not, bind to envelope spikes. A classic example is HIV-1, where many antibodies have been described which bind with high affinity to monomeric gp120 or unprocessed gp160; very few of these, however, show substantial affinity for envelope spikes (reviewed in Parren et al., 1999). Antibody-mediated enhancement of infection is a phenomenon that may support the existence of nonneutralizing but binding antibodies since the antibodies

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involved must bind to virions. However, a key observation here is that enhancement, when described, appears to occur for neutralizing antibodies at subneutralizing concentrations (e.g., Halstead, 1982; Morens et al., 1987). We favor the view that antibody enhancement is an effect that can arise under conditions of low-level antibody coating of virions rather than an indication of antibodies with the ability to bind well to virions but not neutralize. This is discussed in detail below. The phenomenon does suggest, however, a potential problem for some vaccines that induce low levels of neutralizing antibodies A clear example of good evidence for an antibody which binds well to virion envelope spikes without effecting neutralization, at concentrations where it would be expected to completely coat the virion, is provided from studies on rabies virus, as discussed later (Flamand et al., 1993). However, here it appears to be the rabies mutant virus rather than the antibody which has unusual properties. C. EARLY STUDIES ON ANTIBODY NEUTRALIZATION At the end of the nineteeth century, Willem Beijerinck made an important conceptional advance with the recognition that viruses, which he termed the “contagium vivum fluidum,” represent infectious entities distinct from bacteria (Beijerinck, 1898; Van Kammen, 1999). Great advances in understanding of the inactivation of viruses by antibody were then made in the early twentieth century. In a prominent article, Burnet and colleagues reviewed the state-ofthe-art of antibody neutralization in 1937 (Burnet et al., 1937). They argued that neutralization studies of bacteriophage, plant, and animal viruses showed that generally (i) neutralization was reversible, and (ii) more than one antibody was required for neutralization. Based on studies with a number of animal viruses including vaccinia and influenza, they summarized: “virus and antibody combined reversibly, and that the infectivity or otherwise of a given virus particle was determined by the amount of antibody united to it at the time of effective contact with the susceptible cell.” Reversibility indicated that the “union of antibody has no intrinsic inactivating effect on the virus.” The conclusions that neutralization is reversible and requires the binding of multiple antibody molecules to the virion have since been disputed (and rediscovered) in many later publications. In particular, the observation of single-hit kinetics in a number of experimental systems has been interpreted as evidence for neutralization by the binding of a single antibody to a critical site on the virion. In 1956, Jerne and Avegno showed that inactivation of T4 bacteriophage followed first-order kinetics, and inactivation may be the result of a single event. This was plausible, as neutralizing antibodies bind to the tail-plate of the phage, an essential component in the infective process, and the width of the tail-plate is similar to the length of an antibody molecule (Jerne and Avegno, 1956). An influential study which sharply contradicted the conclusions by Burnet et al. (1937) was published by Dulbecco and colleagues also in 1956, who analyzed

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neutralization of Western equine encephalitis virus (WEE) and poliovirus by specific antisera (Dulbecco et al., 1956). It was found that curves describing the logarithm of virus survival as a function of time generally did not display an initial shoulder or lag time, which was interpreted as neutralization following first-order kinetics. After an initial time period, however, the slope of the curves decreased, eventually becoming horizontal, which indicated the existence of a persistent and apparently neutralization-resistant fraction of virus. Dissociation of poliovirus-antibody complexes could not be observed, and there was only a very slight dissociation of WEE-antibody complexes. It was concluded that the attachment of a single neutralizing antibody molecule to a critical site is sufficient to inactivate a virus particle and that neutralization is irreversible (Dulbecco et al., 1956). However, not all results in the manuscript by Dulbecco et al. could be explained by the neutralization model they developed. First, a clear lag time of WEE neutralization was observed when kinetic neutralization experiments were performed at temperatures lower than 37◦ C. Even at 37◦ C, Dulbecco et al. noted that a small lag time may exist, although the experimental conditions used may have made detection difficult. Furthermore, multiplicity curves, which describe the logarithm of virus survival as a function of increasing antibody concentration, did not follow theoretical curves based on the model; experimental curves bent upward at high antibody concentration rather than downward. This deviation could not be completely explained by the persistent virus fraction. The authors concluded that different fractions may be present in the challenge stock consisting of virions with a different number of critical sites or unequal distribution of critical sites (Dulbecco et al., 1956). Krummel and Uhr (1969) recognized that the critical site neutralization models developed often did not fit the data at later time points during the course of neutralization in kinetic experiments. They argued that, for a highly symmetrical virus with a surface array of repeated coat-protein subunits such as the bacteriophage ␾X174 studied, it was likely that multiple antibody molecules were required for neutralization. Kinetic neutralization experiments, in which residual infectivity was assessed using two different methods, indeed indicated that bacteriophage covered with antibody may retain its infectivity. Significantly, they observed an apparent faster neutralization rate if residual infectivity was assessed using a decision tube method rather than the conventional direct plating method. In a kinetic neutralization experiment, antibody and phage are mixed, and samples, drawn at multiple time points, are incubated with bacteria and plated to assess residual infectivity. In the decision tube method, an excess of additional neutralizing antiserum is added a short time after adding the reaction sample to the bacteria. Whereas infectious titers determined with both methods were similar prior to the addition of neutralizing antibody, the decision tube method measured markedly decreased infectious titers compared to the

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conventional plating method at time points after the addition of antibody. As neutralization of ␾X174 phage was effectively irreversible under the conditions used, the difference observed could not be explained by the prevention of reactivation of neutralized phage (e.g., by dissociation of antibody–virus complexes). A plausible explanation is that some phage are partially coated with antibody molecules but not neutralized, and these phage infect bacteria at a slower rate than naked phage. A similar delay in the initiation of bacteriophage-induced lysis in neutralization experiments has previously been interpreted to indicate that partially antibody-coated but infectious phage particles exist (reviewed in Burnet et al., 1937). By entering an infectivity function for such phage–antibody complexes into the mathematical model developed by Krummel and Uhr (1969), the model better matched their data. This clearly indicated that the observed reaction rates of neutralizing antibody with bacteriophage were not first order, even under conditions of antibody excess (Krummel and Uhr, 1969). D. THE CASE OF PICORNAVIRUSES Several mechanisms have been described for the neutralization of picornaviruses, including aggregation, virion stabilization, inhibition of virus attachment, and induction of conformational changes in the viral capsid (see Smith, 2001, for a recent review). The induction of conformational changes (resulting in a large change in isoelectric point) upon antibody binding is the most prominent, and has prompted the most detailed investigations. Most studies have been carried out with poliovirus and human rhinovirus (HRV). Neutralization of poliovirus has been studied extensively. Dulbecco et al. (1956) argued that neutralization of poliovirus is irreversible and follows firstorder kinetics as discussed. Studies performed by Mandel (1961) confirmed that reactivation of virus infectivity after dilution of antibody–virus complexes could not be demonstrated in measurements taken over several months. The apparent irreversibility of the reaction appeared to be due, however, to a very tight binding of antibody to poliovirus rather than the induction of permanent changes in the viral particles, as it was demonstrated that neutralizing rabbit antibody could be eluted from poliovirions at low pH accompanied by quantitative reactivation of virus activity (Mandel, 1961). Mandel (1976) found that the capsid protein of poliovirus may exist in two conformational states, characterized by distinct isoelectric points: state A, with an isoelectric point of about 7, and state B, with an isoelectric point of about 4.5. Neutralizing antibody was found to stabilize the virion in state B. Mandel proposed that binding of neutralizing antibody locked the viral capsid in a noninfectious conformation by rendering it resistant to uncoating following its adsorption to the target cell. Single-hit kinetics in poliovirus neutralization could be explained by the induction of a generalized molecular rearrangement in the viral capsid after binding of a single antibody to a critical site on the capsid molecule. The mechanism is feasible, as the poliovirus capsid

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is a closely packed structure making conformational cooperativity among capsid subunits a possibility (Mandel, 1976). However, the relationship between the conformational state and infectivity was not complete, as infectious virus in the B state could be detected, particularly at low antibody multiplicity (even after subtracting the infectivity assigned to the small fraction of virus naturally occurring in the B state) (Mandel, 1976). A correlation between binding of neutralizing antibody to poliovirus and a shift in isoelectric point has generally held up in studies with monoclonal antibodies (Emini et al., 1983a), although exceptions have been noted (Brioen et al., 1985; Emini et al., 1983a). Changes in isoelectric point following the binding of neutralizing antibody have also been described for another picornavirus, namely HRV(Colonno et al., 1989). However, in this case it is particularly clear that the induced conformational changes do not play a major role in picornavirus neutralization (reviewed in Hewat and Blaas, 2001; Smith, 2001). The most convincing data come from cryo-electron microscopy (cryo-EM) and X-ray crystallography of virus–antibody complexes which show that rhinovirus can be neutralized without the induction of large conformational changes in the virion capsid (Hewat and Blaas, 1996; Smith, 2001; Smith et al., 1993, 1996). Furthermore, as the change in isoelectric point has been observed following binding of a large number of different antibodies against a range of epitopes on rhinovirus, it appears unlikely that all these antibodies would induce a common conformational change required for neutralization. The most likely hypothesis is that the changes in isoelectric point are the result of changes occurring in exposed highly flexible portions of the capsid proteins rather than structural rearrangement of the viral capsid (Che et al., 1998; Smith, 2001). Although, being the result of the binding of neutralizing antibody, these changes are probably mostly irrelevant to the mechanism of neutralization itself. The antibody activity most likely to affect HRV infectivity appears to be inhibition of virus attachment. The strongly neutralizing mAb 17-IA binds to an epitope located on a loop between the B and C strands of the capsid protein VP1 ␤-barrel and situated close to the canyon which harbors the binding site for the cellular HRV receptor ICAM-1 (reviewed in Smith, 2001). mAb 17-IA neutralizes by inhibition of attachment, and it has been demonstrated that attachment of 6–7 antibody molecules per HRV particle is required to reduce attachment by 63% (Smith et al., 1993). Significantly, Colonno et al. (1989) have shown that Fab fragments of antibodies against all four neutralizing epitopes on HRV inhibit attachment, some of which are located more-distal from the HRV receptor binding site. A linear correlation was found when inhibition of attachment was plotted as a function of antibody concentration. Furthermore, the rank order of neutralization potency followed the rank order of inhibition of attachment. The studies on HRV clearly demonstrate that the binding of more than one antibody molecule is required for neutralization, and suggest a role

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for antibody occupancy of binding sites on the virus, as will be discussed in the following sections. Further studies on the neutralization of poliovirus suggest that the neutralization of this picornavirus also requires the attachment of multiple antibody molecules. Particular insight into the relationship between antibody bound to poliovirus and neutralization was provided by Philipson and colleagues (1966) who developed an aqueous polymer phase system to study the interaction between neutralizing antibody and poliovirus. It was found that virions complexed with antibody could be separated from free virions by adding a polymer to the mixture, followed by a simple phase-separation technique. The phase system was more sensitive in detecting antibody binding to the virion compared to conventional neutralization tests, clearly indicating that neutralizing antibody was bound to the virions before neutralization could be detected. Interestingly, binding titers in the phase system method were 2–4 times greater than the serum neutralization titers against poliovirus (Philipson et al., 1966). The significance of this number will become clear shortly. Additional studies by Philipson on the interaction of antibody with poliovirus using countercurrent distribution, furthermore, showed that at low antibody input, virion–antibody complexes separated from free virus retained infectivity (Philipson, 1966). The neutralizing antibody dose is defined as antibody concentration at which virus infectivity is reduced to 37% (1/e) of the infectivity of the nonneutralized virus stock. Icenogle et al. (1983) determined the neutralizing dose for poliovirus in a study using radiolabeled virus and antibody, and demonstrated that the binding of an average of 4–5 antibody molecules to a poliovirus particle resulted in neutralization. Two neutralization models were proposed to explain these results in the context of first-order kinetics: a critical site hypothesis, in which binding to one out of four sites on the virus leads to neutralization, and a stepwise model, in which binding to each site reduces infectivity by 3/4. Both models, however, are in conflict with the studies by Philipson et al. (1966), discussed above, which demonstrated that antibody binding to virions can be detected prior to neutralization. Significantly, the observation that an occupancy of 4–5 antibody molecules per virion is required for neutralization is consistent with the observed differences in binding and neutralization titers in the Philipson studies. The most straightforward conclusion is that the single-hit hypothesis of poliovirus neutralization is not correct, and that the mechanism of neutralization of poliovirus is similar to that of HRV. The overall architecture of poliovirus and HRV are similar, and both viruses have similar proportions consisting of a 30-nm-diameter capsid surrounding the positive-strand RNA genome. The three-dimensional structure of poliovirus in complex with a soluble form of its receptor (CD155) has recently been solved (Belnap et al., 2000; He et al., 2000). The poliovirus receptor CD155 has a footprint similar to that observed for the rhinovirus receptor ICAM-1 bound to

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HRV. Both bind to the canyon, a narrow surface depression on the viral surface. It may therefore not be completely surprising that the stoichiometrical analyses discussed above yield similar results for HRV and poliovirus. The data suggest that the attachment of 4–7 antibody molecules is required for the neutralization of picornaviruses of poliovirus/HRV dimensions. Studies by Horaud and colleagues (Blondel et al., 1986; Crainic et al., 1983; Diamond et al., 1985) have suggested that antibodies may bind to poliovirus without neutralization. This conclusion was made on the basis of studies using neutralizing antibodies which were analyzed for their activity (neutralization and binding) against a range of antigenic poliovirus variants (distinct serotypes and escape mutants). For example, some serotypes appeared to be bound by antibody in an immunoprecipitation assay but were not neutralized. The studies, however, made no distinction between strong and weak binding. Mutations on poliovirus envelope in or near the antibody binding site may result in decreased antibody affinity for the poliovirus mutants in question, resulting in a level of occupancy too low to neutralize the virus (for example, as shown by the studies discussed above). This interpretation is supported by the observation that addition of antiantibody in the studies above did result in neutralization of the mutant viruses (Blondel et al., 1986), presumably because anti-antibody effectively increases affinity (avidity) and raises occupancy to the level required for neutralization (see below). E. SINGLE-HIT VERSUS MULTI-HIT KINETICS 1. Overview Single-hit models are based on the observation that infectivity starts to decrease immediately following addition of antibody to virus. The absence of a detectable lag phase, i.e., a period in which infectivity remains unaffected following antibody addition, has often been taken as supporting evidence for the single-hit model (Dulbecco et al., 1956; Icenogle et al., 1983; Mandel, 1976; McLain and Dimmock, 1994; Taylor et al., 1987). It may be argued, however, that reactions of antibodies of nanomolar affinities for their cognate antigen occur rapidly (e.g., Scicluna and McCullough, 1999), and as samples are being drawn in kinetic studies at intervals of minutes or greater, it may not be possible to measure the surviving virus fraction fast enough to observe the presence or absence of a lag phase under standard conditions (Della-Porte and Westaway, 1977). Furthermore, lag times in neutralization kinetics have indeed been observed in studies with most viruses when the experiments were performed at lower incubation temperature, more dilute antibody concentration, or with antibodies of lower affinity (Burnet et al., 1937; Dulbecco et al., 1956; Lafferty, 1963a; Philipson, 1966; Taylor et al., 1987). This is difficult to reconcile with the single-hit model, as it requires the assumption that the mechanism of

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neutralization changes as temperature, antibody concentration, or affinity are varied. Klasse and Moore (1996, and references therein) have argued that a firstorder neutralization reaction does not give direct proof for neutralization by single-hit kinetics. One antibody molecule neutralizing one virion implies firstorder kinetics, but the reverse is not true. Stoichiometrical analyses (see below) suggest that the binding of more than one antibody molecule to a virus particle is usually required for neutralization. An alternative explanation for the absence of a lag phase to the one discussed above, at least for enveloped viruses, may be found in a heterogeneity of spike density. In virus stocks, virions may exist with a low number of active spikes that may only require one or a few hits for neutralization (Klasse and Moore, 1996). HIV-1 is an example where this hypothesis may be applicable, as shedding of gp120 from the virion results in the formation of inactive spikes (Parren et al., 1999; Sattentau et al., 1995). A plethora of evidence indicates that virus neutralization in general is the result of the attachment of a number of antibody molecules to the infective particle. Many of these arguments have been cogently reviewed by Della-Porta and Westaway (1977). A summary of these arguments, supplemented with evidence for multi-hit neutralization from more recent studies, is presented here. These arguments include: r the occurrence of a persistent fraction of infectious virus r the neutralization of infectious virus–antibody complexes (sensitized virus) by addition of anti-antibody r neutralization of sensitized virus by addition of complement r retardation of infection of sensitized virus r the phenomenon of antibody-mediated enhancement of infection r additive and synergistic effects of antibodies in neutralization 2. The Persistent Fraction of Infectious Virus The presence of a persistent fraction of nonneutralizable virus in most virus preparations has been the source of much debate. The nonneutralizable virus fraction has been attributed to the presence of low-affinity antibodies in antibody preparations (particularly early immune sera), virus heterogeneity, neutralization-interference of nonneutralizing antibodies, virus association with lipids and lipoproteins, and virus aggregation. In some studies, it has been shown that the infectious titer of the persistent fraction can be further reduced by the addition of anti-antibody, implying that this fraction contains virus particles with some level of antibody coating. An early explanation of the nonneutralizable virus fraction attributed the phenomenon to association–dissociation equilibria between virus and antibody (Burnet et al., 1937). Indeed, dissociation may play a role when low-affinity

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antibody preparations are being used, and dissociation of the virus–antibody complexes occurs before or during the addition of the complexes to the target cell (Jerne and Avegno, 1956; Lafferty, 1963b). With high-affinity antibody preparations, however, this does not seem to be an important factor (Dulbecco et al., 1956). It has also been suggested that a low-affinity antibody or nonneutralizing antibody may bind to the virion, preventing the binding of neutralizing antibody and giving rise to a nonneutralizable fraction. In some cases, however, such a fraction is more easily explained in terms of heterogeneity of the virus preparation. Vaccinia virus and rabbitpox virus preparations, for example, contain a small fraction of extracellular enveloped virus (EEV) in an excess of intracellular mature virus (IMV). The EEV contains an additional wrapping membrane and is relatively resistant to neutralization (Ichihashi, 1996; Vanderplasschen et al., 1997). The wrapping membrane however is easily damaged during manipulations thereby exposing the more-neutralization sensitive IMV particle (Ichihashi, 1996; Vanderplasschen et al., 1997). This mechanism, rather than dissociation of low-affinity or nonneutralizing antibody, may explain experiments that show that the persistent fraction in rabbitpox virus can be neutralized following dilution and washing (Lafferty, 1963a). In a study on neutralizing antibodies against mouse mammary tumor virus (MMTV), it was suggested that nonneutralizing mAbs against MMTV gp52 could interfere with the binding and neutralizing activity of a neutralizing mAb (Massey and Schochetman, 1981). This would be one of the only examples of such an activity for a monoclonal antibody. The inhibition described, however, is extremely weak and only apparent at high nonneutralizing mAb:neutralizing mAb ratios. On closer examination, the observed blocking, furthermore, is well within the range of the experimental error of the MMTV pseudotype neutralization assay used (Massey and Schochetman, 1981), and is therefore unlikely to be of any significance. Aggregation of virus may be responsible for many observations of a nonneutralizable virus fraction. Aggregates of virus particles already present in the preparation, or formed by aggregation with antibody, may inhibit access of antibody to virus particles at the inside of a small aggregate protecting it from neutralization. Filtration to remove the aggregate or treatment with anti-antibody to coat the aggregate more extensively can indeed remove the persistent fraction in some instances (Wallis and Melnick, 1967, 1970). 3. Neutralization of Sensitized Virus with Anti-antibody or Complement Neutralization of sensitized virus by anti-antibody or complement components has been cited as an argument against single-hit kinetics, as it indicates that infectious virus–antibody complexes exist (Della-Porte and Westaway, 1977).

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4. Retardation of Infection by Antibody Burnet et al. (1937) noted in their review that infectious antibody–bacteriophage complexes exist, as evidenced by observations in the literature that plaque sizes were reduced in the presence of antibody, which presumably occurred through delayed infection of phages with a low level of antibody attached. Krummel and Uhr (1969) demonstrated reduced infectivity rates for bacteriophages complexed with neutralizing antibody in kinetic neutralization experiments (see above). Westaway (1965) further noted that the rate of penetration of West Nile virus preincubated with neutralizing antibody was delayed, as the virus was susceptible to postattachment neutralization for a longer period of time after adsorption to target cells than nonsensitized virus. These experiments indicate that antibody may combine with virus at an occupancy too low to neutralize the virus but high enough to decrease the rate of entry into the target cell. 5. Antibody-Mediated Enhancement of Infection Antibody-mediated enhancement of infection is a phenomenon that appears to occur for neutralizing antibodies at subneutralizing concentrations (Halstead, 1982; Hawkes and Lafferty, 1967; Morens et al., 1987). Enhancement is very sensitive to the target cell and is dependent on the types and expression levels of Fc and/or complement receptors on the target cell surface. A classical example is enhancement of dengue virus infection, which is dependent on the interaction between virion-bound antibody and Fc receptors expressed on the target cell. In typical assays, neutralization is observed at relatively high concentrations, whereas enhancement is observed at lower concentrations. Using neutralizing antibodies against dengue virus, Morens et al. (1987) indeed showed that the mAb concentration maximizing enhancement was predicted by the neutralization titer. Hawkes and Lafferty (1967) noted that the balance between enhancement and neutralization is dependent on the incubation time of virus and antibody. They observed a shift of the dose response curve as time progressed, and enhancement as well as neutralization occurred at increasingly lower concentrations. Presumably, at early time points, attachment of a small number of antibodies leads to enhancement, and as the reaction progresses, more antibodies attach, eventually leading to neutralization of the virion. This clearly indicates that enhancement and neutralization are two different biological outcomes of the interaction of an antibody with virus at different levels of occupancy. For HIV-1, both Fc receptor-mediated and complement-mediated antibodydependent enhancement of infection have been described (Homsy et al., 1989; Lund et al., 1995; Mascola et al., 1993; Robinson et al., 1988; Schutten et al., 1995; Takeda et al., 1988). In Fc receptor-mediated enhancement, Fc receptormediated endocytosis of virion–antibody complexes may lead to the internalization of virus and infection. A plausible alternative is that binding to Fc receptors

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stabilizes the interaction of virion and target-cell, permitting interaction of the envelope spike and virus receptor at low antibody coating of the virion (Connor et al., 1991). At higher coating, this interaction may be inhibited. Complement receptor 2 (CD21) may mediate enhancement of HIV-1 infection via several proposed mechanisms, either by acting as a receptor for HIV-1 opsonized with antibody and complement or by increasing virus binding to the cell due to an interaction of CD21 with opsonized virus (Boyer et al., 1992; Lund et al., 1995). In addition, virion-bound C1q may directly interact with receptors on the host cell, leading to enhancement of infection (Prohaszka et al., 1997). In some cases, such as HIV-1, the enhancement does not require the Fc part of the antibody molecule (Sullivan et al., 1998b). It is suggested that the low-level coating may trigger conformational changes in the envelope that, for example, favor fusion between the virion and target cell, or such coating may nonspecifically reduce repulsion between the virion and target cell surface. A similar mechanism of antibody-mediated enhancement of infection has been described for Sindbis virus (Flynn et al., 1988). The phenomenon of enhancement indicates that antibodies bind to virions at subneutralizing concentrations and is therefore a strong argument for the importance of antibody occupancy in virus neutralization and neutralization following multi-hit kinetics. High levels of occupancy lead to neutralization, whereas, in the presence of permissive target cells, low levels of occupancy may lead to enhancement of infection. 6. Additive and Synergistic Effects in Neutralization The presence of additive and synergistic effects in neutralization is indicative of a role of antibody occupancy in neutralization of viruses and provides an argument for multi-hit neutralization. Phenotypic mixing experiments, in particular, are informative in this respect. It has been shown that cells dually infected with certain viruses may yield phenotypically mixed virions. These are relatively poorly neutralized by single specificity antisera against each of the parent viruses, but are more effectively neutralized by mixed neutralizing antisera. Cells dually infected with the myxovirus fowl plaque virus (FPV) and the rhabdovirus vesicular stomatitis virus (VSV) produced a virus progeny of which about 20% was composed of virus particles with mixed envelopes. Neutralization of virions carrying mixed envelopes was poor to nonexistent in neutralization assays with anti-VSV and anti-FPV alone. However, strong neutralization occurred when the assay was carried out with a mixture of the two sera (Zavada and Rosenbergova, 1972). Similar observations were made with phenotypically mixed virions of VSV and Sendai virus (Kimura, 1973) and VSV and measles virus (Wild et al., 1975–1976). Additive effects are also observed for antibodies binding to the paramyxovirus Newcastle disease virus (NDV). Binding of single mAbs against the NDV

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hemagglutinin-neuraminidase (HN) glycoprotein leaves a large persistent fraction of nonneutralized virus (Iorio and Bratt, 1983). The persistent fraction appeared to be bound by neutralizing mAb, as it was almost completely neutralized by the addition of anti-antibody (Iorio and Bratt, 1984). It did not seem to contain large aggregates of virus or genotypically distinct viruses (Iorio and Bratt, 1984). Interestingly, the persistent fraction could be neutralized when antibodies against several epitopes on the HN glycoprotein were combined and, in addition, a level of neutralization comparable to a polyclonal immune serum could only be obtained by combining antibodies against four distinct epitopes on NDV (Iorio and Bratt, 1983, 1984). These observations are consistent with some heterogeneity of the NDV HN glycoprotein which may exist in different conformations on the virion surface. Neutralization of La Crosse virus to the level of a polyclonal antibody could similarly only be obtained with combinations of two monoclonal antibodies against the G1 glycoprotein (Kingsford, 1984). These studies suggest a role for antibody occupancy of viral surface binding sites in neutralization of NDV and La Crosse virus. F. NEUTRALIZATION OF VIRIONS BY COATING WITH COMPLEMENT Of interest is that coating virions by early components of the complement system (C1-4) may enhance neutralization without inducing virolysis, which will be discussed below (reviewed by Oldstone, 1975). For HIV-1, it has been shown that the infectivity of plasma virions may be reduced by an in vivo deposition of C3 on the virion surface (Sullivan et al., 1998a). Presumably, the complement coat interferes with virus interaction with the target cell, thereby contributing to neutralization (Parren et al., 1999). G. THE CASE OF ADENOVIRUS Adenovirus is an icosahedral capsid with characteristic long, thin fibers protruding from the 12 vertices. A knob at the end of each fiber serves as the primary receptor attachment site. The penton base protein, located at the base of the fiber, binds to ␣v integrins via an RGD motif (reviewed by Stewart and Nemerow, 1997). An unusual observation is that an antibody directed against the RGD motif neutralizes adenovirus infection as a Fab fragment but not as a whole IgG molecule (Stewart et al., 1997). Cryo-EM of adenovirus complexed with the Fab indicates that the Fab may occupy all five RGD epitopes on the penton base complex. The whole IgG molecule was shown to bind soluble penton base with a stoichiometry of 2.8, indicating that, per penton base, two IgG molecules may bind bivalently and one monovalently. In the intact virus particle, however, the central fiber presumably provides a source of steric hindrance, preventing the IgG molecules but not the Fab fragments from binding to all five sites on the penton base (Stewart et al., 1997). Neutralization of adenovirus by

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an antibody against the penton base protein therefore requires occupancy of all five integrin binding sites. H. THE CASE OF RABIES VIRUS A number of elegant and detailed studies have been carried out by Flamand and colleagues on antibody neutralization of rabies virus. These studies support the general notion that neutralization is the result of coating the virion with antibody molecules (Flamand et al., 1993; Raux et al., 1995). The number of antibody molecules bound per rabies virus particle was determined carefully and correlated with the magnitude of neutralization achieved. It was found that infectivity was completely preserved with fewer than 130 IgG or 30 IgM molecules bound per virion (Flamand et al., 1993). The neutralizing dose, the dose at which infectivity is decreased to 37% (1/e), was between 130 and 320 IgG (40 to 50 IgM) molecules bound per virion depending on the antibody tested. There is a marked threshold effect with a two- to three-fold increase in the number of antibody molecules close to the threshold, leading to a drop in infectious titer of about 1 to 2 logs (Flamand et al., 1993). Three mAbs were identified which bound to the virus without neutralization. Two of these were subsequently shown to bind to a minor population of envelope spikes in an altered acidic conformation of envelope (Raux et al., 1995). Coating of the virion via this altered spike was too incomplete to permit neutralization. If the virion was maintained at lower pH to convert most spikes to the acidic form, then more antibody bound, and the virus was neutralized. One mAb, which effectively neutralized wild-type virus, appeared to saturate the envelope spikes of a neutralization escape variant (1080 molecules of IgG per virion) but did not neutralize it. The antibody-coated virus was still able to attach to target cells. The authors considered it unlikely that this attachment was occurring through envelope, because of the thickness of the antibody shell coating the virus (Flamand et al., 1993). This case appears to constitute a bona fide example of virion coating by antibody without neutralization. Although it is the rabies mutant virus rather than the antibody that seems be an exception to the rule here, it would be very interesting to elucidate how this mutant virus can escape neutralization while being completely coated with antibody. Overall, the studies by Flamand et al. (1993) strongly suggest that there is a correlation between antibody occupancy and neutralization of rabies virus. The experiments show that neutralization occurs at a mean critical coating density of about 225 IgG or 45 IgM molecules bound per virion. I. THE CASE OF HIV-1 We have investigated the relative importance of binding site occupancy and epitope specificity in antibody neutralization of HIV-1 using an extensive panel of neutralizing mAbs against HIV-1 (Parren et al., 1998). Neutralization was studied

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using T-cell line adapted (TCLA) HIV-1 isolates. The strategy adopted was to compare the binding of a number of antibodies to different gp120 epitopes, presented in the form of functional oligomeric gp120 on infected cells, with their capacity to neutralize the corresponding virus. A concentration of mAb yielding half-maximal binding (K50 ) and a neutralization titer (ID50) of similar magnitude would be consistent with antibody occupancy of virion binding sites playing a major role in HIV-1 neutralization. Similar K50/ID50 ratios for different epitopes would suggest that neutralization is independent of the epitope recognized. Indeed, an excellent correlation (r = 0.882; P < 0.0001) between neutralization (ID50) and binding to oligomeric gp120 (K50) was observed. Furthermore, the K50/ID50 ratios fell, in general, within a relatively narrow range for antibodies to different neutralization epitopes, suggesting that occupancy of binding sites on HIV-1 virions is the major factor in determining neutralization, irrespective of epitope recognized (Parren et al., 1998). This is illustrated in Fig. 1 in which the K50/ID50 ratios for a panel of mAbs against five distinct neutralizing epitopes on TCLA HIV-1 isolates are plotted. We proposed a model in which neutralization is effected by coating of the virion with antibody. For attachment of the virus to the target cell to occur and infection to initiate, it is presumed that multiple contacts in a localized area between gp120 and the primary HIV-1 receptor CD4 must be established. Coating of the virus above a critical density would interrupt this process and

FIG. 1. Neutralization of HIV-1 is determined by antibody occupancy irrespective of epitope recognized. The antibody concentration yielding half-maximal binding (K50) and neutralization titer (ID50) and K50/ID50 ratios were determined for a number of neutralizing mAbs against the CD4 binding site (CD4bs), CD4-induced epitope (CD4i), V2 loop, V3 loop, and 2G12 epitopes on gp120 in binding and neutralization assays with HIV-1LAI and HIV-1MN (Parren et al., 1998). Similar K50/ID50 ratios for different epitopes suggests that neutralization is independent of the epitope recognized. This suggests that occupancy of binding sites rather than epitope recognized is the major determining factor in neutralization.

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thereby inhibit infection (Parren et al., 1998 and reviewed in Parren et al., 1999). Schønning et al. (1999) analyzed the stoichiometry of HIV-1 neutralization in an elegant study by use of virions carrying mixed envelope spikes on their surface. Cells were transfected with an HIV-1 clone (A308), which is potently neutralized by a mAb against the V3 loop on gp120, and a mutant of this clone, T321, which resists neutralization by this antibody by a loss-of-binding mutation. The proportion of antigen binding sites on the virion could now be titrated by transfecting plasmids encoding the two clones in different ratios. Neutralization of the mixed envelope virions was performed with a high concentration of neutralizing antibody (500 × the ID50 of the neutralization-sensitive clone), so that all available binding sites would be occupied. Neutralization by virion aggregation did not appear to play a significant role in inactivation. The experiment showed that, as the number of binding sites (and therefore the number of bound antibody molecules) increased, viruses become susceptible to neutralization. Neutralization of 63% of the virions occurs at a level of about 35% occupancy, a level which is consistent with stoichiometric arguments presented below (see Fig. 3). The increase in neutralization is incremental, indicating a multi-hit rather than a single-hit model (Schønning et al., 1999). It has been shown that average, freshly budded HIV-1 virions carry approximately 72 envelope spikes (216 gp120 molecules) on their surface (Gelderblom, 1991). From the analyses above it can be predicted that the attachment of approximately 70 IgG molecules is required for the neutralization of such HIV-1 virions, which would be equivalent to about one IgG molecule/spike. Klasse and Moore (1996) developed a quantitative model of HIV-1 neutralization to explain the large difference in neutralization sensitivity between TCLA strains and primary isolates of HIV-1 (see Moore and Ho, 1995, for reviews of HIV-1 neutralization). The model predicts that neutralization of HIV-1 is due to the reduction of the number of active envelope spikes below a minimum required for the induction of a productive infection. Factors contributing to differences in neutralization sensitivity of different virus/antibody combinations are binding constants of neutralizing antibodies to the envelope spike, the number of spikes on the surface, and the minimum number of spikes required for attachment and fusion (Klasse and Moore, 1996). Possible differences in spike density between neutralization-sensitive TCLA strains of HIV-1 (low active spike densities) and neutralization-resistant primary isolates (high active spike densities) are quoted as an important factor in neutralization resistance (Klasse and Moore, 1996). Differences in spike densities between TCLA strains and primary isolates, however, are controversial (Karlsson et al., 1996; Sullivan et al., 1995). Alternatively, the resistance may be due to the poor accessibility of epitopes on the primary isolate envelope spike (Parren et al., 1999).

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J. STOICHIOMETRY OF NEUTRALIZATION: A MODEL FOR NEUTRALIZATION BASED ON ANTIBODY COATING OF VIRION PARTICLES The stoichiometry of antibody binding at neutralization has been determined for a number of viruses. We briefly summarize the relevant studies focusing on those where stoichiometry was determined by a direct and quantitative method. Methods that determine the amount of antibody bound using purified radiolabeled virion–antibody complexes (e.g., Taylor et al., 1987), or quantitative sodium dodecyl sulfate–polycrylamide gel electrophoresis (SDS-PAGE) (e.g., Flamand et al., 1993), for example, may give reliable data. Methods in which the stoichiometry is extrapolated from kinetic analysis may be inaccurate because of uncertainties with respect to the mathematical models used, particularly in early studies. Rappaport (1970) determined that an average of four antibody molecules bound per neutralized MS2 bacteriophage represented a neutralizing dose. Studies by Collono and Smith and their colleagues have indicated that HRV is neutralized by inhibition of attachment, and that the binding of 6–7 antibody molecules per HRV virion is required to reduce HRV attachment by 63% (Colonno et al., 1989; Smith et al., 1993). Icenogle et al. (1983) determined that neutralization of poliovirus requires the attachment of 4–5 antibody molecules, as discussed above. The picornaviruses poliovirus and HRV therefore require the attachment of an average of 5–6 antibody molecules for neutralization. A study which assessed the number of IgG molecules necessary to neutralize influenza virus showed for two different mAbs (HC2 and HC10) that approximately 70 molecules were required (Taylor et al., 1987). Studies by Flamand and colleagues on rabies virus have shown that an average occupancy of 225 IgG molecules per virion is necessary for neutralization (see Flamand et al., 1993; Raux et al., 1995). Studies on bovine papillomavirus (BPV) determined that the number of antibody molecules required for neutralization were approximately 14, 30, 72, and 36 molecules for the four mAbs 5B6, 3, 6, and 9 studied, respectively (Roden et al., 1994). An average of about 38 bound IgG molecules is therefore required to neutralize a BPV virion. Complexes of mAb 5B6 and 9 with the major capsid BPV protein L1 have been analyzed by cryo-EM. It was shown that mAb 5B6, which apparently requires a slightly lower occupancy for neutralization, binds only to hexavalent capsomers, whereas mAb 9 also binds to pentameric capsomers (Booy et al., 1998). MAb 9 binds monovalently at the tip of the capsomers and is highly contorted, projecting the Fc domain at a 120◦ angle into the space between the capsomers, while the free Fab domain projects outward. MAb 9 provides a dense extended antibody coat, which is consistent with its ability to inhibit attachment of BPV to the target cell. mAb 5B6 binds deeply between the hexavalent capsomers and adopts a more

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linear conformation. MAb 5B6 does not inhibit virion attachment efficiently and likely interferes with a postattachment step (Booy et al., 1998). It is possible that mAb 5B6 may coat the virus more extensively by its lower molecular profile and may therefore be more effective at a somewhat lower level of occupancy. The relatively low number of antibodies required to neutralize some viruses compared to others has generated much discussion. Critical site hypotheses have been widely used to explain neutralization. However, how critical “neutralizationrelevant” spikes differ from other “neutralization-irrelevant” spikes has not been defined, and although molecular rearrangements in the capsid on nonenveloped viruses may provide a possible mechanism, it is unclear how signals would be transmitted between loosely attached spikes in the membrane of an enveloped virus. Some studies have suggested that binding of a neutralizing antibody, for example, to the hemagglutinin (HA) of influenza virus, transmits a signal across the envelope, effecting neutralization (Possee et al., 1982; Taylor et al., 1987). A molecular mechanism for this is unknown. A much simpler hypothesis favored by us is that neutralizing antibodies generally act by coating the virus surface, thereby interfering with critical steps in the infectious pathway, such as obstructing interactions with cell surface receptors or interfering with molecular rearrangements required for virus–cell fusion. Figure 2 provides a schematic representation of the size of the viruses discussed above relative to antibody. The average number of antibody molecules required to neutralize the viruses depicted by increasing size from left to right is 4, 5–6, 38, 70, and 225. A plot of the number of antibodies required for neutralization as a function of the average surface area for each of the viruses is shown in Fig. 3a. The straight line demonstrates that a roughly equivalent fraction of the virion surface, independent of the size of the virion, must be bound by antibody for neutralization to occur. To calculate the average surface area on a virion associated with each antibody molecule at neutralization, we determined the slope of the line in Fig. 3. The line in Fig. 3a can be described by the formula: N = 0.0033 × A, in which N is the number of IgG molecules required for neutralization, and A is the total virion surface area. It follows that each antibody molecule is associated with an average of 300 nm2 virion surface area at neutralization. It is not unreasonable to assume that an antibody molecule can provide steric hindrance for an area this size. A 300-nm2 area is covered by a circle with a radius of about 9.7 nm. The length of an antibody molecule is about 12.5 nm, of which approximately 7 nm is provided by the Fc tail which has a high degree of translational and rotational flexibility relative to the Fab domains. This indicates that it is not unlikely that an antibody, even when bound bivalently, whereby movement of the Fab domains is restricted, could hinder the interaction of virion surface proteins with critical sites on the target cell in an area surrounding the binding site or, alternatively,

FIG. 2. Schematic representation of virion size relative to antibody. Electron microscopic images of each of the viruses considered are depicted on scale relative to the size of an antibody molecule. Viruses with a larger average virion size appear to require the attachment of more antibody molecules to be neutralized. Clearly, however, viruses are generally neutralized well before all available mAb binding sites have been occupied. Saturation of binding sites, for example, only occurred with 30 IgG molecules bound per poliovirus and 1080 IgG molecules bound per rabies virus particle (Flamand et al., 1993; Icenogle et al., 1983). Electron microscopic images were provided by Dr. Linda M. Stannard, University of Cape Town, South Africa (poliovirus, influenza virus and polyoma virus); Dr. H. W. Ackermann, Universite Laval, Quebec, Canada (bacteriophage MS2); Dr. S. K. Vernon, Wyeth Laboratories, Philadelphia, PA (rabies virus). Rabies virus reprinted from Vernon et al. (1972) with permission granted by Academic Press, Orlando, FL.

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FIG. 3. Antibody occupancy of virion surface binding sites at neutralization. To study the observation in Fig. 2 in more detail, we plotted the number of antibodies required for neutralization as a function of the average surface area for each of the viruses shown. A range of virion surface areas is indicated as preparations, particularly of enveloped viruses, are typically heterogeneous with respect to size and shape. (a) The straight line demonstrates a linear relationship. (b) This relationship can be extrapolated to the large pathogen, the intracellular bacterium Chlamydia trachomatis.

hinder the formation of intermediates in the viral entry process such as fusion complexes. We believe that the available data, in agreement with the occupancy model, indicate that antibody-covered virions are either neutralized or not, and that there is no convincing evidence for any intermediate states. Virions covered with antibodies at low occupancy may infect their target cells more slowly, as discussed above. Such particles, however, are still infectious and therefore do not represent a neutralization intermediate, in our view. Once antibody coating proceeds beyond a critical density, which appears to equal a relatively constant number of antibody molecules per unit of virion surface area, the virion is rendered noninfectious. Neutralization is expected to be reversible by dissociation of the antibody–virion complexes in most cases. Neutralization of a virus population can thus be understood in similar terms. The partial neutralization of a homogeneous virus stock at a certain concentration of antibody (e.g., 50%) simply represents a Poisson distribution of antibody molecules attached to virions at a given point in time. The fraction of the viruses coated with antibody molecules below the neutralization threshold is fully infectious, whereas the complementary fraction is fully neutralized. Because antibody typically is in vast molar excess over virus in a neutralization reaction, the average number of binding sites occupied at a given antibody concentration, and thus the fraction of neutralized virus, is predicted to be primarily determined by antibody affinity for the virion.

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The antibody occupancy at neutralization for the intracellular bacterium Chlamydia trachomatis has been determined in a study which is much less quantitative than the studies on viruses discussed above. We include it in our discussion here, as it is the only study of which we are aware to provide an estimate of the number of antibody molecules required to neutralize a nonviral pathogen. The study found that approximately 1000 antibody molecules of the strongly neutralizing mAb UM-4 were required to reduce infectivity of infectious elementary bodies by 50% (Peeling and Brunham, 1991). (A higher number was given for a weakly neutralizing antibody, but we consider this less reliable, since the study assumed that all the antibody in an equilibrium mixture was bound to bacteria.) Interestingly, the C. trachomatis data can be fit by extrapolation of the line described for viruses in Fig 3a (Fig 3b). The occupancy model suggests that one mechanism by which virus could seek to escape neutralization by antibody would be to decrease the number of neutralizing epitopes on the viral surface. Such a mechanism has indeed been described for human cytomegalovirus (HCMV) (Li et al., 1995). A neutralization escape mutant was isolated from a patient who was being treated with a neutralizing human antibody against the HCMV surface glycoprotein gH. The escape mutant was unstable when cultured in the absence of anti-gH neutralizing antibody and displayed a resistant phenotype against a number of distinct neutralizing antibodies and a polyclonal serum against gH. Biochemical analysis revealed that the escape mutant displayed a marked decrease in the amount of gH expressed on the surface. Interestingly, this mechanism of resistance would be consistent with the large differences in susceptibility to neutralization of clinical HCMV isolates, and might be a mechanism by which the virus persists in the presence of neutralizing antibody in the serum (Li et al., 1995). If antibody occupancy indeed plays a major role in neutralization as we suggest, then down regulation of surface-specific molecules on certain viruses may be a more common mechanism of neutralization resistance than currently thought. However, as in the case of HCMV, it may be necessary to culture fresh clinical isolates in the presence of neutralizing antibody to observe these effects, since passaging would allow rapid reversion to wild type. The occupancy model also suggests that Fab fragments may neutralize a viral particle less efficiently than the corresponding whole antibody, as steric hindrance by the Fab is provided at a significantly reduced level. Generally, a ∼1- to 50-fold difference is found in neutralization potency between whole IgG and Fab, which has been explained by differences in avidity (reviewed in Parren and Burton, 1997). Some examples from the literature, however, indicate that a loss of steric hindrance may also play a role in this decrease. Icenogle et al. (1983) showed that treatment of neutralized poliovirus particles at low occupancy (an occupancy of the order of 6 antibody molecules per poliovirus particle reducing infectivity of the virus stock to 28%) with papain to remove the IgG Fc tail reactivated the infectivity. It was shown that the Fab fragments were still bound to the

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virion, however, as addition of an anti-antibody again reduced infectivity to the initial neutralized level (Icenogle et al., 1983). Similar observations were made by Emini et al. (1983b). For the paramyxovirus SV5, Merz et al. (1981) have shown that anti-HN antibody completely neutralized the virus, whereas Fab fragments could only cause partial neutralization, and a relatively large fraction of nonneutralizable virus remained. The neutralization mechanism of this antibody did not appear to involve aggregration or cross-linking, excluding this as the basis for the difference. In agreement with the model proposed here, it has been shown that the neutralization potency of mAbs against the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) correlated with occupancy as potent neutralizing mAbs stained virions in immunoelectron microscopy more strongly than weakly neutralizing mAbs (Weiland et al., 1999). The occupancy model may finally explain the reduction in neutralization rate as a function of time in kinetic experiments. As viruses are more densely decorated with antibody molecules, the reaction rate will start to decrease, as incoming antibody will be sterically hindered by antibody already bound to the virus. Such steric hindrance has been shown to exist by kinetic neutralization experiments on herpes simplex virus (HSV) performed by Ashe and Notkins. These authors demonstrated a drop in the neutralization rate constant as neutralization progressed, the magnitude of which correlated with the level of sensitization (Ashe and Notkins, 1967). Kinetic neutralization studies using HSV presensitized with a neutralizing antibody, furthermore, showed that neutralization rate constants were strongly reduced if the subsequent neutralization reaction was performed with the whole antibody, but were only mildly reduced when performed with the Fab fragment (Ashe et al., 1969). Therefore, under the conditions used, the antibody already bound to the virion reduced the binding rate of whole antibody relative to that of the smaller Fab fragment, presumably by a steric hindrance mechanism. K. A MOLECULAR MODEL FOR PICORNAVIRUS NEUTRALIZATION It has been shown that the attachment of between 4 and 7 IgG molecules can lead to the neutralization of a picornavirus particle. In particular, the binding of about 6–7 IgG molecules to HRV prevents its attachment to the target cell thereby neutralizing the virus. (Colonno et al., 1989; Icenogle et al., 1983; Smith et al., 1993). To visualize how the binding of a small number of IgG molecules to the surface of a 30-nm picornavirus particle can impact the virus–receptor interaction at the cell surface, we prepared a molecular model of a picornavirus with a low number of antibodies bound. Poliovirus capsid was modeled from coordinates in the Protein Data Bank (PDB)(Berman et al., 2000) entry 1dgi (He et al., 2000). Antibody (human IgG1) was modeled from PDB entry 1igt (Harris et al., 1997) and coordinates from Erica Ollmann Saphire (personal communication and

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Ollmann Saphire et al., 2001). Molecular surfaces were calculated with the ◦ program MSMS (Sanner et al., 1996) using C alpha atoms of 4 A radius and a reduced viral surface of 1.4 million triangles. Viral surface covered by antibody ◦ was calculated with python (Sanner, 1999) using a cylinder of 90 A radius penetrating the viral capsid. Surfaces and models were visualized in AVS and PMV (Sanner et al., 1999), while artistic impressions of IgG domain movements were rendered in Adobe Photoshop. In the diagram in Fig 4, three IgG molecules are shown interacting with the visible half of the surface area of a picornavirus particle. This level of coating is close to that predicted to neutralize the virus. The considerable rotational flexibility of the Fc region (with dimensions of 7 × 5.7 × 2.4 nm) is demonstrated for one IgG molecule (Fig. 4a). Taking the flexibility of Fc and the translational flexibility of the antigen-bound Fab fragment into account, the antibody could be thought to hinder the attachment of virus to cellular receptors in a significant area surrounding the antigen-binding site, as shown in Fig 4b. As the initiation of productive infection requires the interaction of virus with a number of receptor molecules in a localized area (see also Fig. 6), it can be envisioned that the binding of a relatively small number of antibody molecules to a picornavirus particle can effectively interrupt attachment and initiation of infection via steric hindrance and geometric constraints. L. THE CASE OF INFLUENZA VIRUS Antibodies against influenza virus neuraminidase (NA) and matrix protein 2 (M2) have been used as an example of binding but nonneutralizing antibodies. The surface of influenza viruses contains three types of spike proteins, namely HA, NA, and M2, inserted in a host cell-derived membrane (Murphy and Webster, 1996). Whereas antibodies against HA neutralize influenza, antibodies against NA and M2 do not (Kilbourne et al., 1968; Webster and Laver, 1967; Zebedee and Lamb, 1988). In the presence of antibodies against NA and M2, however, virus growth in vitro and in vivo is reduced, indicating that these antibodies do recognize these proteins on the surface of the virus or infected cells. The activities observed are most likely the result of reduced virus release and antibody-mediated destruction of infected cells (Murphy et al., 1972; Webster and Laver, 1967; Zebedee and Lamb, 1988). The absence of neutralizing activity of antibodies against NA and M2 can be explained by their abundance and distribution on the viral surface, and is in direct agreement with the occupancy hypothesis. The NA and M2 proteins are far less abundant on the viral surface than the HA protein; generally there are four to five times more HA than NA spikes, and NA spikes furthermore seem to be organized in patches. Each virus usually contains only a few copies of the M2 protein (Lamb and Krug, 1996; Murphy and Webster, 1996). Therefore, although antibodies against NA and M2 may bind to these proteins on the viral

FIG. 4. Picornavirus neutralization. (a) Picornavirus particle with three IgG molecules (gold) attached to approximately half of the virion surface area. Rotational flexibility of the Fc region is demonstrated for one IgG molecule. (b) Region of virus particle surface (green) subject to direct obstruction by a bound IgG molecule. Binding of an average of about 5–6 IgG molecules/picornavirus particle results in neutralization. This number of bound antibody molecules prevents attachment of HRV to target cells. Surfaces were calculated as described in Sanner et al. (1996). This diagram was kindly prepared by Erica Ollmann Saphire.

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surface, the maximum level of occupancy achievable is relatively low and likely to remain below the level required to inhibit infectivity. This is reminiscent of the observations with the minor acid-inducible form of the rabies spike protein described above (Raux et al., 1995). M. NEUTRALIZATION BY ANTIBODIES AGAINST HOST CELL-DERIVED PROTEINS INCORPORATED INTO VIRIONS Many instances of viruses, particularly retroviruses, that incorporate nonviral host cell-derived proteins into their viral envelopes have been described. HIV-1 has been shown to incorporate HLA class I and class II molecules, ICAM-1, LFA1, and CD44 (Arthur et al., 1992, 1995; Capobianchi et al., 1994; Gelderblom et al., 1987; Hoxie et al., 1987; Orentas and Hildreth, 1993; Saarlos et al., 1997). Simian immunodeficiency virus (SIV) and FIV, furthermore, may also incorporate HLA antigens (Arthur et al., 1992; Lee et al., 1982). This may have important consequences, as macaques immunized with human cells or HLA-DR, for example, can be protected from challenge with SIV grown in human host cells (Arthur et al., 1995; Stott, 1994). It has been suggested that cellular adhesion molecules associated with HIV-1 may have a function in increasing adherence to the target cell and thus enhance virus entry; a particularly well-studied example of which is ICAM-1 (Rizzuto and Sodroski, 1997). Antibodies against such host cell-derived molecules can have neutralizing activity; for example, ICAM1-expressing HIV-1 virions can be completely neutralized by an antibody against ICAM-1. Interestingly, the neutralizing antibody activity observed is comparable in magnitude to that of “classic” neutralization. The neutralizing effect, therefore, is far greater than can be explained by the loss of adhesion provided by virion ICAM-1 in contact with its cognate receptor LFA-1 on the target cell (Rizzuto and Sodroski, 1997). Antibodies against HLA class I and II have been shown to neutralize SIV, in which neutralization susceptibility appears to be determined by the number of HLA molecules present on the virion (Vzorov and Compans, 2000). Incorporation of influenza virus HA molecules on phenotypically mixed SIV virions, furthermore, renders the virions susceptible to neutralization by antibodies against influenza HA (Vzorov and Compans, 2000). This was also observed for phenotypically mixed virions of VSV with FPV, Sendai, and measles virus (Kimura, 1973; Wild et al., 1975–1976; Zavada and Rosenbergova, 1972), as discussed above. Neutralization of viruses by antibodies to foreign proteins on the viral surface supports the occupancy model. Clearly, viruses can be neutralized by the binding of antibodies to sites on the viral surface which do not directly serve a function in the infectious process (e.g., influenza HA on SIV). Coating of the viral surface with antibodies against such molecules, and subsequent steric interference with the virion–host cell interaction required to initiate infection, is an attractive hypothesis to explain these observations.

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N. INHIBITION OF INFECTION BY ANTI-RECEPTOR ANTIBODIES If the interruption of viral infection is primarily mediated by an occupancy and steric hindrance mechanism as we propose, then binding of antibodies to the viral receptor on the target cell should also inhibit the initiation of infection. We would predict that the ability of antireceptor antibodies to inhibit infection would then also be a function of occupancy and mostly independent of the epitope recognized. One of the best studied examples is probably the inhibition of HIV-1 infection by antibodies against its primary receptor CD4. CD4 is a molecule of four Ig-like domains. The first two domains project from the cell surface and are connected by a hinge to the last two domains that have been suggested to lie parallel to the cellular membrane (Wu et al., 1997). The gp120binding site has been mapped to the first, most membrane-distal domain of CD4 (Arthos et al., 1989). A large array of mAbs against distinct epitopes on CD4 have been studied for their ability to inhibit gp120 binding to CD4+ cells and HIV-1 infection, and most antibodies in fact were found to inhibit infection (Healey et al., 1990; Sattentau et al., 1986). It is interesting that even antibodies mapped to the third domain, which do not affect the interaction between CD4 and gp120, effectively inhibit infection. A comparison of mAbs against the first and third domains indicated that these antibodies inhibited HIV-1-induced syncytium formation for a range of isolates with similar potency. Inhibition of HIV-1 infectivity required that binding saturation of all available binding sites on the target cell surface was achieved. This inhibition was not correlated with a down regulation of CD4 during the experiment (Healey et al., 1990). The potency of mAbs to the third domain of CD4 is noteworthy, given that this domain is not required for HIV1 infection, as shown by replacement of the last two domains of CD4 with those of CD8, with retention of receptor activity (Bedinger et al., 1988). One antibody, which probably binds to domain 3 (OKT4), did not inhibit HIV-1 infection. However, this may reflect the relatively poor affinity of this antibody for CD4 (Healey et al., 1990; Truneh et al., 1991). One antibody against the fourth domain of CD4 does not appear to block HIV-1 infection (Healey et al., 1990; Truneh et al., 1991). Even though antibodies against the third domain of CD4 inhibit HIV-1 infection, they do not appear to inhibit virion binding to CD4+ T cells (Healey et al., 1990; Ugolini et al., 1997). Interestingly, it has been shown that most anti-CD4 antibodies, irrespective of the epitope recognized, are able to interrupt major histocompatibility complex (MHC) class II-dependent T cell activation. Similar to their HIV-1 inhibitory activity, significant inhibition only occurs at near-saturating antibody concentrations (Merkenschlager et al., 1990). The studies described above appear to show that inhibition of HIV-1 infection by anti-CD4 antibodies is dependent on the saturation of all available binding sites and is mostly independent of epitope recognized. However, a difference

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exists between antibodies against the virus and CD4 in their ability to inhibit virus–cell binding. Whereas all neutralizing antibodies against gp120 inhibit virus attachment, only antibodies which bind in the direct vicinity of the highaffinity gp120 binding site on CD4, and not antibodies to more-distant epitopes, including those in the first domain, inhibit attachment (Truneh et al., 1991; Ugolini et al., 1997). It may be that antibody affinity plays a role in the observed difference, as the inhibitory CD4 mAbs tested were of higher affinity than the noninhibitory CD4 mAbs (Healey et al., 1990; Truneh et al., 1991). Alternatively, it may be a consequence of differences in antibody coating densities. The spike density on the virus, and therefore its antibody coating density, is several orders of magnitude greater than the receptor and resulting antibody coating density of the target cell. It is therefore conceivable that a virus densely coated with a dense antibody cannot approach the target cell sufficiently to initiate binding, whereas viruses are not inhibited in their approach to separated clusters of CD4 molecules with antibodies attached to epitopes distant from the gp120 binding site. Presumably, however, the antibodies attached to CD4 then perturb the necessary spatial organization of the multiple envelope–receptor contacts and conformational changes required to initiate a productive infection event. Notably, it has been suggested that these same antibodies can interrupt the precise spatial organization of CD4-T cell receptor and accessory molecules required for triggering T cell proliferation (Merkenschlager et al., 1990). Following CD4 binding, HIV-1 requires the interaction with a chemokine receptor, belonging to the seven-transmembrane G-coupled receptor family, as an obligatory coreceptor for HIV-1 entry into the target cell. While a large number of chemokine receptors can serve as coreceptors for HIV-1 entry, the majority of isolates use either CXCR4 or CCR5 as their major coreceptor (see Berger, 1997, for a review). A recent study investigated the inhibition of HIV1 infection by antibodies against a number of epitopes on CCR5 (Lee et al., 2000). An antibody against the second extracellular loop (ECL2-A epitope), mAb 2D7, effectively inhibited HIV-1 infection and gp120 and chemokine binding to receptor. All other antibodies studied and directed against a different epitope on ECL2 (ECL2-B epitope), the N-terminus and epitopes involving multiple loops, blocked infection more poorly or not at all, and varied in their ability to inhibit gp120 and chemokine binding to receptor. MAbs against the N-terminus blocked gp120 binding better than chemokine binding, while the reverse was found for antibodies against the ECL2-B epitope. The affinities of these antibodies for CCR5 varied but were equal or greater than that of mAb 2D7 in some cases (Lee et al., 2000). Interestingly, it was found that CCR5 may exist in multiple conformational forms on the cell surface. MAb 2D7, the antibody with the strongest neutralizing activity, recognizes an epitope which is expressed on a much greater proportion of the CCR5 molecules than all the other mAbs tested, which appear to bind to only a fraction of the CCR5 molecules expressed

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(Lee et al., 2000). Therefore, a correlation between inhibition of HIV-1 infection and occupancy of binding sites exists, similar to the studies discussed above. We suggest that most antibodies against CCR5 are not effective in inhibition of HIV-1 infection, as they only recognize a subset of CCR5 molecules on the cell surface and therefore cannot saturate potential HIV-1 coreceptor binding molecules. III. Complement-Mediated Virolysis

The binding of antibodies to the viral surface may lead to the activation of complement, which is dependent on the isotype of the antibody and the spacing of the epitope recognized. Complement activation may lead to virus inactivation at subneutralizing concentrations. A number of enveloped viruses including HIV-1, HCMV, and vaccinia virus (Smith, 1999; Spiller et al., 1997) carry complement regulatory molecules on their surfaces. These are acquired from the host cell membrane during budding (e.g., CD46, CD55, and CD59), or recruited from plasma by attachment envelope glycoproteins, as in the case of factor H and HIV-1 (Saifuddin et al., 1994, 1995; Stoiber et al., 1997; Sullivan et al., 1996). These regulatory molecules promote the resistance of virions to virolysis by inhibiting full activation of the complement cascade and thereby provide the virus with a means to escape inactivation by the binding of relatively low amounts of antibody. Primary isolates of HIV-1 have been found to be more resistant than TCLA viruses to complement-mediated virolysis (Saifuddin et al., 1995; Spear et al., 1990; Sullivan et al., 1996). The resistance of primary isolates to virolysis was found to directly correlate with the poor exposure of antibody binding sites on the primary virus envelope (Takefman et al., 1998). A difference has been noted between HIV-1 primary isolates (grown in PBMC in vitro) and plasma virus in their sensitivities to complement-mediated neutralization (Sullivan et al., 1996, 1998a; Takefman et al., 1998). Plasma virus is considerably more sensitive to antibody-dependent, complement-mediated virolysis (Sullivan et al., 1996; Takefman et al., 1998). An explanation can be found in low expression levels of complement regulatory factors, in particular CD46 and CD55, on the plasma virion surface, compared to on a primary isolate virion (Sullivan et al., 1996). Diminished expression of these complement regulatory molecules on plasma virions may be explained by their decreased expression on CD4+ lymphocytes from HIV-1 infected individuals, since the viral membrane is derived from the cell membrane (Lederman et al., 1989; Weiss et al., 1992). Some viruses, such as orthopoxvirus and herpesvirus, express viral complement regulatory molecules either on their surface or in soluble form that may contribute to viral evasion (e.g., Fodor et al., 1995; Kotwal et al., 1990; Rother et al., 1994; Tortorella et al., 2000). Orthopoxviruses use both these strategies. First, the EEV, which is responsible for cell-to-cell and long-range spread of

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orthopoxvirus infection (Blasco and Moss, 1992), expresses a viral protein on its membrane, B5R, which has homology to complement control proteins and may provide protection against complement-mediated virolysis of EEV (Engelstad et al., 1992; Isaacs et al., 1992; Vanderplasschen et al., 1998). Vaccinia virus, cowpox, and variola virus secrete complement control proteins that inhibit complement activation via inhibition of C3 convertase activity (Kotwal et al., 1990; Rosengard et al., 1999; Tortorella et al., 2000). IV. Antibody-Mediated Phagocytosis

Virions coated by antibody may be eliminated by phagocytosis followed by inactivation in an intracellular compartment within the phagocyte. This process has been described for the picornavirus foot-and-mouth disease virus (FMDV). FMDV sensitized with antibody at subneutralizing concentration was actively phagocytosed in vitro by monocytes and macrophages (McCullough et al., 1988). Phagocytosis in vivo, furthermore, appears to play a role in protection against FDMV infection (see below). V. Antibody-Mediated Cytotoxicity

Antibodies which bind effectively to viral proteins expressed on the surface of infected cells may trigger complement-mediated cytotoxicity (CMC) and ADCC and thereby provide protection against viral infection in vivo. The antibody isotype and spacing of the epitopes are important parameters for the efficient induction of effector functions by antibody. The dominant human IgG isotype IgG1 binds well to IgG Fc receptors and is capable of activating complement via the classical pathway (Burton and Woof, 1992; Parren, 1992). CMC and ADCC can be mediated by neutralizing antibodies (directed against viral proteins also expressed on the virion) as well as nonneutralizing antibodies (directed against viral proteins exclusively expressed on infected cells). The role of CMC and ADCC in protection against viral infection will be discussed in specific examples below. Generally, however, it may be expected that neutralizing antibodies provide stronger protection than nonneutralizing antibodies, as the former can act against free virions as well as against infected cells (see below). A number of viruses, such as HCMV, HSV-1, HSV-2, and varicella-zoster virus (VZV) express virally-encoded Fc receptors on their surface. It has been suggested that viruses decorated with antibody use these receptors to bind Fc, thereby obscuring this part of the immunoglobulin (Ig) molecule from cellular Fc receptors or complement (Lubinski et al., 1998; Tortorella et al., 2000). This indeed would seem an effective mechanism by which virions with neutralizing antibody bound at low occupancy may escape inactivation in vivo.

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VI. Intracellular Neutralization

Polymeric IgA and IgM involved in mucosal immunity may make use of a specialized route to neutralize viruses or prevent viral entry into the host. These antibodies are actively transported over the mucosal epithelium after binding to the polymeric Ig receptor and may, during transport, contact and neutralize transcytosing viruses. Sendai virus added to the apical side of an epithelial cell monolayer was shown to interact intracellularly with polymeric IgA added basolaterally, and viral titers were reduced in cellular supernatant and lysate (Fujioka et al., 1998; Manzanec et al., 1992). Manzanec et al. demonstrated neutralization of influenza virus via a similar mechanism (Manzanec et al., 1995; reviewed in Kato et al., 2000). Studies by Bomsel et al. (1998) have shown that that transcytosis of primary HIV-1 isolates over tight epithelium can be blocked by dimeric immune IgA and IgM. It was shown that basolaterally internalized antibodies met transcytosing virions at an intracellular compartment, thereby redirecting the virions back to the mucosal compartment (Bomsel et al., 1998). Although the affinity for virions of the polyclonal dimeric IgA and IgM, purified from serum from infected individuals, is likely to be low, efficient binding may be occurring by a multivalent interaction at relatively high concentrations within the intracellular compartment. Polymeric IgA and IgM therefore may bind to virus in an intracellular compartment, neutralizing infectivity or redirecting transcytosis. VII. Mechanisms of Antibody Protection in Vivo

A. INTRODUCTION A number of the mechanisms described for antiviral activity in vitro may operate in vivo. These are summarized in Fig. 5. They include mechanisms that act upon free virions, such as neutralization, and those that act upon infected cells, such as CMC and ADCC. As discussed above, in our view, neutralization is mediated by antibodies that bind to envelope molecules on the virion surface and effectively coat the virion, preventing viral attachment and/or fusion. Such antibodies can also bind to envelope molecules expressed on the surface of infected cells and thereby trigger elimination of these cells by Fc-mediated effector systems. Nonneutralizing antibodies can similarly bind to viral proteins expressed on infected cells to produce antiviral activity. These proteins can include envelope molecules in forms that are not expressed or only expressed in relatively low amounts on virions. Finally, there is evidence of special instances where binding of antibodies to infected cells appears to suppress viral replication without leading to cell destruction. For many viruses, there is a correlation between levels of serum antibody neutralizing activity in vitro and in vivo protection. Are these antibody levels responsible for the protection or merely correlated with it? The classical approach

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FIG. 5. Mechanisms of antiviral antibody activity in vivo. Antiviral activity of antibodies can be separated into activity against virions and activity against infected cells. Neutralization is mediated by antibodies that bind well to molecules on the virion surface and effectively coat the virion, thereby preventing viral entry. Antibody-mediated effector mechanisms such as complement-mediated lysis and phagocytosis can aid in virus inactivation. Antibodies against envelope spikes of an enveloped virus may also bind to envelope molecules on the surface of infected cells and thereby trigger elimination of these cells by Fc-mediated effector mechanisms. Nonneutralizing antibodies can similarly bind to viral proteins on infected cells to produce antiviral activity. These proteins can include molecules in forms that are not expressed or only expressed in low amounts on virions.

to this question has been to passively transfer serum antibodies from immune animals to na¨ıve animals and determine the protection provided. In many cases (Chanock et al., 1993), passive transfer does provide protection from disease. There is also evidence in humans of the ability of passively transferred antibodies to protect against disease arising from a number of viral pathogens including RSV, hepatitis A virus, measles virus, poliovirus, VZV, and variola virus (Chanock et al., 1993). In most cases, it has not been determined whether antibodies provide

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sterile protection. It may be that, in many cases, antibodies limit infection, and cellular and innate immunity are also required in protection from disease. Antiviral monoclonal antibodies permit a more quantitative investigation of the relationship between neutralization in vitro and protection in vivo. Clearly, the comparison presents difficulties in that, even if very similar antiviral mechanisms operate in vitro and in vivo, the latter involves a variety of factors for which it is difficult to control, and which may not be easily reproduced in vitro. For instance, the host cell type used in vitro may differ from the activation/maturation state of the cell type infected in vivo. In fact, cell lines are often used in vitro. Other factors and molecules may be influential in vivo but absent in vitro. Viruses with distinct characteristics may be selected in vivo and in vitro, leading to aberrant conclusions; HIV-1 and lactate dehydrogenase-elevating virus (LDV) are striking examples, which will be discussed below. Thus, caution in any comparison is appropriate, but nevertheless it is important to ask how well neutralization of mAbs in vitro correlates with protective activity in vivo. It should be pointed out that a good correlation does not necessarily prove that the mechanisms of in vitro neutralization and in vivo protection are the same. Since neutralization is so strongly correlated with virion binding, any mechanism that was strongly correlated with such binding (e.g., complement-mediated virolysis) could in theory be responsible. However, in many cases, in vivo activity has been shown to be independent of the Fc part of the IgG molecule, implying that the neutralization blocking mechanism is probably paramount. B. RETROVIRUSES For HIV-1, we have reported (Gauduin et al., 1997; Parren et al., 1995, 1997a) that sterile protection of hu-PBL-SCID mice from virus challenge with laboratory-adapted or primary viruses requires serum mAb concentrations one to two orders of magnitude greater than those required for 90% in vitro neutralization, that is, concentrations corresponding to essentially complete virus neutralization in vitro. TCLA viruses, which are much easier to neutralize in vitro, are correspondingly much easier to protect against in vivo. Similar findings with regard to the relationship between neutralization and protection are apparent for challenge of macaques with chimeric SIV/HIV (SHIV) viruses (Baba et al., 2000; Mascola et al., 2000; Shibata et al., 1999). In one instance of mucosal challenge, partial protection was described for a serum mAb concentration corresponding to only about 90% neutralization in vitro (Mascola et al., 1999). Recently, however, we have found that protection from mucosal challenge with an R5 SHIV virus required mAb concentrations capable of neutralizing essentially all of the challenge virus (Parren et al., 2001b). In fact, the results in the macaque model closely paralleled those in the SCID mouse study: Since effector systems are likely to be considerably more efficient in the former than the latter model, the results imply that effector systems are not essential in protection against HIV-1.

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Research on the biology of tumors induced by murine leukemia viruses (MuLV) has shown that antibodies against MuLV antigens expressed on the surface of malignant-infected cells can provide protection against tumor growth. Antibodies against Gross virus cell surface antigens provided complete protection against challenge with syngeneic Gross cell surface antigen-positive leukemia cells. Lysis of these cells by CMC was demonstrated in vitro (Old et al., 1967). The Gross virus cell surface antigen was later found to be identical to MuLV glycogag, which is a glycosylated version of the gag polyprotein that is expressed on the cell surface but is not incorporated into virions (Ledbetter and Nowinski, 1977). It should be noted that the antisera used by Old et al. (1967) also contained neutralizing antibodies against Gross virus, so that it is uncertain that all of the protective effect was mediated by antibodies against glyco-gag. Similarly, however, nonneutralizing mAbs against Moloney MuLV (M-MuLV), which react with a Friend–Moloney–Rauscher virus group-specific antigen expressed on the surface of infected cells but not on virions, protect against tumor growth (Lamon et al., 1987). Pincus et al. (1995) more recently showed that nonneutralizing antibodies against MuLV (glyco-)gag delayed onset of MuLV pathogenesis. An antibody against gag also delayed disease onset as a Fab fragment (albeit less effectively), indicating that clearance of infected cells by Fc-mediated effector functions cannot wholly explain the protective effect. It has been shown that MuLV glycogag plays an important role in viral spread and pathogenesis (Corbin et al., 1994; Portis et al., 1994). Occupancy of glyco-gag on the cell surface by antibody in the form of whole IgG as well as Fab fragment may therefore be able to interfere with glyco-gag function, thereby providing protection by limiting virus spread without directly clearing infected cells. Protection against MuLV challenge as expected is also provided by antibodies against envelope which are more effective than antibodies against gag and show a good correlation between neutralization titer and the degree of protection provided (Pincus et al., 1995). The virus used in these studies was a recombinant Friend MuLV (F-MuLV) with the env gene of the neurovirulent wild-mouse ecotropic virus CasBrE inserted. Antibodies against envelope were also effective against F-MuLV or F-MuLV-infected cells when transferred postchallenge, but then required an Fc portion to be protective. A reduction in the frequency of F-MuLV-producing cells in leukemic spleen could be induced by neutralizing mAbs (IgG2a) which mediated CMC but not others (Britt and Chesebro, 1983). Passive transfer of neutralizing antibodies against F-MuLV envelope furthermore protected against F-MuLV pathogenesis as a whole antibody but not as F(ab′ )2 fragments (Collins et al., 1983). Friend virus has been used as a model to study the mechanisms by which immunization with live attenuated retroviruses protect against subsequent challenge with more virulent strains. The model used is complex but interesting, as

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it is one of the few models of an immunosuppressive retrovirus inducing disease in immunocompetent mice. Challenge is performed with a combination of two viruses: a nonpathogenic replication-competent helper virus (F-MuLV), which contains all the required immunological determinants, and a replicationdefective virus, spleen focus-forming virus (SFFV), required for pathogenicity in adult mice (Dittmer et al., 1998). Immunization with attenuated F-MuLV elicits highly protective immune responses. It has been shown that this protective immune response involves a CD4+ and CD8+ T cell as well as a B cell component, as protective responses could only be transferred to na¨ıve mice by combining all three cell populations in the transplant (Dittmer et al., 1999a). Neutralizing antibodies are present in this response at a level below the concentration required to provide sterile immunity, but nevertheless appear to play an important role in the observed protection against disease, as evidenced by the following. First, protection from plasma viremia is completely correlated with the presence of F-MuLV neutralizing serum antibodies (Dittmer et al., 1999a). Second, passive transfer of a F-MuLV neutralizing antibody to the same level as observed after immunization protects against pathogenicity, as all animals receiving the antibody recovered from Friend virus-induced disease (Dittmer et al., 1999a). Third, complete protection correlated with the presence of both virus-neutralizing antibodies as well as primed cytotoxic T lymphocytes (CTL). Development of protection against persistent infection, in particular, only developed postimmunization after the appearance of neutralizing antibodies (Dittmer et al., 1999b). This series of experiments indicates that neutralizing antibodies below the level required to induce sterile protection can play a critical role in protection against a retrovirus. C. RHABDOVIRUSES—VESICULAR STOMATITIS VIRUS A classical early study was that of Lefrancois (1984) who described a panel of mAbs to glycoprotein G of VSV. Neutralizing mAbs completely protected mice at relatively high doses. Nonneutralizing mAbs also protected, although they were less effective. Neither class of mAbs protected, if given as early as 2 hr postinfection. The nonneutralizing mAbs bound much less effectively than neutralizing mAbs to intact virions. However, the nonneutralizing mAbs could lyse infected cells in the presence of complement. Further, the F(ab′ )2 fragment of a nonneutralizing mAb was not protective, whereas the same fragment of a neutralizing mAb was protective. It was suggested that the nonneutralizing mAbs were likely mediating protection via Fc-dependent effector function activity against infected cells. The behavior of the F(ab′ )2 fragment of the neutralizing mAb suggests that effector systems are not crucial in the protective activity of this antibody. A very interesting feature of this study is the evidence it provides that a certain form of the envelope protein is present at the surface of infected cells, which is essentially absent from virions.

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One of the most detailed comparisons of neutralization and protection is that of Bachmann et al. (1997) who described the properties of a large panel of mAbs to a single antigenic site on VSV-G. The authors reported a good correlation between neutralizing ability of antibody and affinity (avidity) for intact virus particles. However, these two parameters were reported not to correlate with the ability of antibody to protect SCID mice from viral challenge. Protection was said to depend simply upon a minimum serum concentration of antibody independent of affinity. However, most of the antibodies studied fell in a rather narrow affinity range (2–9 × 109 M−1; corresponding to a free energy of binding range of only about 1 kcal/mole), and indeed the minimum serum concentration for complete protection (3/3 mice) occurred over a corresponding narrow range of roughly 20–50 ␮g/ml. An antibody of significantly lower affinity (2.5 × 107 M−1) was found not to protect mice at any of the concentrations used. This contradicted the thesis that neutralization and protection were not correlated, and was interpreted as a “threshold effect” below which the protective behavior of antibody was predictable by the law of mass action. One antibody of somewhat lower affinity (2 × 108 M−1) but apparently disproportionately poor neutralizing ability was able to protect all challenged mice at approximately 20 ␮g/ml. In our view, this latter antibody is the only one that potentially offers a significant challenge to the correlation between affinity/neutralization and protection. D. ALPHAVIRUSES Early studies investigated mAbs to the envelope E1 and E2 proteins of Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalomyelitis (VEE) virus. In a study on Sindbis virus (Schmaljohn et al., 1982), neutralizing anti-E2 and nonneutralizing E1 antibodies were protective. The latter were shown to bind to infected cells but not to virions, and it was suggested that complement-mediated lysis of infected cells might be crucial. Later studies (Schmaljohn et al., 1983) confirmed that the protective epitopes on E1 were only present on infected cell surfaces and not on virions. Neutralizing and nonneutralizing mAbs against the E2 protein of SFV were shown to protect mice from lethal infection, although the former were effective at much lower doses (Boere et al., 1985). Protection was observed for neutralizing mAbs at concentrations expected to saturate the challenge virus, and there was a good correlation between the efficacy of mAb with respect to in vitro neutralization and protection. Both neutralizing and nonneutralizing mAbs bound to infected cells and promoted complement-mediated lysis, although the neutralizing mAbs were more effective. Removal of the Fc part of a neutralizing mAb reduced its neutralizing capacity about 100-fold with a roughly comparable reduction in protective efficacy. Removal of the Fc part of a nonneutralizing mAb caused a complete loss of its protective ability. The results are

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consistent with a crucial role for Fc-mediated effector functions in protection by the nonneutralizing antibody but a lesser role for the neutralizing antibody. A potent anti-E2 neutralizing mAb was found to protect mice against lethal challenge with VEE (Mathews et al., 1985). However, the corresponding F(ab′ )2 fragment was not protective, although its neutralizing potency was the same as the parent mAb. The importance of Fc effector functions for protection was inferred, although the much shorter half-life of F(ab′ )2 fragments in vivo may have contributed to its inefficiency. A nonneutralizing anti-E1 mAb was found to protect at much higher concentrations, presumably through its binding to infected cells, since the antibody did not bind to intact virions. E. ARENAVIRUSES Passively administered immune serum, maternal antibodies, and anti-envelope glycoprotein (G1) mAbs have been shown to protect mice against lethal challenge with lymphocytic choriomeningitis virus (LCMV) (Baldridge and Buchmeier, 1992). Protection with mAbs required concentrations leading to essentially complete neutralization of virus in vitro. It appears that complete protection required antibody activity not only against free virions but also against infected cells. Thus F(ab′ )2 fragments of a protective mAb retained neutralizing activity in vitro but were not protective in vivo. Furthermore, an IgG1 anti-GP1 mAb with more potent neutralizing activity than protective anti-GP1 IgG2a antibodies was not protective. The activity against infected cells did not involve complement, since C5-deficient mice were also protected by the IgG2a mAbs. An interesting advantage of passively transferred neutralizing antibodies in LCMV infection is that, by effectively reducing the viral challenge, they can attenuate the subsequent CTL response (Wright and Buchmeier, 1991). This can be crucial in modulating the potentially destructive effects of an “over-vigorous” T cell-mediated immune response. F. CORONAVIRUSES Several early studies looked at the ability of mAbs to protect mice against lethal challenge with murine hepatitis viruses (MHVs). The ability of a panel of 13 mAbs given i.p. to protect mice against intracerebral challenge with MHV4 showed that 3 potently neutralizing mAbs specific for the E2 glycoprotein were protective at high dose (Buchmeier et al., 1984). MHV-4 infection was not blocked completely, but viral loads in liver, brain, and central nervous system (CNS) were lower than in unprotected mice, and demyelination rather than fatal encephalomyelitis resulted. One potently neutralizing anti-E2 mAb was not protective. However, this mAb recognizes an epitope which is subject to rapid neutralization escape (Dalziel et al., 1986), and the challenge virus may have contained escape variants of unusual fitness in vivo (M. J. Buchmeier,

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personal communication). Nonneutralizing anti-E1 membrane glycoprotein and anti-N nucleoprotein mAbs were nonprotective. Another study investigated the activities of 20 mAbs specific for the E2 protein of MHV-4 (Wege et al., 1984). Three mAbs that potently neutralized virus, inhibited cell fusion, and bound well to infected cells protected against intracerebral challenge. All other mAbs including some that neutralized more weakly and some that did not neutralize but bound well to infected cells were not protective. A further study assessed the activities of 4 anti-E1 mAbs. Only one was neutralizing and then only in the presence of complement. This mAb was protective. Of the 3 nonneutralizing mAbs, 1 was protective. The neutralizing mAb was equally effective in C5deficient or decomplemented mice, suggesting that complement was not crucial for protection. Monitoring of the viral load in liver, spleen, and brain allowed the effects of mAbs in i.v. challenge of mice with MHV-2 to be explored quantitatively (Nakanaga et al., 1986). Protection against lethal challenge was provided by a potent neutralizing anti-E2 mAb, a nonneutralizing anti-E2 mAb, and a nonneutralizing anti-nucleoprotein (NP) mAb. However, the neutralizing mAb resulted in much lower viral loads in the tissues of infected animals, particularly in the brain where viral titers were 3–4 orders of magnitude lower for the neutralizing mAb-treated than the nonneutralizing mAb-treated mice. Furthermore, the neutralizing mAb was able to protect against a challenge dose of 30,000 pfu compared to the usual dose of 30 pfu. Protection by the anti-NP is presumably mediated by activity against infected cells that express NP on the surface, and this was confirmed for MHV-3 challenge (Lecomte et al., 1987). A comparison of a potent neutralizing IgG and the corresponding F(ab′ )2 and Fab fragments showed that all three molecules could neutralize the neurotropic MHV-A59 virus, and all three could protect mice against lethal intracerebral challenge at very high doses (producing serum titers approximately 104 times 90% neutralization titers in vitro). The efficiency of protection of a given molecule correlated well with its potency of neutralization and affinity for antigen (Lamarre and Talbot, 1995). A single-chain Fv fragment of the same mAb could neutralize virus but was ineffective at protection, presumably because of its extremely short in vivo half-life (6 min) (Lamarre et al., 1997). G. FILOVIRUSES A recent study (Wilson et al., 2000) describes the in vitro and in vivo effects of a panel of mAbs to the envelope glycoprotein of Ebola virus. The majority of mAbs protected mice against challenge with a mouse-adapted Ebola virus. None of the protective antibodies neutralized the virus in a plaque assay in the absence of complement. In the presence of complement, a subset of antibodies neutralized the virus. For these antibodies, complete protection was observed at a serum concentration approximately an order of magnitude greater than the

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concentration required for 80% neutralization in vitro. A caveat to note here is that neutralization is carried out on plaquing virus, although most infectious particles are nonplaquing, as revealed by the correspondence of the challenge dose of 300LD50 to only 10 pfu of virus. Using a potent human neutralizing anti-Ebola antibody (Parren et al., 2001a), we found that protection against a guinea pig-adapted virus in a guinea pig model required serum antibody concentrations corresponding to essentially complete virus neutralization in vitro (Maruyama et al., 1999; Parren et al., 2001a). In this case, the viral challenge was much higher (104 pfu; >105 LD50) than the previous example (10 pfu; 300 LD50). H. FLAVIVIRUSES Early studies showed that neutralizing mAbs against the envelope glycoprotein and nonneutralizing antibodies against the nonstructural glycoprotein of yellow fever (YF) virus could protect mice against lethal intracerebral challenge (Gould et al., 1986; Schlesinger and Chapman, 1995). Neutralizing mAbs were more effective and could protect against more neurovirulent strains of YF virus, against which the anti-NS mAbs were ineffective. More recently, it was shown that the F(ab′ )2 fragments of a strongly neutralizing IgG2a mAb were equally effective at neutralization but did not mediate protection in vivo (Schlesinger and Chapman, 1995). However, incubation of F(ab′ )2 virus complexes with a rabbit IgG antimouse IgG resulted in protection. In contrast, the corresponding rabbit F(ab′ )2 fragment did not afford protection. In addition, an IgG1/IgG2a switch variant was suggested to have the specificity of the parent IgG1, but the Fc of IgG2a was more effective in protection than the parent IgG1. The results imply a role for Fc-mediated effector systems in protection against YF. A recent study (Forthal et al., 1993) on the development of humoral immunity to tick-borne encephalitis virus (TBEV) provides some novel insight into how antibodies to virions and to infected cells can be important. Passive transfer of polyclonal neutralizing antibodies at doses expected to completely neutralize virus in vitro protected mice from lethal TBEV challenge. However, despite the high doses used, protection was not sterile and some viral replication occurred. Transfer of sera from these mice could confer complete protection from disease on na¨ıve mice. Interestingly, transfer of T-enriched spleen cells from the mice could not confer protection on na¨ıve mice. The specificity of the transferred antibodies was shown to be for the nonstructural protein (NS1) which is absent from the virion, implying that antibody activity against infected cells can confer protection. Antibody-dependent enhancement (ADE) of TBEV can be demonstrated in vitro, and it has been suggested that postexposure Ig prophylaxis, which is widely used in nonvaccinated individuals who are bitten by ticks in areas where the virus is endemic, might worsen the outcome of infection. However, studies in

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mice with antibodies that did show ADE in vitro gave no evidence of enhanced infection in vivo under a range of conditions (Kreil and Eibl, 1997). It should be noted that an in vivo relevance of antibody-mediated enhancement of infection is uncertain in many cases. The flavivirus dengue virus, however, forms a clear example of a virus for which a strong in vivo enhancement by antibodies against envelope glycoprotein has been demonstrated (Halstead, 1979, 1982). I. HERPESVIRUSES Passive transfer of immune rabbit anti-IgG was shown to protect mice against lethal challenge with HSV type 1 (HSV-1) after corneal inoculation and HSV-2 after subcutaneous inoculation (Oakes and Lausch, 1981). Protective serum neutralizing titers were approximately 1:50. The corresponding F(ab′ )2 preparation was equally effective at neutralization in vitro and protection in vivo. However, interestingly, only the IgG preparation showed activity in established infection. In another model of HSV-1 infection, mice were inoculated with virus in the footpad and then treated 4 hr later with a single dose of immune rabbit IgG or five daily doses of the corresponding F(ab′ )2 preparation (McKendall, 1985). Both treatments led to serum neutralizing antibody titers of 1:16. IgG treatment markedly, and F(ab′ )2 treatment moderately, reduced footpad viral titer, viral spread to sciatic nerve and spinal cord, and the establishment of latency. The IgG effects were also apparent in C5-deficient mice. A further study looked at the activity of immune mouse and human IgG against HSV in nude mice (Hayashida et al., 1982). Relatively high concentrations of antibody (1 ml of serum neutralizing titer >1 : 32 given i.p.) were required to inhibit the development of skin lesions and to prolong survival of lethally infected mice. Purified F(ab′ )2 fragments were ineffective even when given repeatedly to maintain neutralizing titer. IgG was effective in C5-deficient mice. The results are consistent with the notion that ADCC becomes crucial in protection against HSV if sterile protection is not provided at challenge. Many studies have looked at the ability of mAbs to protect against HSV infection. Early papers showed that both neutralizing anti-gD and gC mAbs and nonneutralizing anti-gD, gC, gB, gD, and gE mAbs could protect against lethal footpad challenge in mice (Balachandran et al., 1982; Dix et al., 1981). The latter study found that a mixture of mAbs had an efficacy corresponding roughly to the sum of that of the individual mAbs and that protection was equally apparent in C5-deficient mice. A good correlation was reported between protection and the titers of the nonneutralizing mAbs in an ADCC assay. It was suggested that ADCC could provide protection against lethal HSV infection. In the murine zosteriform spread model, infection is initiated at a cutaneous site and spreads to the peripheral nervous system, from which the virus (HSV-1) reemerges and infects regions of the epithelium remote from the inoculation

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site. The model mimics the course of human herpetic disease. High doses (0.2–5 mg/mouse) of anti-gD, gC, and one gB mAb offered partial protection in the model (Mester et al., 1991). It was reported that protection correlated better with in vitro ADCC than neutralization activity. In addition, F(ab′ )2 and Fab fragments of two protective anti-gC mAbs were nonprotective. Investigation of the ability of a panel of anti-gD mAbs to protect against HSV-2 challenge showed that a mAb, neutralizing virus even in the absence of complement, was the most effective (Ishizaka et al., 1995). Of a series of mAbs that neutralized in the presence of complement, the order of efficacy of a series of switch variants was IgG2a>IgG2b>IgG1. A human neutralizing antigD mAb was shown to completely protect nude mice against lethal challenge with HSV-1 using a high iv dose (450 ␮g/mouse) (Sanna et al., 1996). The same mAb applied topically could protect C57Bl/6 mice against vaginal challenge with much lower doses of mAb (400 ng/mouse) (Zeitlin et al., 1996). IgG and F(ab′ )2 fragment were approximately equivalent in topical protective ability, with the Fab fragment being somewhat less effective. J. ORTHOMYXOVIRUSES—INFLUENZA VIRUS A detailed study (Mozdzanowska et al., 1997) has compared the in vitro neutralization and in vivo protective activities of a number of mAbs to influenza virus HA. The mAbs were chosen to have a range of neutralizing activities. In the presence of complement (1.6% serum), neutralizing ability was differentially enhanced from 2- to 75-fold. The serum antibody concentration conferring protection was two to three orders of magnitude higher than the concentration required for 50% neutralization in vitro in the presence of complement. However, the rank order of protective ability was not well predicted by the order of neutralizing ability. K. PICORNAVIRUSES McCullough et al. (1986) have shown in passive transfer studies that a good correlation exists between neutralization and protection against FDMV, as only antibodies which neutralized a certain FDMV challenge isolate strongly or moderately provided protection. Interestingly, however, these antibodies protected at concentrations 10- to 60-fold below those required for in vitro neutralization of FDMV. The enhanced protective ability of these antibodies appeared Fc-mediated, as conversion of the mAb into F(ab′ )2 fragments reduced protective ability, whereas their neutralizing ability was mostly retained (McCullough et al., 1986). Neutralization titers were not affected by the addition of complement, suggesting that CMC did not play a significant role (McCullough et al., 1988). The effect rather appeared due to the induction of phagocytosis of sensitized virus, as impairment of phagocytosis by silica treatment abrogated the enhanced protection. In silica-treated mice, neutralizing antibody concentrations

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well over the 90% neutralization titers were required for protection, indicating that in the absence of phagocytosis, protection correlates with neutralization of all the challenge virus. It is likely that efficient protection against FDMV infection by neutralizing antibody at low occupancy is provided by an efficient phagocytosis of sensitized virions, as has been demonstrated for FDMV in vitro (McCullough et al., 1988, 1992 ). L. PARAMYXOVIRUSES Many studies have looked at the activities of antibodies against RSV, and one anti-RSV mAb is in clinical use for the prophylaxis of RSV disease. Passively transferred polyclonal antibody administered i.p. to produce a serum neutralizing titer of 1:380 or greater provided sterile protection in the lungs of cotton rats challenged with RSV (Prince et al., 1985b). Similarly, high doses of anti-F or anti-G glycoprotein mAbs reduced the virus titers in the lungs of cotton rats challenged with RSV to undetectable levels (Walsh et al., 1984). From 15 antiRSV mAbs, including 10 to the F glycoprotein, only 2 were neutralizing in vitro, and these were the only mAbs that were completely protective in mice (Stott et al., 1984). In contrast, a nonneutralizing anti-G mAb that protects SCID mice against intranasal RSV challenge at a dose of 5 mg/kg has been described (Corbeil et al., 1996). Protection is reduced in decomplemented or C5-deficient mice, suggesting complement is important in the antiviral activity. In bovine RSV, two neutralizing anti-F mAbs administered by the intratracheal route at moderate dose (0.4 mg/kg) protected against infection, whereas a nonneutralizing mAb was not protective (Thomas et al., 1998). An anti-F IgA at 0.5 mg/kg introduced intranasally to mice prior to RSV challenge greatly reduces virus titers in the lungs (Weltzin et al., 1994). In human phase III trials, the enhanced activity of one mAb relative to another has been attributed at least in part to its better neutralizing ability in vitro (Johnson et al., 1999). The antiviral activity of a panel of nine mAbs to the F protein of Sendai virus has been compared in vitro and in vivo (Mochizuki et al., 1990). None of the mAbs were neutralizing in standard assays, but two were potently neutralizing in the presence of complement. These mAbs protected very young mice against lethal infection and allowed them to thrive in terms of body weight gain, when given at high dose. Three other mAbs showing weak or no neutralization in the presence of complement were also protective. Two of these mAbs were of the IgG1 isotype, which lead to the suggestion that ADCC rather than complement might be important in protection. M. REOVIRUSES Passive transfer of a neutralizing mAb against bluetongue virus (BTV) has been shown to protect mice and sheep from disease, whereas nonneutralizing mAb did not protect. A neutralizing antibody titer of 1:20 protected the

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sheep from disease, but did not provide sterile immunity, as an increase in neutralization titers was observed 8 to 9 days postchallenge. The results are in strong contrast with the inability of 100-fold higher neutralizing antibody titers to clear virus from sheep with existing infection (Letchworth and Appleton, 1983). The impact of antibody on established infection is discussed in detail below. N. NEUROPATHOGENIC LACTATE DEHYDROGENASE-ELEVATING VIRUS AND HIV-1: CAUTIONARY NOTES ON THE DIFFICULTIES OF COMPARING IN VITRO AND IN VIVO ANTIBODY ACTIVITIES Infection of mice of certain strains with neuropathogenic LDV results in a fatal paralytic disease through interaction with endogenous retroviruses. The infection of anterior horn neurons by LDV and the development of disease are prevented by anti-LDV antibodies. The mechanism by which motor neurons were protected from infection by LDV was unclear as in addition to neutralizing antibodies also nonneutralizing (polyclonal) antibodies prevented neuron destruction and disease. In addition, protection occurred in the absence of any apparent effect of antibody on LDV replication in a subpopulation of macrophages known to be the primary permissive host cells. The resolution to this paradox is a lesson with regard to the ability of viral quasispecies to mislead (Chen et al., 1999). It appears that neuropathogenic LDV isolates contain both neuropathogenic and nonneuropathogenic quasispecies. Using biological clones, it was shown that the nonneuropathogenic species were about 100 times more resistant to in vitro neutralization than the neuropathogenic species. Some antibodies therefore do not neutralize nonneuropathogenic viruses and are scored as “nonneutralizing” by in vitro assays. These antibodies do, however, neutralize neuropathogenic viruses and therefore are protective in vivo. The paradox described above appears to be due to LDV heterogeneity. It is of interest that mixed virus populations with distinct neutralization properties have also been described for another arterivirus: PRRSV (Weiland et al., 1999). T-cell line adapted strains of HIV-1 represent a striking example of how studying neutralization of viral variants selected in vitro may lead to aberrant conclusions with respect to the neutralizing responses in infection. The adaptation of HIV-1 to growth in CD4+ T cell lines selects for variants that are readily neutralized by soluble CD4 and a large spectrum of different mAbs. By contrast, plasma virus or viruses which have only been passaged in primary cultures of activated peripheral blood mononuclear cells (PBMC) are mostly resistant to neutralization by these same ligands (reviewed in Moore and Ho, 1995; Parren et al., 1999). An explanation for this phenomenon may be the high expression levels of heparan sulfate proteoglycans on the surface of T cell lines, which through an interaction with gp120 may select for HIV-1 viruses with unusual

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strongly basic gp120 V3 loops (Moulard et al., 2000). The envelope spike of these TCLA viruses adopts a much more open configuration, which provides access to a range of epitopes which are inaccessible on naturally occurring HIV1 isolates. Consequently, sera from HIV-1 infected individuals, as well as sera from vaccinees immunized with recombinant HV-1 envelope subunits, typically contain high titers of neutralizing antibodies against TCLA strains of HIV-1, whereas the more relevant titers against HIV-1 primary isolates are usually very poor. O. FURTHER CAUTIONARY NOTES ON COMPARING IN VITRO AND IN VIVO ANTIBODY ACTIVITIES: THE POSSIBLE EFFECTS OF VIRUS CHALLENGE DOSE AND ANATOMICAL CONSIDERATIONS It is in the nature of passive transfer studies that typical virus challenge doses are relatively large to ensure that all control animals become infected. It is sometimes argued that, in some human infections, challenge doses could be smaller and protection achieved at lower antibody concentrations than indicated from animal studies. For example, SIV or SHIV experiments in macaques usually involve virus challenge doses of 10 AID50 (50% animal infectious dose) or more. However, the typical human challenge dose with HIV-1 is probably 0.01 AID50 or less, since the frequency of infection is about 1:100 or less, depending upon the nature of exposure. This has been interpreted to indicate that relatively low vaccine-induced serum antibody concentrations compared to in vitro neutralization titers may offer protective benefit against HIV-1 infection. From a thermodynamic standpoint, this argument appears to have little merit, since the amount of serum antibody will be in vast molar excess over the challenge virus in most scenarios, and the extent of antibody coating of virus (and therefore neutralization) should be determined by the binding constant of antibody to virus rather than the number of challenge particles. However, there are no studies of which we are aware that have directly addressed the ability of antibody to protect at very low challenge doses. For a given virus, the existence of factors during low dose natural infection processes outside the laboratory that complicate the extrapolation from animal studies to humans cannot be categorically excluded. Anatomical considerations may also complicate the interpretation of in vivo protection data. Antibody serum concentrations can be readily measured. For the most part, tissue antibody concentrations are not measured, and the concentration of antibody that may in fact be responsible for blocking infection at a tissue site is not known. It is possible that the high serum antibody concentrations required for protection in some instances reflect the difficulty of achieving protective antibody concentrations at a critical tissue site. If some antibodies are better able than others to diffuse to tissue sites, they may therefore show enhanced in vivo relative to in vitro activity.

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VIII. Mechanisms of Antiviral Antibody Activity in Established Infection

Although there is evidence that antibody can impact upon a number of established viral infections (Chanock et al., 1993), there are little quantitative data relating in vivo and in vitro activities. In the data reviewed below, we consider antibody given 1 day or more after virus as having activity against “established” infection as opposed to the prophylactic activity considered above. This is clearly a somewhat artificial distinction, and antibody generally appears to have diminished activity the further infection progresses. Elegant studies show that SCID mice infected with influenza A for 1 day can be cured using neutralizing mAbs to viral HA with a close correlation between prophylactic and curative activity (Mozdzanowska et al., 1997). The data suggest that the curative effect is mainly due to the neutralizing activity of antibody against free virus, with some contribution from activity against infected cells. This conclusion is supported by investigation of two nonneutralizing mAbs, one to viral NA and one to M2 (Mozdzanowska et al., 1999). Both mAbs reduced pulmonary viral titers in established infection by 100- to 1000-fold, but they failed to clear infection even at high dose in combination. It might be predicted that neutralizing antibody would have activity against a virus such as influenza that does not propagate via cell-to-cell spread and is cytopathic for infected cells. The presence of high levels of neutralizing antibody should eventually terminate infection. However, for viruses that do propagate via cell-to-cell spread, antibody would be expected to be less effective, since higher concentrations of neutralizing antibody are generally required to inhibit infection by this route than are required to inhibit infection by free virions (Hooks et al., 1976; Pantaleo et al., 1995). Indeed, for HIV-1, hu-PBL-SCID mouse studies suggest that the virus replicates unhindered in a significant proportion of cases in the presence of serum concentrations of a single mAb that are largely protective if administered prior to virus challenge (Poignard et al., 1999). In the remainder of cases, neutralization escape occurs rapidly, showing that the mAb does exert some pressure on virus replication in those animals. If a cocktail of mAbs is administered at high dose (50 mg/kg) in established infection, then neutralization escape is rapidly apparent. However, as the serum mAb concentrations wane to around 10 times 90% in vitro neutralization levels, the wild-type neutralizationsensitive virus reemerges. This suggests that the escape variants are less fit than the wild type, and establishes a high threshold of serum antibody to impact upon propagation of infection. Another study shows that passive transfer of neutralizing polyclonal anti-SIV antibody has a very modest effect upon SIV replication in macaques (Binley et al., 2000). The effect of antibody on established retrovirus infection has also been investigated in the F-MuLV system (Hasenkrug et al., 1995). High doses of neutralizing mAb could induce recovery, but success was dependent upon the presence of

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both CD4+ and CD8+ T lymphocytes. The results strikingly demonstrate the ability of antibody and two populations of T cells to work together in a way which is not fully understood at this time. RSV is a virus that, from its name, one might expect to propagate via cellto-cell spread. However, although syncytia are observed in vitro, they have not been observed in vivo, and the mode of infection propagation is unclear. Because the tracheal epithelium is only sparsely infected at any point in time, however, it is unlikely that cell-to-cell spread is a major mechanism of spread in vivo (McIntosh and Chanock, 1990). In any case, a number of studies have shown that neutralizing antibody can have a therapeutic effect. Early work in the cotton rat model showed that passively administered polyclonal immune antibody resulting in serum neutralizing titers in the range 1:400–1:1000 could drastically reduce the virus titers in the lungs by as much as a factor of 104 (Prince et al., 1985a). Later work focused more on intranasal inoculation when relatively small quantities of neutralizing antibody were found to be effective in cotton rats, mice, and monkeys (Crowe et al., 1994; Hemming et al., 1985; Prince et al., 1987, 1990; Weltzin et al., 1996). Therapeutic effect could be achieved with F(ab′ )2 (Prince et al., 1990) and Fab fragments (Crowe et al., 1994) of neutralizing antibody, suggesting that it was a direct result of interaction between antibody and virus. In the case of Fab fragments, viral loads in the lungs of infected mice could be reduced by a factor of almost 104 by as little as 13 ␮g of protein introduced intranasally. A number of studies show that antibody can be highly effective against established CNS infection in rodent models, preventing disease or death. Administration of large amounts of hyperimmune anti-HSV-1 serum (0.5 ml/mouse) completely protected na¨ıve animals from illness when given up to 24 hr following footpad challenge (Lubinski et al., 1998). As the time interval from challenge to antibody administration was increased, the incidence of illness increased to 25% at 24 hr, 62% at 72 hr, and 86% at 96 hr. MAbs at high dose given 24 hr postexposure were similarly shown to protect against HSV-1-induced ocular disease in mice, with protection occurring for nonneutralizing as well as neutralizing mAbs, suggesting, as expected, the importance of activity against infected cells. Protection against intracerebral challenge of mice with YF virus was shown to occur even when mAbs were given i.p. several (3–5) days after virus inoculation when peak infectious virus titers and histopathological evidence of infection were present in brains. Nearly complete protection (eight of nine animals) was noted for one mAb given at a dose of approximately 1.5 mg/kg 4 days after cerebral challenge. Protection was apparent for neutralizing and nonneutralizing mAbs. Furthermore, some of the nonneutralizing mAbs were shown to inhibit viral replication in vitro in a neuroblastoma cell line, hinting at a novel mechanism of antibody protection.

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A thorough study showed that both polyclonal antibodies and mAbs can protect against neurally spreading reovirus type 3 (Dearing ) in mice (Virgin et al., 1988) even when given several days after cerebral or footpad challenge. For instance, a potent neutralizing mAb given at approximately 8 mg/kg 1 week postchallenge led to the survival of roughly half of the animals infected. Protection was apparent with both neutralizing and nonneutralizing polyclonal antibodies, but a nonneutralizing mAb was far less effective than the neutralizing mAb. Serum complement was not required for antiviral activity. Attempts to investigate the activity of F(ab′ )2 fragments in comparable experiments were thwarted because of the short half-life of F(ab′ )2, so that even daily administrations did not maintain serum levels. This careful work emphasizes that, unless F(ab′ )2 levels are specifically monitored, conclusions drawn from comparing IgG and F(ab′ )2 should be treated with caution. Further studies show that high doses of neutralizing mAbs can protect mice against lethal challenge with Theiler’s murine encephalomyelitis virus (Fujinami et al., 1989) and neurotropic measles virus (Liebert et al., 1990) when given 2 days and 5–8 hr postexposure, respectively. In the latter case, nonneutralizing mAbs are ineffective. Further, the latter study again provides evidence of the ability of antibody to restrict viral replication inside an infected cell by binding to viral antigen. This is an interesting and potentially very important phenomenon. It was first described by Fujinami and Oldstone (1979, 1980) for measles virus-infected cells, and has been extensively investigated by Griffin and co-workers for Sindbis virus infection of neurons (Levine et al., 1991). Treatment of persistently infected SCID mice with mAbs to the E2 glycoprotein of Sindbis virus results in the gradual noncytopathic removal of viral RNA by a process which is independent of complement and T cells. Antibodies can also clear Sindbis virus from persistently infected neuronal cell cultures. The isotype of antibody is not important, but bivalency is required (Ubol et al., 1995). It is suggested that clearance involves a novel mechanism triggered when antibody cross-links viral protein expressed on the surface of infected cells. A similar mechanism has been proposed for antibody activity against rabies-infected neural cells (Dietzschold et al., 1992). This mechanism is distinct from earlier studies that described the ability of antibody to modulate antigen expression at the surface of infected cells and thereby reduce the efficiency of virus budding (e.g., Chesebro et al., 1979). Finally, nonneutralizing IgA mAbs can resolve an ongoing rotavirus infection, apparently by interaction between antibody and virus during transcytosis (Burns et al., 1996). SCID mice, infected with rotavirus for at least 2 months, were transplanted subcutaneously with hybridomas secreting mAbs to VP4 (an outer capsid viral protein) and VP6 (a major inner capsid viral protein). Only two nonneutralizing IgA mAbs to VP6 were capable of resolving chronic infection. These mAbs were not, however, active when presented directly to the luminal

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side of the intestinal tract, suggesting their mode of action is during transcytosis as described in vitro (Bomsel et al., 1998; Manzanec et al., 1992). Neutralizing IgA mAbs to VP4 did not resolve infection. IX. Observations with Nonviral Pathogens

We note that a correlation between inactivation of infectivity of a pathogen and antibody coating extends beyond the viruses on which we have focused in this review. For example, as discussed above, a correlation exists between neutralization of the intracellular bacterium Chlamydia trachomatis and coating of the infectious elementary bodies with antibody (Peeling and Brunham, 1991). Complement may enhance neutralization of opsonized C. trachomatis (Megran et al., 1988). Passive transfer of an antibody against an outer surface protein of Borrelia burgdorferi, which strongly stained nonpermeabilized B. burgdorferi cells, completely protected mice from challenge (Mbow et al., 1999). Protection against the parasite Trypanosoma cruzi by antibody, furthermore, has been correlated with antibodies that bind to living trypomastigotes in immunofluorescence (Heath et al., 1990). X. Conclusions

We began with an assertion that some general rules describing the in vitro and in vivo activities of antibodies against viruses can be discerned from the literature. We now review our interpretation of the data as a whole and its significance for these rules. There has been much discussion of the mechanisms of neutralization of viruses in vitro. A prominent opinion has been that antibodies can act at many different stages of the infectious process, including post-viral entry to the target cell, that several different mechanisms may operate in concert, and that critical sites on the virion surface must be occupied for neutralization (Dimmock, 1993). We disagree. We believe that the data are consistent, in the vast majority of cases, with a simple occupancy model essentially as initially proposed by Macfarlane Burnet in 1937. According to this model, neutralization occurs when a sizable fraction of available sites on the virion are occupied by antibody, leading to inhibition of virus attachment or interference with the entry (fusion) process. The relatively large bulk of the antibody molecule, very roughly similar to that of a typical envelope spike for an enveloped virus, is suggested to be critical (Fig. 6). The model is consistent with a number of observations. First, we have shown here that there is a roughly linear relationship between the surface area of a virus and the number of antibody molecules bound at neutralization. This number is approximately that predicted to effectively coat the virion particle, given the

FIG. 6. Model for proposed interactions between envelope spikes and neutralizing IgG. The molecules depicted are drawn to scale assuming that an IgG molecule has roughly the same molecular weight as the monomer of a typical trimeric envelope spike. The model explains how antibody coating of the virus surface may interrupt infection without occupying all available binding sites. (a) Envelope spike with an IgG molecule bound. Additional binding sites on the envelope spike are still available and the binding of additional antibodies to distinct epitopes or recombinant soluble receptor molecules may not be inhibited. An antibody to the V3 loop on HIV-1 gp120, for example, will not inhibit binding of soluble CD4. (b) Envelope spike with various numbers of IgG molecules associated. Coating of virion spikes with antibody, irrespective of the epitope(s) recognized, interferes with the initiation of a productive infection, as the establishment of multiple critical contacts with membrane receptors required for infection is inhibited by steric hindrance and geometric constraints (see also Parren et al. (1998)).

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size of the antibody molecule. Second, any antibody that binds well to and coats the virion surface should neutralize the virus, as is almost universally found. Nonneutralizing antibodies should not coat the virus, although they may bind at lower levels of occupancy. Heterogeneous envelope spikes provide an opportunity for binding to virions without neutralization because of low occupancy, as discussed above for rabies virus and the FPV/VSV mixed virions. Third, the model predicts that the precise epitope recognized by the antibody on the virion surface should not be crucial. Rather, the number of antibody molecules bound per unit area, which will be determined by the affinity of antibody for virionexpressed antigen, will be most important. This is precisely that which is found for HIV-1, as discussed above. Antibody enhancement of viral infection in vitro receives much attention, although its significance in vivo has only been demonstrated for dengue virus infection. It appears to occur in the presence of subneutralizing concentrations of neutralizing antibodies. The model described above suggests that this is a phenomenon arising from low occupancy of virion sites, as discussed earlier. The question whether some antibodies bind well to the virus without neutralization is often raised. In particular, the concern is expressed that nonneutralizing antibodies might compete with the binding of neutralizing antibodies to virus, thereby interfering with neutralization. First, we strongly question the evidence for binding but nonneutralizing antibodies. Generally, there is a very strong correlation between occupancy and neutralization, and most examples of such nonneutralizing antibodies can indeed be explained by poor virus binding, or even a failure to appreciate that antibodies which bind well to isolated envelope or capsid molecules do not necessarily bind well to the virus particle. Some convincing but isolated examples exist, such as the rabies mutant virus, which merit further investigation. Second, to our knowledge, there is no convincing and confirmed evidence for a nonneutralizing antibody interfering with neutralization. Virion coating but non-neutralizing antibodies therefore do not appear to play a significant role (if any) in the humoral response against viruses. Antibody activity in vivo can arise through binding to virions or virion products on infected cells. Protection by antibody at the level of sterile immunity requires activity against free virions, i.e., neutralizing antibodies. Several studies with mAbs show that sterile immunity is only achieved when serum concentrations of the challenged animal are of the order of two to three orders of magnitude higher than in vitro neutralization titers, that is, serum concentrations capable of neutralizing essentially all of the challenge virus. Expressed in another way, serum neutralizing titers of 1:100–1:1000 are required for sterile protection. In a number of cases (discussed above), even such high levels of neutralizing antibody do not provide sterile protection, although they do prevent disease. Most vaccines do not elicit very high neutralizing titers, especially over an extended period, but then they probably do not provide sterile protection. Rather, it seems

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likely that they reduce the effective challenge to such a level that infected cells can be controlled by cellular immunity and possibly by antibodies. There are a number of cases (discussed above) where in vivo protection by neutralizing antibodies is independent of the presence of the Fc part of the molecule. In these cases, it seems likely that the mechanism of protection in vivo is essentially equivalent to neutralization in vitro. In a number of other cases, it appears that F(ab′ )2 fragments, that are equally active as whole IgG molecules at neutralization in vitro, are ineffective at protection. Further, neutralizing mouse IgG1 switch variants are ineffective at protection, whereas IgG2a molecules are effective. In many of these examples, protection is independent of complement, suggesting that protection by neutralizing antibodies requires activity against infected cells as well as free virions. This requirement, in some cases, for activity against infected cells as well as virions may contribute to observations of incomplete correlation between neutralization and protection for neutralizing antibodies, although other factors such as the use of different cell types in vitro and in vivo should be considered. It is worth emphasizing, though, that most neutralizing antibodies protect at appropriate concentrations, and antibodies assessed as the most potent in neutralization assays in vitro generally are the most effective at protection in vivo. There are numerous examples of protective activity exhibited by nonneutralizing antibodies. This activity appears to be directed at infected cells, and generally appears to be somewhat less potent than that of neutralizing antibodies. For instance, cases are described above where neutralizing antibodies are protective against higher challenge doses or more pathogenic viruses than nonneutralizing antibodies. In many cases, protection by nonneutralizing antibodies is shown to depend critically on the Fc part of the antibody molecule and to occur in complement-deficient mice, suggesting that ADCC (or phagocytosis) may be crucial in clearing antibody-complexed infected cells. It should be noted that protection with nonneutralizing antibodies is mostly restricted to protection against enveloped viruses. What is the significance of these conclusions for vaccine design? In the first case, the time-honored focus on eliciting neutralizing antibodies is well justified. Serum neutralizing antibody titers of the order of, or greater than, 1:100 provide the greatest likelihood that antibody alone can protect against viral challenge. In many cases, antibody and cellular responses may cooperate to protect, although, with notable exceptions (Dittmer et al., 1999a), this is an underexplored area. The model described above asserts that neutralization is determined by the extent of coating of virus by antibody. At a given antibody concentration, this is in turn determined by the affinity of antibody for the antigen on the virion surface. Hence, the model predicts that a vaccine should simply aim to elicit antibodies of the highest affinity for the virion surface antigen. This is most directly achieved by immunization with molecules identical to or as similar to the viral surface

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antigen as possible. This may not be easy. We have argued that a number of enveloped viruses have evolved surface proteins of low immunogenicity (Burton and Parren, 2000). Subunit envelope proteins appear, in a number of instances, to elicit antibody responses that show little reactivity with the form of the envelope (usually oligomeric) expressed on the virion surface (Parren et al., 1998; Roben et al., 1994; Sakurai et al., 1999; Sattentau and Moore, 1995). Another interesting question is the value of eliciting antibodies targeted to infected cells rather than virions. There are examples, such as arenaviruses, where complete passive protection appears to require antibodies to infected cells as well as virions. Of course, neutralizing antibodies could fulfill both roles by binding, for instance, to envelope molecules on virions and infected cells. However, it is also possible that epitopes expressed on virions are not expressed optimally on infected cells, for ADCC for example, and then the induction of nonneutralizing antibodies may be beneficial. This is a factor worthy of consideration in vaccine design, particularly using subunit proteins to elicit antibodies. Finally, could passively administered antibodies be used in the treatment of acute viral diseases? The studies discussed above suggest high doses would be required to have any reasonable chance of efficacy. However, given the availability of human antibodies from new technologies such as transgenic mice and phage display (Burton and Barbas, 1994; Green et al., 1994; Lonberg et al., 1994; Winter et al., 1994), and the ability to produce large amounts of such antibodies relatively cheaply in culture systems (e.g., Verma et al., 1998), larger animals (e.g., Pollock et al., 1999), or plants (e.g., Fischer et al., 1999), antibody intervention in acute viral disease may become increasingly realistic. Antibodies are already used in certain situations in a postexposure mode to prevent, e.g., disease due to Junin virus (Argentine hemorrhagic fever), rabies virus, and TBEV. However, the evaluation of antibodies in humans following the appearance of symptoms in infections due to viruses such as RSV, dengue, and hanta would be of great interest. ACKNOWLEDGMENTS We thank Drs. Kim Hasenkrug and Michael Buchmeier for reviewing the manuscript. We thank Erica Ollmann Saphire for preparing the molecular models of poliovirus. We are grateful to Drs. Linda M. Stannard and H. W. Ackermann for providing electron microscopic images. We acknowledge the financial support of the National Institutes of Health under Grant numbers AI33292 and HL59727 (to DRB); AI40377 and AI48494 (to PWHIP).

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ADVANCES IN IMMUNOLOGY, VOL. 77

Mouse Models of Allergic Airway Disease CLARE M. LLOYD,* JOSE-ANGEL GONZALO,† ANTHONY J. COYLE,† AND JOSE-CARLOS GUTIERREZ-RAMOS† *Leukocyte Biology Section, Biomedical Sciences Division, Imperial College of Science,

Technology, and Medicine, London SW7 2AZ, United Kingdom; and †Millennium Pharmaceuticals, Cambridge, Massachusetts 02139

I. Introduction

Asthma is a clinical syndrome characterized by intermittent episodes of wheezing and coughing. The diagnosis is confirmed by abnormal lung physiology, including reversible airway obstruction and airway hyperresponsiveness (AHR) to spasmogenic stimuli. The fact that the clinical and biological manifestations of asthma are extremely heterogeneous reflects the multitude of causative and aggravating factors, as well as the presence of many underlying pathophysiological mechanisms. Pathological manifestations have been found to include airway inflammation, remodeling, and mucus hypersecretion. The airways of patients with even mild asthma are inflamed, and some data suggest that the severity of asthma parallels the degree of this inflammation (Broide et al., 1991; Pare and Bai, 1995; Peters, 1990). In addition, the localization and activation of specialized leukocytes correlate with the temporal phases of airway obstruction and enhanced bronchial responsiveness to spasmogenic stimuli. Among the leukocytes considered causative players in the development of bronchial inflammation, eosinophils are thought to be critical, since eosinophilia is a common feature of asthmatic airways and eosinophils have been demonstrated to cause mucosal injury. However, evidence points to the fact that although eosinophils are largely responsible for asthmatic symptoms, their function is largely under the control of specialized subsets of chronically activated memory T cells sensitized against an array of allergenic, occupational, and viral antigens that home to the lungs after appropriate antigen exposure or viral infection. In asthmatics, CD4+ T cells producing interleukin 4 (IL-4), IL-5, and IL-13 have been identified in bronchoalveolar lavage (BAL) and airway biopsies. Evidence for their functional involvement stems from the fact that T helper 2 (Th2) cells are present in the airways and that Th2-derived cytokines are required for the development of airway eosinophilia and immunoglobulin E (IgE) production. Despite the fact that particular leukocytes and mediators have been implicated as causative agents in asthma, the mechanisms responsible for the initiation and maintenance of allergic inflammation remain poorly defined. Animal models, including guinea pigs, monkeys, rats, and mice, have been used to study the pathogenesis of asthma. Mouse models of allergic lung disease 263 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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have been utilized to dissect the complex pathophysiological mechanisms underlying the asthma phenotype. Mice are increasingly attractive for such studies for a number of reasons. The progress in outlining pathways and mediators in murine immunology has been substantive, and a plethora of technologies and reagents to manipulate these pathways have been developed. Moreover, technology has been developed that gives us the ability to manipulate the mouse genome by production of transgenic lines, as well as ablation of specific genes and thus their protein products, through homologous recombination. Therefore, it is possible to dissect inflammatory pathways to investigate the functional roles of particular mediators or cells. A wealth of research activity has shown that mice can be induced to display a range of the pathophysiological features that are hallmarks of the human disease. Mice have been shown to develop inflammatory infiltrates in the lungs, both in peribronchiolar tissues (as shown in lung sections) and in the airway lumen (collected in bronchiolar lavage). Although eosinophils are generally the most prolific cell type within these infiltrates, lymphocytes are also present in significant numbers. Lung sections show, too, that there is an increase in mucus secretion from the bronchoepithelial surface. Analysis of serum reveals that mice show an increase in both total and allergen-specific IgE, as well as increased IgG2a titers. This Th2-type profile is reflected in the cytokines generated within the lungs, IL-4 and IL-5 being present in significantly greater quantities than interferon ␥ (IFN-␥ ). Many investigators have also documented changes in lung function following allergen provocation, using a variety of techniques. The physical properties of the lungs can be assessed after their removal from the host, mice can be anesthetized and attached to instruments to allow recording of airway mechanics, or alternatively, mice can remain unanesthetized and unrestrained while function is measured by plethysmography. These techniques have been used to determine airway hyperreactivity to ␤2 -agonists before and after provocation with allergen. The variety of protocols that have been used to induce pulmonary eosinophilia, bronchial hyperreactivity, and mucus hypersecretion is tremendous. In this chapter, we review the models that have been used, in an attempt to identify the immunological and pathophysiological mechanisms underlying the asthma phenotype. The use of animal models has enabled us to highlight specific pathways and has given us the opportunity to study the function of these pathways in vivo. The challenge is to connect these pathways observed and identified in animal models to the equivalent in human airway disease. A. ACTIVE IMMUNIZATION MODELS Active immunization models rely on the delivery of an antigen to replicate a sensitization and challenge phase, in order to mimic the allergic response to exogenous or innocuous stimuli. This protocol involves preimmunization with the allergen before a sensitization phase in which the allergen is introduced to the target organ, in this case the lungs—intranasally, as an aerosol,

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or via the trachea. This basic protocol induces a pulmonary eosinophilia, generally in conjunction with an increase in circulating IgE levels. However, there are countless variations to this protocol that have profound consequences for the range of pathophysiological features developed. This method has been used with a variety of antigens and protocols, varying from complex microorganisms to simple proteins and chemicals. Although all of these methods ultimately produce a pulmonary inflammation, we have observed that the type and degree of inflammation are widely variable. In this section, we review the relative merits of different antigens, as well as critical parameters of various immunization protocols. 1. Selection of Antigen Antigens used to initiate pulmonary eosinophilia range from simple protein antigens to complex microorganisms. The immune response to these different allergens is likely to be very different and will affect the ensuing tissue inflammation. For example, complex microorganisms such as parasites will have a much higher number and range of antigenic epitopes available during T cell receptor priming compared to those for a soluble protein antigen. These and other factors are important when comparing the effects of particular antigens in a protocol. a. Complex Microorganisms. i. Fungi. Allergic airway inflammation may occur after sensitization from spores from fungi such as Aspergillus or Candida (Kauffman et al., 1995; Pacheco et al., 1998). Patients may develop an allergic eosinophilia or, in the case of Aspergillus fumigatus, two different forms—as asthma with increased serum IgE titers or hypersensitivity pneumonitis with increased serum IgG and low IgE titers (Kurup and Kumar, 1991). In mice, sensitization and intratracheal challenge with A. fumigatus induce pulmonary eosinophilia, lavage IL-4 and IL-5 production, and increased serum IgE titers in conjunction with heightened AHR (Grunig et al., 1997; Lukacs et al., 1999; Shibuya et al., 1999). ii. Parasites. Due to the specialized structure of the lung, it is a target for the trapping of parasites during phases of the life cycle. Some parasite infections are associated with tropical pulmonary eosinophilia caused when larval stages of the life cycle pass through or get trapped in the lungs. The immunological reaction to the worm (in the case of filarial infection) or the egg (in the case of schistosomal infection) bears some resemblance to the pathophysiological reaction to allergen, and this has been exploited in animal models. iii. Schistosoma models. The immunological response to surface antigens of the Schistosoma mansoni egg stage has been exploited to establish an animal model of some facets of asthma. Intratracheal delivery of parasite antigen to presensitized mice was found to elicit a Th2 response and pulmonary inflammation that resolved after 3–4 days (Lukacs et al., 1994). Soluble egg antigens induce an antigen-specific eosinophil recruitment to the lungs—in conjunction

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with IL-4, tumor necrosis factor ␣ (TNF-␣), and C–C chemokines macrophage inflammatory protein 1␣ and RANTES—as well as AHR (Lukacs et al., 1996; Padrid et al., 1998; Wynn and Cheever, 1995). This model has been exploited to characterize Th2 and Th1 responses in the lungs. Lung granulomas were elicited by beads coated with purified protein derivative of Mycobacterium bovis to induce a Th1-type response or with soluble egg antigens for a Th2-type response (Chensue et al., 1995). The type 1 granuloma was composed mostly of mononuclear cells and is largely dependent on IFN-␥ and TNF-␣, whereas the type 2 granuloma is eosinophil rich and is largely dependent on IL-4. This model has proved useful in determining the role of cytokines and chemokines in granuloma formation (Chensue et al., 1997; Lukacs et al., 1996). iv. Worm models. Adult filarial parasites reside in the lymphatic organs of the host and chronically release large numbers of larvae into the bloodstream, some of which become trapped in the lungs. In humans, this can lead to a pulmonary eosinophilia associated with symptoms that resemble the pathophysiological features of chronic asthma (Ong and Doyle, 1998; Ottesen and Nutman, 1992). Mouse models have been used to try to replicate this condition, and a variety of studies have shown that infection with worms such as Nippostrongylus, Angiostrongylus and Necator, Trichinella, Taenia, and Trichuris leads to eosinophilic lung inflammation (Hall et al., 1998; Wilkinson et al., 1990). In some cases, this is also associated with an increase in bronchial hyperreactivity and a rise in Th2-type cytokines. b. Protein Antigens. Soluble protein antigens are widely used to elicit allergic pulmonary inflammation and range from simple proteins such as ovalbumin (OVA) to complex, environmentally relevant antigens such as cockroach or house dust mite proteins. The immune response to these antigens is much more controlled and reproducible, since a defined amount of antigen can be delivered at a particular site. Thus, it is perhaps easier to establish a more stable model than if using an intact biological organism. The most commonly used protein antigen is chicken egg OVA, the use of which models late-phase events such as eosinophilia, and in some protocols, AHR in vivo. OVA is an important human allergen and has the advantage of reliably inducing in mice antigen-specific IgE responses that are largely dependent on IL-4. The majority of investigators have found that sensitization and subsequent challenge with OVA result in a significant increase in the number of eosinophils and lymphocytes to both the peribronchiolar tissue and the BAL. These increases occur in conjunction with a significant increase in levels of Th2-type cytokines (IL-4 and IL-5) as well as serum IgE levels. The use of this type of model by a range of investigators has been successful in defining the role that individual cells and immune pathways play in mediating the eosinophilic response. A comparison of several of the models currently in use in the literature

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using OVA as the allergen is depicted in Table I. Although all of the protocols use the same allergen, they vary in terms of route of administration, the use of adjuvant, and the frequency of challenge. These variations give rise to very different pathophysiological symptoms. Other investigators have tried to establish models using more environmentally relevant antigens, such as those proteins derived from cockroaches or house dust mites. Sensitivity to cockroach antigen is a common problem in inner cities and for those living in crowded, lower socioeconomic areas (Rosenstreich et al., 1997). A model has been established to determine the specific responses associated with sensitivity to cockroach antigens. In this model, the allergic responses to intratracheal cockroach antigens in sensitized mice included allergen-specific airway eosinophilia and significantly altered airway physiology concomitant with eosinophilic airway inflammation (Campbell et al., 1998, 1999). Similarly, sensitization and challenge of mice with the house dust mite Dermatophagoides farinae have been found to elicit pulmonary inflammation (Coyle et al., 1996b; Yu et al., 1996; Yasue et al., 1998). Intranasal challenge with D. farinae in previously sensitized mice induces pulmonary edema, inflammatory cell recruitment to the lungs, eosinophilia, production of cytokines, and AHR (Yu et al., 1996). This eosinophilia was also found to be CD4+ T cell dependent. c. Chemical Compounds. A model of industrial asthma has also been established using toluene diisothiocyanate (TDI), a low-molecular-weight compound known to cause occupational asthma in 5–10% of exposed workers. Mice sensitized subcutaneously and challenged intranasally with TDI show tracheal hyperreactivity to carbachol. This tracheal hyperreactivity was found to be lymphocyte dependent but IgE independent and was not associated with leukocyte infiltration of the airways (Scheerens et al., 1996). Exposure over a prolonged period elicited TDI-specific IgE antibodies and in vivo AHR (Scheerens et al., 1999). 2. Parameters of Immunization Protocol a. Route of Immunization. There is a wealth of data to show that sensitization/challenge models reproduce facets of the human asthmatic condition; however, subtle differences in the basic protocol can have drastic effects on the development of pathophysiology, and the interpretation of results becomes critical. This is especially important when inhibitory reagents or genetically modified animals are used to outline the potential functional importance of selected molecules. Of particular importance in this context is the choice or route of administration of antigen for either sensitization or challenge. The basic active immunization protocol relies on a sensitization phase to induce peripheral priming of the immune system followed by antigenic challenge directly to the target organ. In the case of pulmonary inflammation, the lungs can be targeted by intranasal inoculation, aerosolization of the antigen, or installation by intratracheal

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injection. This route of antigen administration and the combination of challenges have been shown to be critical to the final pathological outcome. In an analysis of the role of systemic versus local administration of OVA in inducing pulmonary allergic responses, Zhang and colleagues (1997) found that a combination of systemic and local exposure to OVA resulted in a maximal and reproducible induction of responses. These responses included AHR in vivo, production of allergen-specific IgE, peri-airway infiltration with eosinophils and their appearance in BAL, and increased expression of Th2 cytokines in the local lymphoid. A protocol using subcutaneous sensitization with alum-precipitated OVA combined with multiple intranasal doses of OVA in normal saline is also effective in inducing AHR, eosinophilic inflammation of the lung parenchyma, and increased total IgE (Eum et al., 1995). One report suggests that the site of antigen delivery is critical, in that the pulmonary environment promotes preferential Th2-type differentiation (Constant et al., 2000). In this study, an antigen/mouse combination was used that, in almost all conditions of immunization previously examined, is strongly biased toward priming for Th1 CD4+ T cells. However, administration of Leishmania major parasites to B6 mice via the intranasal route preferentially induced a Th2-dominated response. These included an influx of lymphocytes and eosinophils into alveoli, as well as the induction of Th2-type foci of inflammation around pulmonary blood vessels and airways. In addition, high levels of Th2-associated cytokines (IL-4 and IL-5) were generated when lung-draining lymph node and tissue cells were restimulated with L. major lysate. Although this study demonstrated that the lung environment favors Th differentiation using antigen given solely via the airways, we and others (Stampfli et al., 1998; Zhang et al., 1997) have found that mice given OVA solely by the intranasal route produced neither local responses (e.g., AHR or BAL eosinophilia) nor systemic responses (e.g., plasma IgE). These results contrast with those of a study in which OVA delivered repeatedly by nebulization generated circulating levels of IgE and increased AHR but not pulmonary eosinophilia (Renz et al., 1992). These differences probably reflect differences in the vehicle, dose, or frequency of allergen challenge and illustrate once more the diverse outcome possible using various protocols. b. Adjuvant. One other major difference in protocols is the use of an adjuvant during the priming phase to boost the immune response to the allergen in use. This seems to be particularly important when using OVA, since this is a relatively simple protein structure and is not particularly antigenic when used alone. Specifically, the use of aluminum compounds (alum) is associated with the induction of Th2 responses (Brewer et al., 1996; Grun and Maurer, 1989). OVA alum was found to induce IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in IL-4R␣–and Stat6-deficient mice (Brewer et al., 1999a).

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c. Dose. A major factor in the elicitation of an effective T cell response is in the dose of antigen used in the priming stages. As shown in Table I, the choice of antigen dose for the sensitization step varies 10-fold. A thorough review of the process of induction of Th1 and Th2 effector T cells described a conflict in findings relating to whether Th1- or Th2-type responses are elicited by high versus low doses of antigen (Constant and Bottomly, 1997). There are reports suggesting that priming with high-dose antigen leads to humoral responses, whereas low-dose antigen precipitates cell-mediated immunity (Parish and Liew, 1972). However, the reverse has also been documented, whereby mice given repeated low doses of antigen develop Th2-like responses with high IgE production (Wang et al., 1996). Constant and Bottomly (1997) concluded that there is no clear-cut conclusion regarding dose and the type of immunity developed but pointed out that the type of antigen used is critical. They remarked that the studies in which low-dose Th1 responses were elicited used parasite antigens, whereas low doses of soluble proteins tended to elicit a Th2-type response. Thus, it may be that the antigen itself can influence the nature of the immune reaction. Antigen dose is also an important issue in the context of immunotherapy. It has been suggested that a suitable immunotherapy for T cell–mediated disease may be immunization with immunodominant T cell epitopes (Yssel et al., 1994). Immunotherapy with suboptimal doses of OVA has been found to down-regulate AHR and BAL eosinophilia with concomitant decreased production of Th2 cytokines (Janssen et al., 1999). However, the same study found that immunotherapy with an immunodominant epitope of OVA aggravates AHR and increases BAL eosinophilia (Janssen et al., 1999). d. Genetic Background. Perhaps one of the most striking differences in the development of a complete set of features of the asthmatic syndrome rests in the genetic background of the mouse strain used. One particular facet of the pulmonary allergic response that seems to be genetically restricted is AHR. Not only does allergen-induced AHR vary among different strains of mouse, but it seems that native AHR is also dependent on the background strain of the mouse, consistent with the hypothesis that AHR is a heritable trait (reviewed in Drazen et al., 1999). Levitt and associates (1990) measured AHR in nine different strains of commonly used laboratory mice and found that the AKR/J and A/J strains showed the greatest degree of airway responsiveness, while the C57BL/6J, SJL/J, and C3H/HeJ strains were the least responsive. Moreover, there was a 6-fold difference in AHR for acetylcholine between the most divergent strains (A/J and C3H/HeJ). Subsequent studies have confirmed these data (Chiba et al., 1995). Further studies have been carried out to determine the genetic variability in AHR as well as cellular and antibody production following antigen challenge in multiple strains of mice. After choosing two strains in which they found widely differing responses to acetylcholine stimulation under naive conditions,

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Wills-Karp and Ewart (1997) measured responses in A/J and C3H/HeJ mice after antigen challenge. They determined that inbred strains of mice are genetically predisposed to be susceptible (A/J) or resistant (C3H/HeJ) to the bronchoconstrictor effects of cholinergic agonists under inflammatory and noninflammatory conditions. Interestingly, these data conflict with a study by Brewer and colleagues (1999b) that measured pulmonary pathophysiology and serum IgE responses in mice of 12 different inbred strains following allergen challenge. The results of the study showed that the intravenous methacholine dose required to reduce lung conductance by 50% varied by 3-fold depending on the strain used. Moreover, BAL eosinophils ranged from 3% to 91% of total cells, while tissue eosinophilia varied from being not detectable to being widespread and severe. OVA-specific IgE concentrations ranged from <3 ng/mL to 455 ng/mL in different strains. In contrast to the data from Gavett and colleagues (1994), the study by Brewer’s group found A/J mice to be resistant to OVA-induced pulmonary inflammation and AHR. This may reflect differences in the method of airway exposure—sensitization with OVA and phosphate-buffered saline and challenge in the trachea in one case and challenge with OVA/alum and aerosolized challenge in the other. Another study contrasted the responses of two commonly used strains in the literature (Zhang et al., 1997). Several OVA-induced allergen challenge protocols were used to determined the importance of the route of allergen administration and genetic background in modulating the physiological, inflammatory, and immunological features characteristic of allergen-induced asthma. C57BL/6 mice showed significantly decreased responses compared with BALB/c mice for all parameters of allergic pulmonary disease examined, with the exception of airspace eosinophilia. The two strains demonstrated intrinsic differences in airway mechanics, with C57BL/6 mice showing lower basal dynamic compliance than did BALB/c mice. In agreement with earlier studies, naive C57BL/6 mice required higher doses of methacholine to achieve a 50% decrease from the basal value for either lung conductance or dynamic compliance than did BALB/c mice. Following allergen provocation, BALB/c mice demonstrated greater hyperreactivity than C57BL/6 mice for both of these parameters. All of these studies emphasize the heterogeneity of the response to allergen and the importance of a range of factors in the development of a pathophysiological outcome. BALB/c mice tend to be high IgE responders and allergic protocols in this strain exhibit a more Th2-skewed response, compared to the same protocol used in C57BL/6 mouse. Thus, allergic protocols result in higher levels of IL-4 and IL-5 in BALB/c compared to C57BL/6 mice. As described earlier, the latter strain also tends to be less hyperreactive compared to BALB/c mice. Another feature of the allergic response that seems to have a significant genetic component is IgE-stimulated mast cell activation. Linkage analysis in the progeny of an A/J × C57BL/6 cross shows that the mice of the C57BL/6 strain are unable to

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produce mast cell protease 7 (Ghildyal et al., 1994; Hunt et al., 1996). Because of this deficiency, it has been speculated that the C57BL/6 mouse is resistant to the induction of mast cell–dependent AHR (Drazen et al., 1996). This point adds further weight to the importance of the choice of genetic background before induction of an allergen protocol. B. OVERVIEW OF CURRENT PROTOCOLS The number of active immunization models elicited by OVA described in the literature is tremendous. Inevitably, these lead to a variety of pathophysiological outcomes. We have summarized some of the basic protocols with their main features in Table II. These basic protocols have been modified by other investigators but represent the variations possible in the parameters of immunization described above. Pathophysiological end points measured by investigators vary, as does the time at which measurements are taken. Some investigators describe a time course of events following the final allergen challenge, but the majority concentrate on one time point only. Most investigators measure BAL eosinophilia, but we have found it important to examine tissue sections to obtain a clearer picture of leukocyte migration following challenge. In comparing the outcomes of the models depicted in Table I, the use of alum initiates a more robust inflammatory response, with leukocyte recruitment increased severalfold after protocols B–F compared with protocol A. Similarly, features such as AHR or cytokine production tend to be increased to a greater extent with the use of alum. The degree of increase is also a facet of the background strain used, since C57BL/6 mice give less pronounced Th2 responses compared to BALB/c mice. We have found that protocol A initiates an acute but transient eosinophilia and AHR, whereas protocol D elicits an enhanced sustained inflammation and AHR. Protocol C does not involve a sensitization step and thus does not result in a particularly defined pulmonary inflammatory response. Other investigators have found that some sort of preliminary sensitization step is critical to induce a strong allergen response (Schwarze et al., 1997; Stampfli et al., 1998). The number of allergen provocations during the challenge phase also serves to increase the degree of inflammation. We have found that multiple (about five to seven) allergen challenges result in maximal inflammation. 1. Recommendation The likelihood of obtaining a robust, Th2-dependent pulmonary inflammation is enhanced by a protocol carried out in BALB/c mice, utilizing two peripheral priming steps in adjuvant followed by multiple local allergen challenges. We have found that this is sufficient to elicit tissue and lavage eosinophilia, Th2 cytokine production, and increased antigen-specific IgE production as well as AHR to methacholine.

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TABLE II MODULATION OF ACTIVE IMMUNIZATION MODELS Molecules Cytokines IL-4 IL-5 IL-5 IL-5 IFN-␥ IFN-␥ R GM-CSF IL-10 IL-10 IL-12 IL-12 IL-13

IL-13

IL-13 Adhesion molecules ICAM-1 ICAM-2 VCAM-1 P-selectin

L-selectin Leukocytes Lymphocytes T cells CD4

Treatment

Effect

Neutralization by antibody Neutralization by antibody Neutralization by antibody Gene knockout

Decreased AHR, no effect on Eos Decreased eosinophilia, no effect on AHR Decreased AHR and eosinophilia Decreased eosinophilia and AHR Enhanced eosinophil and CD4 T cell infiltration Prolonged eosinophilia and T cell response Inhibition of cell recruitment and AHR Inhibition of cell recruitment Decreased AHR, eosinophilia, and BAL IL-4 and IL-5 Decreased eosinophilia, BAL IL-4 and IL-5 Inhibition of AHR, decreased mucus production, no effect on cell recruitment Inhibition of AHR, decreased mucus production, eosinophil recruitment Induction of AHR, eosinophilia, mucus and IgE

Neutralization by antibody Gene knockout Gene knockout Recombinant cytokine Gene transfer Recombinant cytokine Recombinant cytokine Soluble IL-13␣ 2– IgFc fusion protein Soluble IL-13␣ 2– IgFc fusion protein Recombinant protein Gene knockout Gene knockout Hypomorphic mutant mice Gene knockout Gene knockout Gene knockout SCID RAG-1 CD3ε transgenic Anti-CD3 antibody Anti-CD4 antibody Gene knockout

Reference Corry et al. (1996) Corry et al. (1996) Hamelmann et al. (1999a) Foster et al. (1996) Iwamoto et al. (1993) Coyle et al. (1996a) Zuany-Amorim et al. (1995) Stampfli et al. (1999a) Gavett et al. (1995) Iwamoto et al. (1996) Wills-Karp et al. (1998)

Grunig et al. (1998)

Wills-Karp et al. (1998)

Eosinophilia abolished Prolonged eosinophilia and AHR Eosinophilia abolished

Gonzalo et al. (1996a) Gerwin et al. (1999)

Eosinophilia delayed Decreased AHR and eosinophilia No effect on eosinophilia

Gonzalo et al. (1996) De Sanctis et al. (1997)

Decreased AHR and eosinophilia Eosinophilia abolished Eosinophilia abolished Decreased eosinophilia Decreased eosinophilia and AHR Eosinophilia abolished

Gonzalo et al. (1996a)

Gonzalo et al. (1996a) Corry et al. (1996) Gonzalo et al. (1996a) Gonzalo et al. (1996a) MacLean et al. (1996) Gavett et al. (1994) Gonzalo et al. (1996a)

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TABLE II (Continued) Molecules CD8

Treatment

B cells

Anti-CD8 antibody Gene knockout Gene knockout

NK cells

Depleting antibodies

Mast cells Dendritic cells

Deficient mice Conditional depletion

TCR-␣/␤ lymphocytes TCR-␥ /␦ lymphocytes

Gene knockout, depleting antibodies Gene knockout

Immunoglobulin/R receptor IgE Gene knockout IgE

Non-anaphylactogenic antibody

CD23

Blocking antibody and knockout mice

Chemokines Eotaxin

Neutralizing antibodies

Eotaxin

Gene knockout

MCP-5

Gene knockout Neutralizing antibodies

MCP-1

Neutralizing antibodies

MCP-3

Neutralizing antibody

RANTES

Receptor antagonist

MIP-1␣

Neutralizing antibodies

MDC

Neutralizing antibodies

IL-8 receptor

Gene knockout

Effect

Reference

Eosinophilia decreased No effect on eosinophilia Enhanced eosinophilia and AHR Decreased eosinophilia, BAL cytokines Inhibition of eosinophilia Decreased pulmonary T cell numbers, IgE, and BAL IL-4 and IL-5 Eosinophilia and IgE abolished Eosinophilia, IgE response, and AHR attenuated

Hamelmann et al. (1996a) Gonzalo et al. (1996a) Korsgren et al. (1997), MacLean et al. (1999) Korsgren et al. (1997)

No effect on eosinophilia or AHR Inhibition of eosinophil recruitment and IL-4 and IL-5 production Inhibition of eosinophilia

Lloyd et al. (2001)

Decrease in eosinophil recruitment Partial reduction in eosinophil recruitment No effect Reduction of tissue eosinophil recruitment and AHR Reduction in AHR and lavage and tissue eosinophilia Reduced lavage eosinophilia Reduction in tissue and lavage eosinophilia Partial reduction in eosinophilia and AHR Reduction of tissue eosinophil recruitment and AHR Increased B cell recruitment and serum IgE, decreased AHR

Gonzalo et al. (1996)

Coyle et al. (1996) Lambrecht et al. (1998)

Schramm et al. (2000) Schramm et al. (2000)

Coyle et al. (1996b)

Coyle et al. (1996b)

Rothenberg et al. (1997) Yang et al. (1998) Jia et al. (1996), Gonzalo et al. (1998) Gonzalo et al. (1998)

Stafford et al. (1997) Gonzalo et al. (1998) Gonzalo et al. (1998) Gonzalo et al. (1999)

De Sanctis et al. (1999)

(continued)

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TABLE II (continued) Molecules Surface receptors Fas

Treatment Blocking antibody

T1/ST2

Blocking antibody

CD28

CTLA4–Ig treatment

CD86 (B72)

Blocking antibodies

CD80 (B71) ICOS

Blocking antibodies Ig fusion protein

Effect Clearance of eosinophils from the lungs Decreased eosinophilia, IgE, and IL-5 Decreased AHR, inflammatory cell recruitment Inhibition of eosinophil recruitment, IgE, Th2 cytokines, and AHR Partial inhibition of eosinophilia Decreased eosinophilia, AHR, and lavage IL-4 and IL-5

Reference Tsuyuki et al. (1995) Coyle et al. (1999) Krinzman (1996), Tsuyuki et al. (1997) Tsuyuki et al. (1997)

Tsuyuki et al. (1997) Coyle et al. (2000)

The table summarizes the work of different investigators in blocking or potentiating immunological pathways during ovalbumin-induced active immunization models. The treatment column signifies the method by which particular molecules or cells were blocked or potentiated. The effect column denotes the resulting change in phenotype after blockage or overexpression. For further details of each experiment, see individual references. AHR, Airway hyperresponsiveness; BAL, bronchoalveolar lavage; CTLA, cytotoxic T-lymphocyte-associated antigen 4; EOS, eosinophils; GM-CSF, granulocyte–macrophage colony–stimulating factor; ICAM, intercellular adhesion molecule; ICOS, inducible T cell costimulator; IFN, interferon; Ig, immunoglobulin; IL, interleukin; MCP, monocyte chemotactic protein; MDC, monocyte derived chemokine; MIP, macrophage inflammatory protein; NK, natural killer; RAG, recombination activating gene; SCID, severe combined immunodeficiency; TCR, T cell receptor; Th, T helper, VCAM, vascular cell adhesion molecule.

C. MODULATION OF ACTIVE IMMUNIZATION MODELS The development of this series of allergic inflammatory models has enabled investigators to look at the functional contribution that a range of cells, mediators, and molecules make to the development of features of the asthmatic syndrome. The immunological response to allergen has been dissected by using neutralizing antibodies or genetic mutants to eliminate the contribution of particular populations of cells, molecules, and mediators. Table II summarizes the results of functional analysis in vivo of a range of cells and molecules thought to be critical in the immune response to antigen. It is important to point out that the choice of end point measured by investigators varies tremendously. For a particular cell or molecule to be unequivocally implicated in the induction of the allergic response, it is important to examine the physiological response by measuring AHR and the pathological response in the lavage and tissue. Interestingly, there are several findings that are directly contradictory, for example, the role of IL-5 in AHR and the role of CD8 cells, IgE, or eotaxin in eosinophilia. The varying impacts of these cells and molecules can be explained in part by differences in the choice of background or type of model used. Some protocols elicit a more robust response than others and are more difficult to attenuate; other protocols may be initiated in a strain that is less responsive than in another, conflicting, report.

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D. PASSIVE TRANSFER MODELS Because the role of critical mediators in the development of pulmonary inflammation has been defined in active immunization models, several groups have sought to further investigate the relationship between particular mediators and the development of inflammation by passive transfer into naive mice. Since the induction of an immune response is undoubtedly the result of multiple effector mechanisms, a passive transfer system allows the impact of one particular effector pathway to be examined in the absence of other systems. 1. Antibody Transfer Oshiba and colleagues (1996) used such a model to investigate the relationship between IgE and IgG1 and AHR using murine B cell hybridomas secreting OVA-specific IgE or IgG1 antibodies. Passive transfer experiments were performed in which naive mice received two doses of anti-OVA IgE, IgG1, IgG2a, or IgG3 before subsequent challenge with nebulized OVA. Thus, the effects of allergen-specific subclass antibodies on immediate hypersensitivity and AHR was assessed. The experiments by this group showed that passive sensitization with either IgE or IgG1 caused immediate cutaneous hypersensitivity and played an essential role in the development of AHR after specific allergen challenge to the airways. Although these antibodies were unable to alter AHR when delivered alone, when combined with limited airway exposure to antigen they elicited eosinophil infiltration of the lungs, increased eosinophil peroxidase activity, and altered AHR (Hamelmann et al., 1999b; Oshiba et al., 1996). 2. Lymphocyte Transfer The discovery that the pulmonary eosinophilia characteristic of the allergic response is controlled by CD4+ T cells has led to the development of a number of models designed to study subsets of these cells in greater detail. Moreover, these models are designed so that the T cells are passively transferred to naive mice so that the specific response of T cells to allergen can be studied in the absence of a peripheral priming response to antigen. Effector T cells can be divided into distinct subsets based on their cytokine profiles and functional properties. Th1 cells characteristically produce IFN-␥ and generally contribute to host defense against pathogens, whereas Th2 cells produce IL-4 and IL-5 and are associated with allergic reactions involving IgE, eosinophils, and basophils (Mosmann et al., 1986). It is possible to artificially polarize CD4 cells toward either a Th1 or Th2 phenotype in vitro using cytokine cocktails. Incubation of cells in an environment of IL-12 and anti–IL-4 leads toward a Th1 phenotype, whereas addition of IL-4 and anti–IL-12 predisposes to a Th2 phenotype. a. Transfer of CD4+ T Cells. The role of CD4 cells in the induction of pulmonary inflammation has been investigated in great detail using a variety of

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protocols based around a common theme—the passive transfer of effector cells into naive mice. The aim of all of these experiments has been to determine the effector population responsible for the initiation and development of inflammation and AHR. Highly purified CD4+ cells isolated from the spleens of sensitized donor mice then transferred to naive mice confer pulmonary eosinophilia and AHR (Hogan et al., 1998). In an elegant study, Wise and associates (1999) have identified a population of cells that actually mediate inflammation. Spleen cells from OVA-sensitized mice were cultured in vitro for 3 days in the presence of OVA. These authors found that cultured, antigen-stimulated cells, but not freshly isolated cells, promoted eosinophilia after a single OVA challenge after transfer to naive mice. Interestingly, the kinetics and magnitude of this inflammatory response in the adoptive transfer system were nearly identical to the response in mice receiving the conventional OVA sensitization and challenge. As previously described in other adoptive transfer systems in the mouse and rat, this antigen-specific pulmonary inflammation occurs in the absence of increased IgE levels found when the mice are directly sensitized and challenged (Cohn et al., 1998; Watanabe et al., 1995). Detailed positive and negative selection analysis indicates that the cells responsible for the transfer are found within the CD4highCD62LlowCD25+ phenotypic subset, which accounts for <1% of the cultured cells. Thus, this study determined that extensive lung inflammation can be induced in naive mice by the transfer of <100,000 cells. The acquisition of the capacity to promote eosinophilic lung inflammation that occurs during the 3-day culture period is associated with the initiation of IL-5 production by these cells, suggesting that the capacity to secrete IL-5 may be the critical factor that distinguishes the cultured cells from the freshly isolated splenocytes. b. Transfer of T Cell Clones. Kaminuma and colleagues (1997) generated a panel of OVA-reactive T cell clones from lymphoid cells of immunized BALB/c mice. T cell clones or unprimed CD4+ cells were transferred intravenously to naive mice and after 24 hr were challenged with OVA. Cytokine analysis of the panel of clones revealed several that secreted IL-5 and IL-4 upon antigen stimulation in vitro, one at a level 10-fold higher than all of the others. Interestingly, this was the only clone that, when transferred to naive mice in vivo, induced BAL eosinophilia, AHR, and BAL IL-5 and IL-4 production. The airway eosinophilia was suppressed by the administration of a neutralizing antibody for IL-4. The authors concluded that effector T cells were critical in the development of eosinophilic inflammation. A similar study found similar results by passively transferring conalbumin-specific Th2 clones to naive mice and comparing parameters of allergic lung disease observed with those in mice sensitized and challenged with OVA (X. M. Li et al., 1998). Although the Th2induced responses in naive mice showed pulmonary eosinophilia, mucus production, and AHR, they occurred to a lesser degree and extent than in sensitized

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and challenged mice. These changes were seen in the absence of serum IgE production. c. Transfer of Effector Th Cells. Transfer systems using well-defined subsets of effector T cells have been established in order to dissect the role of Th in the development of various features of allergic airway disease. A series of studies by Cohn and co-workers (1997) attempted to carefully examine the role of Th subsets and their cytokines in the development of different pathophysiological parameters. The aim of these studies was to determine whether Th2 or Th1 cells in isolation could reproduce some of the inflammatory changes associated with the asthmatic response, and whether this would lead to pathological changes such as mucus secretion. To accomplish this, the authors generated in vitro polarized cells from CD4 cells isolated from transgenic mice in which all the T cell receptors are specific for an immunodominant OVA peptide (Murphy et al., 1990). Populations of effector T cells were biased to a Th1 or a Th2 cytokine phenotype by culture in specific cytokines. It was demonstrated that after transfer to naive mice and daily aerosolized antigen challenge, Th1 or Th2 cells are recruited to the lungs and result in neutrophilia or eosinophilia, respectively (Cohn et al., 1997). Despite similar degrees and localization of lung inflammation, Th2 cells, but not Th1 cells, induced mucus secretion. The role of Th2 in mucus secretion was further dissected using IL-4–deficient mice. IL-4 was found to be critical for the primary phase of mucus induction, but once inflammation is established Th2 cells were found to stimulate airway epithelial mucus in the absence of IL-4 (Cohn et al., 1997). Further work showed that Th2 cell transfer also induced AHR, whereas Th1 cells did not (Cohn et al., 1998). IL-4 was not essential for the induction of AHR but was critical for the migration of eosinophils from lung tissue into the airway lumen. Moreover, IL-5, eosinophilia, nor mast cells are essential for mucus production, although signaling via IL-4R␣ is critically important for Th2 stimulation of mucus production (Cohn et al., 1999a). d. Potential Modulatory Effect of Effector Populations. This body of work has demonstrated a critical role for Th2 cells in the induction of a range of features of the asthmatic syndrome. However, Th1 cells are also present in biopsies from asthma patients, but their role is unclear (Cembrzynska-Nowak et al., 1993; Krug et al., 1996). One theory proposes a protective role for Th1 cells, stemming from evidence that Th1 cells inhibit the proliferation and development of Th2 cells (Abbas et al., 1996). Successful immunotherapy has been shown to increase levels of IFN-␥ in asthmatic patients (Durham et al., 1996; Jutel et al., 1995; Varney et al., 1993). In addition, infection of mice with M. bovis– bacille Calmette–Guerin ´ has been shown to suppress allergen-induced airway eosinophilia (Erb et al., 1998). An alternative theory suggests that Th1 cells may potentiate the inflammatory response because of the pro/inflammatory effects of Th1 cytokines (Holtzman et al., 1996; Krug et al., 1996). Thus, the role of Th1

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cells is ill defined, and a shift in the balance between Th2 and Th1 may relieve symptoms or, alternatively, make them worse. A number of recent studies have set out to define the mechanisms by which Th1 cells affect Th2 responses in the lungs, using passive transfer of in vitro polarized Th1 or Th2 cells. Randolph and colleagues (1999a) used passive transfer of effector cells during an active immunization model to determine whether it was possible to modulate airway inflammation by T cell subtypes. By intracellular cytokine staining and flow-cytometric analysis, they determined that both Th1 and Th2 cells are recruited to the lungs during allergen challenge in mice, with Th1 cells predominating early in the response and Th2 cells increasing later. Shifting the balance between phenotypes by increasing the number of Th1 cells via passive transfer resulted in enhanced inflammation. Interestingly, this effect was observed regardless of whether the cells were transferred before sensitization or after inflammation was established. Th1 cell transfer also resulted in increased recruitment of both Th1 and Th2 cells to the lungs. In contrast, transfer of Th2 cells had no effect. Thus, their results indicate that rather than being protective, an increase in Th1 cells may potentiate pulmonary inflammation. Similar results were observed by Hansen and co-workers (1999), who assessed the capacity of Th1, Th2, or Th0 cells to counterbalance the effect of Th2 cells. Th2 cell transfer to naive severe combined immunodeficiency (SCID) mice induced severe inflammation, but the addition of Th1 cells failed to counterbalance the associated AHR. However, Th1 transfer did reduce the number of airway eosinophils as well as decrease intrabronchiolar mucus production. Th1 cells themselves caused severe airway inflammation, but not AHR. In contrast to these results, Th1 and Th2 cells have been found to cooperate during allergic inflammation. Randolph and associates (1999b) found that the Th2 cells transferred alone to naive BALB/c mice caused minimal inflammation but required the presence of Th1 cells to promote a robust eosinophil-rich inflammatory response. Moreover, Th2 cells did not accumulate in the airway if transferred alone, although Th1 cells were readily recruited to the lungs— even in the absence of Th2 cells. When transferred together, both Th1 and Th2 cells were found in the airways, in conjunction with eosinophils. This is in direct contradiction to work by others and by us. We found that both Th1 cells and Th2 cells transferred alone induce pulmonary inflammation and are readily recruited to the lungs after challenge (Cohn et al., 1997; Coyle et al., 1999; Lloyd et al., 2000). A similar passive transfer system using influenza virus hemagglutinin–specific Th1 or Th2 cells induced similar pathology—with Th1 transfer inducing neutrophilia and Th2 transfer promoting eosinophilia (L. Li et al., 1998). In this case, cotransfer of hemagglutinin-specific Th1 cells did not inhibit antigen-induced lung eosinophilia. The only instance in which a positive regulatory role for Th1 cells was detected was in another cotransfer study. Delivery of both Th2 and Th1 cells resulted

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in a marked decrease in airway eosinophilia and mucus staining (Cohn et al., 1999b). This effect was abolished when signaling through the IFN-␥ receptor was inhibited by the use of IFN-␥ receptor knockout recipient mice, suggesting that development of airway eosinophilia requires activated Th2 cells and can be inhibited by IFN-␥ receptor secreted by Th1 cells. In the absence of IFN-␥ receptor signaling, Th1 cells induced mucus production but not eosinophilia. All of the above studies were designed to investigate the role of Th1 cells in the pathogenesis of a predominantly Th2-mediated phenomenon: pulmonary allergic inflammation (summarized in Table III). Although these studies have used a similar technique in their investigations—namely, the passive transfer of in vitro polarized subsets of effector T cells—a confusing picture remains of the role of Th1 cells. This can be explained in part by subtle differences in the experimental protocols used by the investigators. One fundamental problem is the lack of Th2 TABLE III TRANSFER OF EFFECTOR T CELL POPULATION IN MICE Cells Transferred CD4 T cells CD4 T cells from sensitized donors OVA stimulates spleen cells T cell clones OVA clones into naive BALB/c mice plus OVA challenge Conalbumin clones into naive BALB/c mice plus concanavalin A challenge Effector Th cells (OVA specific) Th2 cells into BALB/c mice, 7 × OVA Plus Th1 cells Th2 cells into SCID mice, 3 × OVA Plus Th1 cells Th2 cells into BALB/c mice, 4–6 × OVA Th1 cells into BALB/c mice, 4–6 × OVA Th1+Th2 cells into BALB/c mice, 4–6 × OVA

Effect

Reference

Eosinophilia, AHR

Hogan et al. (1998)

Eosinophilia, AHR

Wise et al. (1999)

BAL eosinophilia, IL-4, and IL-5

Kaminuma et al.

BAL eosinophilia, mucus AHR

X. M. Li et al. (1998)

BAL eosinophilia, mucus, AHR amelioration BAL eosinophilia, AHR No amelioration No effect

Cohn et al. (1997) Cohn et al. (1999b) Hansen et al. (1999) Hansen et al. (1999) Randolph et al. (1999b)

Th1 trafficking to the lungs

Randolph et al.(1999b)

BAL eosinophilia

Randolph et al. (1999b)

The protocols are summarized to compare methods and resulting phenotypic features of asthma. For details of experiments, see text and individual references. AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; IL, interleukin; OVA, ovalbumin; SCID, severe combined immunodeficiency; Th, T helper.

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migration to the lungs following allergen challenge in the study by Randolph and colleagues (1999b). In this case, Th2 cells required the cooperation of Th1 cells to accumulate in the lungs, causing only minimal inflammation if transferred alone. This is in direct contrast to all of the other studies, in which a significant Th2 cell infiltration is seen after transfer of cells to naive mice. The preparation of the populations of effector T cells is likely to be critical in determining their fate in vivo. The stage of differentiation as well as the activation state at the time of transfer are both likely to be important in inducing maximal inflammation. The T cell populations in the Randolph study are prepared by giving one round of polarizing cytokines before freezing the sample. The cells were then restimulated with IL-2 before transfer in vivo. In contrast, our work and that of Hansen’s group (1999) has been completed using cells that have undergone at least three rounds of stimulation with polarizing cytokines, producing mature, terminally differentiated effector T cells. Although Cohn and associates (1996, 1997, 1999) have also used T cells after one round of polarization, this may be somewhat compensated for by the use of a longer challenge period in vivo—seven antigen challenges during 1 week, compared to one to three in other studies. These differences in T cell protocols are likely to be critical in interpreting the data from experiments when the results are seemingly in conflict. 3. Recommendation To produce a robust Th2-mediated inflammation, consisting of increased tissue and BAL eosinophilia together with BAL IL-4 and IL-5 and AHR, we recommend that the T cells that are transferred are mature, terminally differentiated cells. This type of model, we believe, represents a useful means of determining the role of particular T cell–associated molecules in the absence of the influence of the peripheral immune system. In addition, a well-characterized model would be useful to test therapies aimed to diminish the inappropriate Th2 response to antigen and could determine whether they would then shift the balance to a Th1-dominant phenotype, which would not be advantageous. This model can also be combined with novel technologies that engineer T cells to artificially produce molecules. This method has been used to determine that T cells secreting tumor growth factor ␤ regulate Th2-mediated AHR and inflammation (Hansen et al., 2000). E. GENE TRANSFER SYSTEMS 1. Transgenic Models One of the most compelling reasons to use mice in generating and analyzing models of human disease is the fact that genetic technology gives us the ability to manipulate the mouse genome by production of transgenic lines, as well as ablation of specific genes and thus their protein products, through homologous

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recombination. Many investigators have used this technology to determine the function of cytokines in vivo. The role of IL-4 in allergic inflammation was assessed by fusing the IL-4 gene to an Ig promoter/enhancer region and introducing this into transgenic mice (Tepper et al., 1990). Overexpression of IL-4 in various lymphoid organs elicited a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion with pathological characteristics of an allergic reaction. Similarly, a definitive role for IL-5 in eosinophil differentiation was outlined by the generation of IL-5 transgenic mice (Dent et al., 1990). IL-5 transgenic mice appeared normal, aside from having splenomegaly and increased circulating eosinophil counts. Eosinophilia was also present in the lungs, spleen, bone marrow, peritoneum, lymphoid organs, and gut. These studies are useful in that they confirm functional aspects of the molecules studied; however, this effect is necessarily general, since the gene is widely expressed throughout the immune system. It is interesting to note that both of these studies used promoters designed to induce expression throughout the immune system, yet eosinophilia was described in both the lungs and the gut (Dent et al., 1990). These studies, however, tell us little of the specific role of molecules in the development of lung injury.

2. Tissue-Specific Expression Systems More recent studies have taken advantage of a well-characterized lung-specific regulatory element to drive expression in the epithelium of adult animals (Hackett and Gitlin, 1992). This method takes advantage of the fact that ∼50–70% of epithelial cells in the trachea, bronchi, and bronchioles of mice are Clara cells, which produce a 10-kDa protein referred to as CC10 (Rankin et al., 1996). a. IL-4. Expression of IL-4 driven by the CC10 promoter induced pathological pulmonary changes. This tissue-selective expression of IL-4 elicited an inflammatory response characterized by epithelial cell hypertrophy, with the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils but without AHR (Rankin et al., 1996). b. IL-5. Adult mice engineered to express IL-5 in their lung epithelial cells develop pathological changes in their lungs, which include eosinophil invasion of the peribronchiolar spaces, eosinophil hypertrophy, goblet cell hyperplasia, and increased mucus production (Lee et al., 1997). The mice showed evidence of eosinophil migration to the airway lumen comparable to that seen in asthmatic patients. Most interestingly, perhaps, even in the absence of antigen-induced inflammation, these mice still display AHR in response to methacholine challenge. Thus, in this way, the main features of an allergic pulmonary inflammation can be reproduced in mice by the expression of a single molecule.

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c. IL-13. IL-13 is a cytokine that has attracted a significant amount of attention as a player in the pathogenesis of asthma, since it is secreted in large quantities by Th2 cells. Additionally, it has been found to play a direct role in tissue eosinophilia, mucus metaplasia, and AHR in allergen challenge models of asthma (Grunig et al., 1998; Wills-Karp et al., 1998). Similarly, its overexpression in the lung epithelium causes a mononuclear and eosinophilic inflammatory response, mucus cell metaplasia, airway fibrosis, eotaxin production, airway obstruction, and nonspecific AHR (Zhu et al., 1999). These results confirm a central role for IL-13 in the multiple features that are pathognomonic of asthma. d. IL-9. IL-9 is a pleiotropic cytokine produced by Th2 cells and is thought to be a susceptibility gene for asthma (Nicolaides et al., 1997). More recently, IL-9 has been shown to directly stimulate mucin production in respiratory epithelial cells (Longphre et al., 1999). IL-9 transgenic mice were generated using the CC10 promoter (Temann et al., 1998). Mice exhibited a striking eosinophilsand lymphocyte-rich pulmonary inflammation, a striking lung mast cell hyperplasia, epithelial cell hypertrophy, and subepithelial collagen deposition. Although mice showed normal baseline airway resistance, their response to inhaled methacholine was markedly increased. e. IL-11. Similarly, the potential effector functions of IL-11 were assessed using the same technique. IL-11 expression in lung epithelium manifested as nodular peribronchiolar mononuclear infiltrates, with B cells present in larger numbers than T cells, and substantial airway remodeling with subepithelial fibrosis (Tang et al., 1996). Evaluation of airway function demonstrated that transgenics had increased airway resistance as well as nonspecific airway responsiveness to methacholine compared to nontransgenic littermates. These studies specifically demonstrated one aspect of asthma that has previously not been adequately studied: airway remodeling. Tang’s group went on to further refine the system by using an inducible element in the expression system so that expression of the transgene is externally regulatable. This system was designed to mitigate the fact that expression of a transgene is initiated in utero and is constitutively expressed thereafter. Thus, the ability of the system to model the waxing and waning nature of a disease such as asthma is limited. Regulation of the transgene is achieved by incorporating a reverse tetracycline trans-activator, so gene expression could be turned on by incorporation of the drug in the animal’s water. This system was used to show that IL-11 caused abnormalities dependent (large alveoli) and independent (airway remodeling and peribronchiolar nodules) of lung growth and development (Ray et al., 1997). 3. Transient Gene Transfer Models Gene transfer has also been accomplished during allergen challenge models using a delivery system that uses adenoviral vectors that express a transgene in a

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dose-dependent manner in a specific tissue over an extended period. Expression is transient but avoids the pathology that may be observed in transgenic mice— particularly important if the transgene is found to be lethal. This technique has been used to determine whether repeated allergen exposure by aerosol in conjunction with localized granulocyte–macrophage colony–stimulating factor (GM-CSF) expression can lead to a Th2-driven eosinophilic inflammation in the airways. Repeated passive exposure by aerosol generally leads to a state of T cell–mediated immunological tolerance (McMenamin and Holt, 1993; McMenamin et al., 1994; van Halteren et al., 1997). However, mice that received aerosolized OVA after delivery of a GM-CSF–expressing adenoviral vector exhibited marked airway inflammation characterized by airway eosinophilia and goblet hyperplasia (Stampfli et al., 1998). Migration of eosinophils was preceded by an increase in both IL-4 and IL-5. Interestingly, airway eosinophilia induced after GM-CSF can increase local antigen presentation, thus facilitating the development of an antigen-specific eosinophilic inflammatory response. This is an important study, since the investigators have tried to model the occurrence in humans, whereby antigen is introduced to the host by way of the airway, generally without prior peripheral sensitization. Coexpression of IL-10—a cytokine with described immunoregulatory capacities—inhibited this GM-CSF–driven airway inflammation (Stampfli et al., 1999). This finding suggests that the development of an immunotherapeutic strategy based on IL-10 warrants further consideration (Stampfli et al., 1999). F. VIRAL INFECTION Asthma exacerbations and bronchial hyperreactivity in nonasthmatics can be triggered by viral respiratory tract infections (Busse, 1990). Viral infections can also predispose individuals to the development of asthma by mechanisms that are presently undetermined. Among the respiratory viruses implicated in childhood asthma are respiratory syncytial virus (RSV), the most common respiratory virus in infants. Epidemiological evidence suggests that viral respiratory infections may contribute to allergic sensitization (Frick et al., 1979). Mouse models have proved invaluable for investigating the immune response to human RSV. Intranasal infection of mice with in vitro–cultured RSV results in viral replication within the lungs, causing nonfatal bronchiolitis. Transient alveolar lymphocyte influx is followed by focal inflammation initially comprising mononuclear cells (Taylor et al., 1984). Preimmunization with recombinant viral proteins has shown that selected peptides elicit divergent immune responses. Sensitization with the G (attachment) or F (fusion) protein leads to lung hemorrhage and an influx of polymorphonuclear cells, but only G protein sensitization leads to eosinophil infiltration (Openshaw, 1995). Cell transfer experiments have shown that CD4 T cell responses are important in eliciting respiratory distress and weight loss in this model (Alwan et al., 1993). In another study, CD8 cells were found to be critical in the recruitment of eosinophils to the lungs and in the

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development of AHR (Schwarze et al., 1999b). Viral bronchiolitis has also been induced in rats by infection with Sendai virus, which initiates episodic, reversible airway obstruction and AHR in conjunction with chronic airway inflammation and airway wall remodeling (Kumar et al., 1997; Uhl et al., 1996). The association between viral infection and asthma has attracted attention as epidemiological evidence suggests that respiratory viruses are the most important trigger for acute asthma symptoms and may contribute to allergic sensitization (Busse, 1990; Frick et al., 1979; Gern et al., 1999). It has been speculated that infections early in life (i.e., during the first year) may influence the development of a Th2 environment, thus predisposing the individual to atopy (Gern et al., 1999). Experimental parainfluenza virus in rats has shown that time of infection is critical. Infection during weaning as opposed to during the neonatal period leads to chronic episodic, reversible airway inflammation and remodeling (Kumar et al., 1997). This work highlights the importance of windows in time that determine the outcome of host-environment interactions (Gern et al., 1999). Several investigators have used mouse models to examine this relationship between viral infection and allergy. RSV infection in mice leads to AHR in the acute phase and enhances the effects of airway sensitization to allergen after infection (Schwarze et al., 1997). Mice infected with RSV and then subsequently challenged with aerosolized OVA exhibited more pronounced AHR and lung eosinophilia that was found to be IL-5 dependent. Cell transfer experiments showed that T cells (CD8 cells in particular) are critical in mediating RSVinduced development of lung eosinophilia and AHR following allergic sensitization (Schwarze et al., 1999a,b). The relationship between early viral infection and development of asthma is a particularly important one, in light of the increasing incidence of childhood asthma, and there is a particular need to further develop models to investigate the relationship in greater detail. G. LIMITATIONS OF MODELS This chapter has described a variety of animal models designed to investigate the pulmonary allergic response. For the most part, these models have highlighted the importance of particular cells or pathways likely to be critical in the development of pathophysiology. However, there are limitations to these models that must be remembered when interpreting results. In particular, the choice of a model is critical. Conflicting results regarding the role of mediators such as IL-5 or IgE can be explained in part because of intrinsic differences in the experimental protocols used for different studies. Some critics have argued that animal models are limited because they do not faithfully represent the human disease. In particular, it is argued that murine models do not demonstrate key features of asthma pathophysiology such as epithelial changes, eosinophil degranulation, and plasma exudation (Gleich and Kita, 1997; Persson et al., 1997).

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This may be due to the fact that most investigators look for eosinophilia in the lavage and tissue and perhaps do not complete an exhaustive pathological analysis of the lungs. Qualitative and quantitative information regarding structural and histological changes to the lungs would be useful in the majority of models. Other criticisms of mouse models of asthma stem from the lack of mucosal inflammation and eosinophil recruitment to the epithelial layer in some models, both of which are characteristic of human asthma (Persson et al., 1997). In addition, most mouse models are relatively short term, with allergen rarely given for longer than 2 weeks, so lungs are devoid of the chronic inflammatory and epithelial changes that typify human asthma. Temelkovski and colleagues (1998) have tried to overcome these limitations by developing a novel protocol that involves chronic inhalation challenge of OVA-sensitized mice by controlled exposure to low mass concentrations of aerosolized antigen. They have succeeded in eliciting acute-on-chronic inflammation, in which mice exhibit abnormalities of the airway epithelium similar to those observed in human asthma, as well as AHR. II. Conclusion

While mice can never wholly replicate the features of human asthma, given the significant differences in the microanatomy and physiology of the respiratory system, the value of well-characterized models with defined pathophysiological end points cannot be underestimated. Animal models are of vital importance to identify critical pathways to target for potential therapies. They provide corroborative functional evidence for particular cells or mediators identified in clinical samples. Moreover, models are of strategic importance in the testing and screening of novel therapeutic compounds. In the future, as therapeutic agents are identified, in vitro disease models will be vital to understand how therapies work in vivo. It may be necessary to manipulate these models to potentiate particular pathways and parameters of human disease and test the efficacy of compounds in vivo. Thus, our knowledge of each particular facet of the allergic response in mice in vivo is critical for our ultimate understanding of the human disease. REFERENCES Abbas, A. K., Murphy, K. M., and Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature 383, 787–793. Alwan, W. H., Record, F. M., and Openshaw, P. J. (1993). Phenotypic and functional characterization of T cell lines specific for individual respiratory syncytial virus proteins. J. Immunol. 150, 5211– 5218. Brewer, J. M., Conacher, M., Satoskar, A., Bluethmann, H., and Alexander, J. (1996). In interleukin4–deficient mice, alum not only generates T helper 1 responses equivalent to Freund’s complete adjuvant, but continues to induce T helper 2 cytokine production. Eur. J. Immunol. 26, 2062–2066.

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Tsuyuki, S., Tsuyuki, J., Einsle, K., Kopf, M., and Coyle, A. J. (1997). Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness. J. Exp. Med. 185, 1671–1679. Uhl, E. W., Castleman, W. L., Sorkness, R. L., Busse, W. W., Lemanske, R. F. J., and McAllister, P. K. (1996). Parainfluenza virus–induced persistence of airway inflammation, fibrosis, and dysfunction associated with TGF–beta 1 expression in brown Norway rats. Am. J. Respir. Crit. Care Med. 154, 1834–1842. van Halteren, A. G., van der Cammen, M. J., Cooper, D., Savelkoul, H. F., Kraal, G., and Holt, P. G. (1997). Regulation of antigen-specific IgE, IgG1, and mast cell responses to ingested allergen by mucosal tolerance induction. J. Immunol. 159, 3009–3015. Varney, V. A., Hamid, Q. A., Gaga, M., Ying, S., Jacobson, M., Frew, A. J., Kay, A. B., and Durham, S. R. (1993). Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergen-induced late-phase cutaneous responses. J. Clin. Invest. 92, 644–651. Wang, L. F., Lin, J. Y., Hsieh, K. H., and Lin, R. H. (1996). Epicutaneous exposure of protein antigen induces a predominant Th2-like response with high IgE production in mice. J. Immunol. 156, 4077–4082. Watanabe, A., Mishima, H., Renzi, P. M., Xu, L. J., Hamid, Q., and Martin, J. G. (1995). Transfer of allergic airway responses with antigen-primed CD4+ but not CD8+ T cells in brown Norway rats. J. Clin. Invest. 96, 1303–1310. Wilkinson, M. J., Wells, C., and Behnke, J. M. (1990). Necator americanus in the mouse: Histopathological changes associated with the passage of larvae through the lungs of mice exposed to primary and secondary infection. Parasitol. Res. 76, 386–392. Wills-Karp, M., and Ewart, S. L. (1997). The genetics of allergen-induced airway hyperresponsiveness in mice. Am. J. Respir. Crit. Care Med. 156, S89–S96. Wills-Karp, M., Luyimbazi, J., Xu, X., Schofield, B., Neben, T. Y., Karp, C. L., and Donaldson, D. D. (1998). Interleukin-13: Central mediator of allergic asthma. Science 282, 2258–2261. Wise, J. T., Baginski, T. J., and Mobley, J. L. (1999). An adoptive transfer model of allergic lung inflammation in mice is mediated by CD4+CD62LlowCD25+ T cells. J. Immunol. 162, 5592– 5600. Wynn, T. A., and Cheever, A. W. (1995). Cytokine regulation of granuloma formation in schistosomiasis. Curr. Opin. Immunol. 7, 505–511. Yang, Y., Loy, J., Ryseck, R. P., Carrasco, D., and Bravo, R. (1998). Antigen-induced eosinophilic lung inflammation develops in mice deficient in chemokine eotaxin. Blood 92, 3912–3923. Yasue, M., Yokota, T., Suko, M., Okudaira, H., and Okumura, Y. (1998). Comparison of sensitization to crude and purified house dust mite allergens in inbred mice. Lab. Anim. Sci. 48, 346–352. Yssel, H., Fasler, S., Lamb, J., and de Vries, J. E. (1994). Induction of non-responsiveness in human allergen-specific type 2 T helper cells. Curr. Opin. Immunol. 6, 847–852. Yu, C. K., Lee, S. C., Wang, J. Y., Hsiue, T. R., and Lei, H. Y. (1996). Early-type hypersensitivityassociated airway inflammation and eosinophilia induced by Dermatophagoides farinae in sensitized mice. J. Immunol. 156, 1923–1930. Zhang, Y., Lamm, W. J., Albert, R. K., Chi, E. Y., Henderson, W. R. J., and Lewis, D. B. (1997). Influence of the route of allergen administration and genetic background on the murine allergic pulmonary response. Am. J. Respir. Crit. Care Med. 155, 661–669. Zhu, Z., Homer, R. J., Wang, Z., Chen, Q., Geba, G. P., Wang, J., Zhang, Y., and Elias, J. A. (1999). Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103, 779–788. Zuany-Amorim, C., Haile, ´ S., Leduc, D., Dumarey, C., Huerre, M., Vargaftig, B. B., and Pretolani, M. (1995). Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice. J. Clin. Invest. 95, 2644–2651.

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ADVANCES IN IMMUNOLOGY, VOL. 77

Selected Comparison of Immune and Nervous System Development JEROLD CHUN Department of Pharmacology; Neurosciences Program; Biomedical Sciences Program; School of Medicine; University of California, San Diego; La Jolla, California 92037

I. Introduction

The developing mammalian nervous system shares many similarities with the formation of the immune system. Many of these similarities are at the level of homology or analogy, such as the concept of “memory,” but others extend into actual identity, as is the case for a number of different molecules, possibly reflecting their use in related functions. The purpose of this chapter is to review neural development for an immunological audience, with an aim of selecting a few comparative examples in which immunological information may provide insight into neurobiological developmental mechanisms, and perhaps vice versa. Emphasis is placed on neurogenesis within the developing mammalian cerebral cortex, as compared to immunological events in the thymus. Two aspects of current interest receive particular attention here: programmed cell death (PCD, or apoptosis) and the hypothesis that some form of DNA rearrangement occurs within the nervous system, especially in light of recent studies on nonhomologous end-joining (NHEJ) molecules. In each major section, a neurobiological feature is discussed, and germane aspects of immunological development are also considered. II. Major Cellular Components of the Nervous System

The primary constituents of the mature nervous system are neurons and glia. In addition, there are a large number of other cell types that contribute to normal brain function, such as endothelial cells, meningeal cells that form an outer covering of the brain, and choroid plexus cells that produce cerebrospinal fluid, which are not discussed further here. Still other cell types also exist during development of the brain which appear transiently, such as cells from macrophage lineages that have phagocytic functions. In considering the cellular constituents of the brain, it is important to note the developmental age, since the complement of cells varies for particular developmental stages. A. EFFECTOR CELLS: NEURONS Neurons come in a bewilderingly wide range of sizes and shapes (Stevens, 1979). These differences were first noted in the late nineteenth century by 297 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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early neuroanatomists/histologists such as Golgi and Ramon ´ y Cajal, who began to categorize neuronal types based on their histological appearance (Ramon ´ y Cajal, 1901, 1960). Indeed, the Golgi technique of silver precipitation to visualize single neurons, along with many neuronal descriptions, such as “granule” and “pyramidal” neurons, are in use to this day; many modern techniques, such as filling live neurons with fluorescent dyes, have both confirmed and augmented the concept that neuronal morphologies are complex. Neurons are the primary effector cells of the nervous system by way of their electrical excitability and synaptic contacts. By definition, neurons are postmitotic, and it is of note that despite their expression of a range of potentially oncogenic molecules, mature neurons remain postmitotic, as further evidenced by the lack of central nervous system (CNS) neoplasms that contain pure populations of neurons. Related to their morphological differences, neurons also have distinct anatomical connectivities, physiological properties, neurotransmitter identities, and probable differences in a variety of other molecules, such as the protocadherins (Hirano et al., 1999; Kohmura et al., 1998; Obata et al., 1995, 1998; Sago et al., 1995; Wu and Maniatis, 1999). Anatomical differences in neurons are not intuitive from a molecular immunological perspective, since there are no specific molecular markers such as T cell receptors (TCRs) or immunoglobulins (Ig’s) to mark these differences. However, the actual location of a single neuron in threedimensional space contributes to its virtually unique identity. For example, visual space is mapped precisely and topographically onto the cerebral cortex (Shatz, 1987; Shatz and Luskin, 1986), and the neuron representing this point in space, along with its shape and physiology, has a unique identity. To maintain a high degree of organization necessary to interpret the visual world, mature neuron cell bodies are not motile, and their synaptic contacts are relatively stable. However, there can be remodeling of the system at the level of synaptic contacts (Mooney et al., 1996), a phenomenon that may account for synaptic “plasticity” (Katz and Shatz, 1996; Shatz, 1990b, 1996), which includes features of learning. It is also worth noting that a typical neuron does not simply receive a single input from a single cell, as is often represented diagrammatically for didactic purposes. In fact, a single CNS neuron can receive on the order of 10,000 synapses and be connected to 1000 other neurons (Stevens, 1979). The importance of these attributes for this discussion is that neurons are, in reality, extremely heterogeneous (some would argue unique from neuron to neuron), stationary cells that are further capable of synaptic plasticity as well as connected in complex manners to many other neurons. Considering that the human brain contains on the order of 1011 neurons with the aforementioned connectivities and phenotypic differences, the brain achieves a degree of complexity that compares favorably with that calculated for Ig’s and TCRs. The effector cells of the immune system, lymphocytes, are quite different compared to neurons. In particular, the primary effector functions of the immune

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system involve interactions between antigen and antigen receptors of highly mobile cells that maintain the ability to proliferate through molecularly defined developmental stages (Darnell et al., 1986). The existence, for example, of B cell tumors of pro–B, pre–B, and mature B cell lineages underscores this capability (Whitlock et al., 1985). In addition, cell–cell communication is not required for terminal stages of effector function, as seen by the role of soluble Ig’s. From this comparison, the mature effector cells of the nervous system—neurons—are really very different from T cells and B cells in many respects. What stands out in this comparison is the multifaceted complexity of neurons that, in degree, is similar to the molecular complexity of lymphocytes achieved through the formation of antigen receptors. B. NONEFFECTOR CELLS: GLIA The other major cell type in the brain, glia, can be distinguished morphologically, biochemically, and molecularly from neurons and are traditionally viewed as supporting cells for neurons (Mission et al., 1991; Rakic, 1988). It is estimated that there are nearly 10 times more glia than neurons within the CNS (Truex and Carpenter, 1971). Two general types of glia are the astrocytes (Truex and Carpenter, 1971), which are often closely apposed to blood vessels, and myelinproducing oligodendrocytes (Raine, 1997), or the peripheral nervous system (PNS) counterpart of oligodendrocytes, the Schwann cells (Brockes et al., 1979; Weiner and Chun, 1999). In general, glia are not in synaptic contact with neurons and thus do not participate directly in the effector functions of neurons. Glial cells maintain the ability to proliferate. Astrocytes (Alberts et al., 1983; Brockes et al., 1979) express a marker protein called glial fibrillary acidic protein that can also be expressed by an embryonic counterpart known as radial glia (Rakic et al., 1974; Woodhams et al., 1981). In addition to these classical glia, a third type of glia are microglia, which are actually derived from a macrophage lineage (Hiremath et al., 1998; Truex and Carpenter, 1971). Perhaps the clearest similarity of glia to immunological cells are those involved in antigen presentation in the immune system. It is likely that an extensive number of mechanisms utilized by antigen-presenting cells are maintained in nervous system microglia, including the involvement of cytokines and growth factors (Otero and Merrill, 1994). Moreover, there is evidence that some astroctytes are themselves capable of in vivo antigen presentation (Williams et al., 1995). III. Embryonic Divisions of the Nervous System

A. NEURAL TUBE AND NEURAL CREST Neurons and glia of the nervous system arise from two classical subdivisions of the nervous system that reflect distinctions in their embryological origin (Cowan, 1979; Langman, 1981). This distinction arises around the time of neural tube

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closure, at which point cells of the neural tube give rise to the CNS, whereas cells arising from the excluded lip of the closing tube—the neural crest—give rise to the PNS (Fig. 1). The nervous system takes form at a point preceding formation of the primitive streak. In humans, this occurs by embryonic day 18 (E18); in the mouse (gestation about 20 days), this occurs around E7. The presence of the primitive streak in the dorsocaudal (tail) portion of the embryo marks the morphological beginning of the cephalad (head) location of the neural plate, which is simply a thickening of the proliferating ectoderm. Within hours in mice (days in humans), neurulation commences through inductive effects of the underlying mesoderm. The morphological result of induction is the formation of the neural groove that arises by cell proliferation in the lateral edges of the neural plate. The increase in cells laterally creates neural folds that bend toward one another and eventually fuse at the midline to form two important structures: the neural tube, which gives rise to the CNS proper, and the neural crest, which gives rise to the PNS (Fig. 1), along with a multitude of other nonneural tissues, discussed in the following section. B. PERIPHERAL NERVOUS SYSTEM The cells of the neural crest migrate to a variety of locations throughout the embryo to form the PNS, along with melanocytes; facial tissues, including odontoblasts and cartilage cells; some of the meninges (coverings of the brain); and other tissues as well (LeDouarin, 1980). By comprising the PNS, the neural crest gives rise to the dorsal root neurons of the dorsal root or sensory ganglia, Schwann cells that are responsible for myelination of peripheral axons, the sympathetic and parasympathetic ganglia (or parts of the autonomic nervous system), and the chromaffin cells of the adrenal medulla. Two remarkable features of the neural crest are their pluripotency—giving rise to a variety of tissues—and their capacity to migrate into the surrounding mesoderm. An additional group of neurogenic regions that are embryogically distinct from the neural crest and the CNS are referred to as placodes (from the Greek plateform), such as the olfactory placode. These regions appear to be most closely akin to the neural crest in being distinct from the neural tube and possessing cells with substantial migratory capacity, such as those giving rise to neurons producing gonadotropin-releasing hormone. The remainder of this chapter focuses on the CNS; however, it should be noted that the overwhelming majority of studies on “neuronal” cell lines in the literature are of most relevance to the PNS, where the overwhelming majority of such cell lines (e.g., neuroblastomas) arise. C. CENTRAL NERVOUS SYSTEM With the formation of the neural tube, cell proliferation (hyperplasia) and increases in cell size (hypertrophy) produce a decidedly asymmetrical and enlarging

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Neural Plate

A Ectoderm

B

Neural Groove Neural fold Somite

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Neural Crest Neural Tube

D

Central Canal

Spinal Cord (White Matter)

Spinal Cord (Gray Matter)

FIG. 1. Embryonic derivation of the peripheral nervous system (PNS) and central nervous system (CNS). Transverse section through an early embryo reveals the neural crest cells, which migrate away and give rise to the PNS. In contrast, the CNS derives from closure of the neural groove to produce the neural tube. Adapted from Cowan (1979).

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FIG. 2. Embryonic development, nomenclature, and general organization of the neural tube. Names of tube divisions are as indicated. The cerebral cortex derives from the prosencephalon/ telencephalon, growing in cell number and size to form its adult-like appearance (D). Adapted from Cowan (1979).

tube (Fig. 2) that moreover develops several distinctive kinks and bends, whose names are not critical here. The most proximal and eventually predominant (at least in mass) portion of the embryo is called the telencephalon, which gives rise to the basal ganglia, hippocampal formation, and the cerebral cortex. Immediately caudal to it is the diencephalon, which gives rise to the thalamus, epithalamus, and subthalamus. Next to it is the mesencephalon, or midbrain, which gives rise to the tectum. Next comes the metencephalon, which gives rise to the cerebellum and the pons. The final portion is called the myelencephalon, which gives rise to the medulla and is continuous with the spinal cord. In humans, all of these embryonic regions are discernible by the first trimester (by about E12 in the mouse). Contrasting with the nervous system, the immune system arises embryologically from extraembryonic stem cells that are more generally part of hemopoiesis, occurring through 10 weeks’ gestation in humans (E12 in the mouse)

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(Cooper and Nisbet-Brown, 1993). During this period, colonization of endoderm derivatives for T cells and B cells takes place. T cell precursors colonize the thymic anlage (primordium) around E10–E11 in the mouse, the anlage forming from the third and fourth pharyngeal pouches. A similar time course of colonization occurs for B cell precursors; however, they populate the fetal liver, an endodermal outgrowth of the embryonic hepatic diverticulum. By E15, they are also found in the spleen, and before birth, can be found in their adult-like location, the bone marrow. The embryonic origin and developmental compartments for both T cells and B cells are completely distinct compared to the nervous system that arises from the embryonic ectoderm. The majority of the nervous system originates from the telencephalon: it will eventually comprise ∼85% of the total mass of the brain (Truex and Carpenter, 1971). The focus on its major component, the cerebral cortex, thus serves both as an illustrative example and as a paradigm for most of the CNS in exploring developmental issues. IV. Embryonic Development of the Cerebral Cortex

A. ORGANIZATION The embryonic cortex arises from the proximal portion of the closed neural tube (first called the prosencephalon, Fig. 2), which later becomes the telencephalon mentioned above. At its earliest stages, it consists of a single layer of pseudostratified epithelium (Fig. 3) that forms a single sphere, then later as the paired hemispheres that give rise to the forebrain. By examining a magnified cross-section of the telencephalic wall, several obvious parts can be distinguished using simple, histological stains. The inner portion of the epithelium (toward the bottom of Fig. 3) that is in contact with the developing ventricles is aptly referred to as the ventricular zone (VZ). This is the zone of cell proliferation from which the vast majority of all neurons and glia of the cerebral cortex are derived. Somewhat nonintuitively, this region historically is considered apical. The outer, or basal, surface of the brain is also referred to as the pial surface because it is surrounded by the pia mater, consisting of supporting cells of neither neuronal nor glial lineage (probably derived from the neural crest). Cells in the epithelium, at least while the layers of cells in the epithelium are few, appear to have attachments to both the pial and ventricular surface. While the VZ remains present throughout neurogenesis, the histologically distinguishable layers, as seen in a tissue section extending from the pial to the ventricular surface, change with time (Fig. 4). This change reflects the generation of more superficial embryonic cell layers that are formed by the production and migration of postmitotic neurons and by the ingrowth and outgrowth of axons. The

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CP

IZ

VZ

V

V

V

FIG. 3. Embryonic zones in the developing cerebral cortex. A section from the cerebral wall is enlarged to reveal the three major embryonic zones that are visible by standard histology. The ventricle (V) is surrounded by the ventricular zone (VZ), the zone of cell proliferation during neurogenesis. The intermediate zone (IZ) sits superficial to the VZ and contains growing processes and migrating postmitotic neurons. The cortical plate (CP) contains differentiating postmitotic neurons that will remain in the adult as the cortical gray matter. The microscopic components in these zones are shown in Fig. 4.

latter process, combined with cell migration, produces a region just superficial to the VZ, which is aptly named the intermediate zone (IZ). Just superficial to the IZ is the cortical plate (CP), which consists of postmitotic neurons that have reached their adult-like location. Only the CP persists into adulthood, transformed into the six-layered cerebral cortex. The VZ is essentially lost, while the IZ becomes a zone of axon fibers commonly referred to as white matter.

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D MZ

CP

C MZ

CP IZ

B

A

MZ

IZ

IZ

SZ

SZ

VZ

VZ

MZ

VZ

VZ

FIG. 4. Microscopic anatomy of the embyronic cortical zones. These change with time as development proceeds (increasing age from left to right). (A) The early proliferative stage consists of a pseudostratified epithelium. (B and C) With development, the cerebral wall increases in width. Some cells, with processes stretching from the ventricular surface (black bar) to the pial surface (top of each illustration) represent radial glia that are used by some young neurons during migration to the pial surface. (C and D) With further development, more mature neurons (black triangular shapes) begin to differentiate superficially in the cortical plate (CP), along with the ingrowth/outgrowth of axons in the intermediate zone (IZ; lines parallel to the black bars). MZ, Marginal zone; SZ, subventricular zone; VZ, ventricular zone. Adapted from Cowan (1979).

Once located within the postmitotic CP, a prominent feature of cortical development can be seen: the formation of cellular layers based on the “birthdate” of a neuron. Newly postmitotic neurons do not assemble randomly within the CP, but assume histologically visible layers. The normal adult cerebral cortex has six layers, and cells within each layer have distinguishable morphologies and anatomical projections. In addition, the neurons within a layer are not generated randomly but instead form in an inside-out manner. Thus, the oldest cells are located closest to the ventricle, while the younger cells must migrate past the older ones to take a more superficial position (closer to the pial surface). In this way, the neurons of the cerebral cortex form cellular layers on the basis of their birthdate, in addition to layers that have both functional and anatomical specificity.

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FIG. 5. Interkinetic nuclear migration of neuroblasts within the ventricular zone (VZ). Proliferating neuroblasts undergo characteristic shape changes linked to the cell cycle. After mitosis, the daughter cells can either continue proliferating, repeating this process, or can become postmitotic and migrate superficially to differentiate in the cortical plate (CP) (see Figs. 3 and 4). IZ, intermediate zone; V, ventricle.

B. NEUROGENESIS Within the proliferative VZ, two cellular events must occur. First, a sufficient number of progenitors must be produced over a limited period. Second, postmitotic neurons must also be produced. To achieve this, a neuroblast can assume one of three general cell fates. It can reenter the S phase to again undergo mitosis, can become permanently postmitotic, or can undergo PCD. The former two fates constitute the historical view of how neurons of the cortex form and are examined here first, with recent data on cell death treated in more detail below. A characteristic of cell proliferation that is associated with neurogenesis is termed interkinetic nuclear migration (Fig. 5), in which the shapes of neuroblasts change in concert with phases of the cell cycle (Sauer, 1935; Seymour and Berry, 1975). As a given cell undergoes DNA synthesis (Angevine and Sidman, 1961; Sidman and Rakic, 1973; Sidman et al., 1959), its nucleus is positioned above (basal to) the ventricular surface, while the cell itself has a fusiform morphology. Following the S phase, the nucleus moves apically while retracting its basal process until reaching the ventricular surface. This results in a rounding up of the cell body, whereupon it undergoes mitosis. With the completion of mitosis, the neuroblast regains a fusiform morphology to continue proliferation to maintain or expand the blast population, depending on the stage of development. The resulting two daughter cells (Zhong et al., 1996) produced by mitotis can, alternatively, become postmitotic, migrating in a basal direction to locate just beneath the pial surface to form the future cerebral cortical gray matter, which at this age is the CP. The outlines of this phenomenon have been known since the 1930s; however, the significance of it for neurogenesis remains obscure (Chun, 1999). A problem faced by migrating neurons is that they must find their way from the VZ to their more superficial location. This problem is exacerbated as

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development proceeds because of the increasing width of the cortical wall (i.e., increasing ventricle-to-pial distance) caused by the accumulation of postmitotic neurons and the development of afferent and efferent fiber systems (see below). One mechanism that seems to be used by migrating neurons is their migration to the pial surface along a special kind of glial cell (which is also generated from the VZ). These radial glia (Culican et al., 1990; Rakic et al., 1974; Woodhams et al., 1981) (e.g., Fig. 4C) span the long distances between pia and ventricle and appear to serve at least partially as a substrate along which the neurons can migrate. However, nonradial migration also clearly occurs during formation of the cortex, as do neuronal contributions from noncortical areas. Thus, to reach its final location, a newly postmitotic neuron must interact extensively with an array of different cells and cell types. The primary technique that has allowed the demonstration of these neurogenic phenomena is use of labeled nucleotide analogues that are incorporated into DNA during the S phase of the cell cycle within the VZ, combined with visualization of cells in tissue sections using histology. Classical techniques that were used in the 1950s and continue to be used today utilize [3H] thymidine incorporation followed by tissue autoradiography (Angevine and Sidman, 1961; Berry and Rogers, 1965; Caviness and Sidman, 1973; Chun and Shatz, 1988, 1989a,b; Sidman et al., 1959). Labeled cells can be detected using photographic emulsions overlaid on tissue sections. After photographic developing, location of 3 H-labeled ␤-particle emission is marked by silver grain deposition over a labeled cell and can be seen using bright- or dark-field microscopy. More common today is the use of bromodeoxyuridine (BrdU) that is incorporated like thymidine and that can be visualized using immunohistochemistry (Blaschke et al., 1996, 1998; Pompeiano et al., 2000; Takahashi et al., 1995; Weiner and Chun, 1997b, 1999). The neurogenic formation of the cortex has parallels with T cell development in the thymus (Weissman, 1967, 1973). The nomenclature for the thymus as compared to the CNS is potentially confusing, since the less mature T cells are present in the thymic “cortex.” Like cells in the CNS VZ, cells in this outer thymic region can be labeled by brief pulses of nucleotide analogues, following which labeled cells can be studied (e.g., by tissue autoradiography or by BrdU immunohistochemistry). The outermost portion of the thymic cortex, a rim of cells on the order of eight cell diameters, is similar to the proliferative VZ. Using [3H]-thymidine, what are now known to be CD4+CD8+ double-positive cortical thymocytes were shown to be “born” in this discrete zone, whereupon they migrate centripetally to the mature location of single-positive thymic T cells, the medulla. Unlike most of the CNS, proliferation and maturation of T cells represent a continual process that occurs throughout most of life. However, the existence of CNS stem cells (Gage et al., 1995) raises the interesting possibility that related mechanisms may function at other developmental stages, if on a reduced scale.

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C. INITIAL PROCESS OUTGROWTH A hallmark of the CNS is its precise connections among the appropriate populations of neurons (Shatz, 1987, 1996; Shatz et al., 1988). To allow the development of these connections, two criteria must be met: (i) the growing axons must find the general target population, and (ii) a fine-tuning of the connection must be made so that the axons can synapse on the precise target cell. The first criterion appears to be met, at least in part, by the growth of young axons that interact with chemoattractants and chemorepulsive molecules (Dreyer et al., 1967; Tessier-Lavigne, 1992). This occurs along specified routes that consist of extracellular matrix or cell surface molecules (Goodman, 1994; Goodman and Shatz, 1993; Grenningloh et al., 1990). Some of these molecules, such as fibronectin, laminin, and proteoglycans as well as Ig superfamily members, probably involve the creation of a permissive (or nonpermissive) surface on which axons can travel, something like a highway that allows cars to travel but imparts little information about direction. Other molecules may serve to impart information—continuing the analogy, like a road sign or stoplight. By this kind of growth, axons can reach their target populations; this large body of information has been the subject of numerous excellent reviews (Goodman, 1994; Tessier-Lavigne, 1995; TessierLavigne and Zipursky, 1998). The second task is making the exact synaptic connection to the appropriate target cell. A popular model postulates that the “activity” of the axons—their propagation of action potentials and resultant synaptic function—allows axons to make these appropriate connections (Penn and Shatz, 1999; Shatz, 1990a). Evidence in favor of this hypothesis comes in part from studies using tetrodotoxin (a sodium channel blocker) to block activity. When this is done for the retinogeniculate pathway (from the retina to the thalamus), axons fail to sort out into their appropriate target layers, although the axons do reach the general target population and neurons appear to be normally organized. A recent report, however, complicates this view. Genetic deletion of Munc 18-1, which is required for neurotransmitter release from synaptic vesicles, results in a remarkably normal organization of the embryonic CNS (Verhage et al., 2000), suggesting that at least this form of activity is nonessential for normal development. Key mechanisms for forming initial groupings of cells and initial connections remain an active area of discovery. One intriguing possibility is a role for a large gene family encoding cell surface molecules, the protocadherins, in the formation of synaptic connections (Kohmura et al., 1998). These molecules likely have cell–cell recognition functions like the cadherins do (Takeichi, 1991; Takeichi et al., 1990a,b), which may be consistent with a more elaborate system of cell–cell interactions in organizing the early nervous system. By comparison, the immune system does not require precise connections between cells, nor do nervous system synapses function in cell–cell recognition. There do exist a plethora of Ig superfamily molecules with obvious relevance to

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both immune and nervous systems (Williams and Barclay, 1988). Indeed, some are expressed or are present within both systems, such as Thy-1 (Brockes et al., 1979; Lancki et al., 1995; Williams and Barclay, 1988), cadherins (Muller et al., 1997; Takeichi, 1991), major histocompatibility complex (Corriveau et al., 1998; Germain, 1994), Ig’s (Schatz, 1997; Weiner and Chun, 1997a), or signaling molecules such as fyn (Yasunaga et al., 1996) that interact with protocadherins (Kohmura et al., 1998). Whether there is truly shared functionality for these molecules that is common to the immune and nervous systems remains an open question, but whatever the case, the cellular substrates (e.g., synapses on postmitotic neurons for the nervous system) appear to be quite distinct. V. Ventricular Zone Neuroblast Programmed Cell Death

PCD is a feature common to all multicellular organisms. It has a wellcharacterized and essential role in the immune system, perhaps best exemplified by cell selection of appropriate T cells within the thymic cortex. Estimates on the extent of PCD in the thymus are in the range of 97% (Egerton et al., 1990; Shortman and Scollay, 1994; Shortman et al., 1990). The major mechanism for this intrathymic death within the thymic cortex appears to be by “neglect” because of a failure of T cells to undergo positive selection (Surh and Sprent, 1994), whereas “negative selection” constitutes a comparatively minor form that takes place in the thymic medulla. An interesting historical note is that controversy existed for decades following the proposal that intrathymic cell death actually took place (Metcalf, 1966), and the lack of death was championed by anatomists, who noted the absence of histological evidence for intrathymic cell death (Poste and Olson, 1973). The vast majority of normal PCD in the thymus takes place within the thymic cortex, overlapping with regions of thymic cell production, based on S-phase incorporation of labeled nucleotides such as [3H] thymidine (Surh and Sprent, 1994; Weissman, 1973). It is further notable that data obtained using [3H] thymidine was a mainstay of many studies supporting the conclusion that PCD did not take place in the thymus (discussed in Shortman and Scollay, 1985, 1994; Shortman et al., 1990). It was not until the use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (Gavrieli et al., 1992), which identified free DNA ends associated with PCD, that intrathymic cell death was demonstrated (Surh and Sprent, 1994). What is the role of PCD in the developing CNS? Pioneering studies had identified cell death as a significant mechanism in matching neuronal number to target tissues (Hamburger, 1975; Hamburger and Levi-Montalcini, 1949; Hamburger and Oppenheim, 1982), and this form of PCD occurred among postmitotic neurons, likely utilizing mechanisms of synaptic competition and target-derived growth factors (Davies and Lumsden, 1984). This type of cell death appears to occur throughout the nervous system (Chun and Shatz, 1989a,b; Chun et al.,

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1987; Verhage et al., 2000). However, the theoretical existence of earlier developmental phases of neuroblast cell death in the VZ was largely ignored or dismissed (discussed in Blaschke et al., 1996). Indeed, elaborate mathematical models have been constructed that must assume there is no PCD during neurogenic phases of development (Caviness et al., 1995). Contrasting with postmitotic neurons, VZ neuroblasts have more similarities with immunological cells such as double-positive T cells within the thymic cortex. Neuroblasts do not have synaptic contacts at this stage of development, can proliferate, migrate, and have comparatively simple morphologies. Another common feature shared by cortical thymocytes (Turka et al., 1991) and VZ neuroblasts (Chun et al., 1991) is expression of recombination-activating gene 1 (RAG1; discussed further below), although the role of RAG1 in the nervous system remains obscure. These superficial similarities between thymocyte development and VZ neurogenesis led us to ask whether, like the thymus, PCD operated in the embryonic cerebral cortex. To address this possibility, two techniques were developed and tested on normal and induced PCD using a variety of tissue and cell systems, including the thymus. In situ end-labeling plus (ISEL+) (Blaschke et al., 1996, 1998; Chun and Blaschke, 1997; Pompeiano et al., 1998, 2000; Staley et al., 1997; Zhu and Chun, 1998) is a much more sensitive variation of what was used to demonstrate intrathymic cell death, TUNEL. The second independent technique developed is based on ligation-mediated polymerase chain reaction (PCR) of blunt, 5′ phosphorylated DNA ends that are produced during apoptosis in the form of nucleosomal ladders that can be specifically amplified from apoptotic cells or tissues, including the normal thymus (Blaschke et al., 1996, 1998; Chun and Blaschke, 1997; Staley et al., 1997; Zhu and Chun, 1998). As an aside, these ends are identical to those generated by RAG cleavage (Schlissel et al., 1993). Both techniques produced results that supported or extended PCD results obtained in the thymus using TUNEL, as well as in other tissues. Examples of thymic labeling in dexamethasone-treated (induced PCD) or normal thymus are shown in Fig. 6. Notable in these figures is an increase in the number of dying cortical thymocytes compared to results obtained with TUNEL (Surh and Sprent, 1994), consistent with the ≈10-fold greater sensitivity of ISEL+ as compared to TUNEL (Chun, 1998; Chun and Blaschke, 1997) and in closer agreement to previous estimates of cortical T cell PCD (Egerton et al., 1990; Scollay and Shortman, 1985; Shortman and Scollay, 1985, 1994; Shortman et al., 1990). An example of what is observed in the embryonic cerebral cortex using ISEL+ is shown in Fig. 7. Consistent with PCD identified in the thymic cortex, dying cells were present in the expected postmitotic neuronal compartments (IZ and CP). Most strikingly, PCD was also present in the VZ, the region of cell proliferation. This provided the first evidence for VZ neuroblast PCD, comparable to the evidence that was used to demonstrate intrathymic PCD (Surh and Sprent, 1994).

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FIG. 6. ISEL+ (in situ end-labeling plus) identifies induced and normal cell death in the thymus. (A and B) Dexamethasone-induced programmed cell death in the thymic cortex (low magnification in A, higher magnification in B). (C and D) Normal thymic cell death identified in the thymic cortex. Med, Medulla.

As already noted above, the concept of intrathymic PCD was met with strong denial by histologists, and an essentially identical response, now repeated some 15–20 years later, has occurred for VZ PCD because, here too, there is a lack of histologically identifiable cell death. The caveats associated with deriving conclusions based purely on histology and the use of nucleotide incorporation are obvious, based on past studies of the thymus. Indeed, histological techniques do not accurately identify PCD that is known to occur in other model systems, such as the extreme example of the small intestinal villus, where an entire villus population of cells dies and is replaced every few days (Pompeiano et al., 1998). The demonstration of significant PCD among VZ neuroblasts by ISEL+ and ligation-mediated PCR has been independently supported by mouse null mutations in several pro–cell death genes. A major prediction from our data was that if it were possible to block neuroblast PCD, many more neuroblasts should then be present. This would appear as both an enlarged VZ and an increase in the total size of the brain. Several laboratories with immunological interests have created mice null for pro–cell death genes (e.g., caspases). Remarkably, the effects of deleting these genes, particularly individual caspases, did not result in

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FIG. 7. ISEL+ (in situ end-labeling plus) identifies dying cells in the embryonic cerebral cortex, especially apparent in the ventricular zone (VZ). (A) Low-magnification view of the cerebral wall, pial surface to the top. Note the presence of dying cells in the cortical plate (CP), the intermediate zone (IZ), and the VZ. (B) Higher magnification of VZ dying cells.

obvious effects to immunological tissues. However, in dramatic contrast, deletion of caspase 3 (Kuida et al., 1996; Woo et al., 1998), caspase 9 (Hakem et al., 1998; Kuida et al., 1998) and Apaf1 (Cecconi et al., 1998; Yoshida et al., 1998) all resulted in increased neuroblast numbers and enlarged brains at ages that preceded the generation of most postmitotic neurons. In addition, one would expect a loss of ISEL+-labeled cells in caspase-null mice, and indeed, this has been formally demonstrated, at least for caspase 3 (Pompeiano et al., 2000). It is somewhat surprising that these caspase deletion studies did not produce an analogous increase of cell number and size in immunological tissues known to undergo PCD (e.g., the thymus). Whether this represents caspase-independent PCD or the requirement for deletion of multiple caspases simultaneously is unknown. However, the observation that deletion of a single pro–cell death gene produces a nervous system phenotype but not a related immunological phenotype, despite the operation of extensive PCD in both systems, underscores differences in how PCD is regulated in these two systems. This major difference is tempered by observations using ISEL+ on caspase 3−/− embryos, where it is

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notable that the block in neuroblast PCD is far from complete, approximating a 30% reduction in dying cells (Pompeiano et al., 2000). Therefore, despite the obvious phenotype, a majority of PCD in this system remains and is not dependent on caspase 3 alone. As in the thymus, whether this majority of neuroblast PCD reflects caspase-independent mechanisms or the operation of multiple caspases remains to be determined. One interesting corollary of the results of caspase deletion is that it demonstrates differential susceptibilities of VZ neuroblasts to alterations in cell death machinery; that is, not all cells are affected. This may be due to the known developmental heterogeneity within the embryonic cortex, or possibly molecular heterogeneity of individual neuroblasts, at least with respect to their employed PCD pathways. These possibilities remain open questions that have relevance to recent, intriguing data on the immunological and nervous system defects observed in mouse null mutations of NHEJ genes. VI. Nonhomologous End-Joining and DNA Rearrangement

Most molecules and cellular functions common to both the immune and nervous systems are also shared with other cell types. However, a mechanism fundamental to the normal development of B and T lymphocytes—somatic DNA rearrangement, which includes both V(D)J recombination and heavy-chain class switching—has thus far distinguished lymphocytes from all other somatic cells. In the immune system, failed or inappropriate DNA rearrangement results in PCD. V(D)J recombination in lymphocytes is necessary in the assembly of antigen receptor genes encoding Ig’s and TCRs. Two events are necessary in order for DNA rearrangement to occur: DNA cleavage, then joining of cleaved free ends to produce the rearrangement. The enzymatic machinery required for the first part of the reaction requires the recombinase proteins RAG1 and RAG2 (Schatz, 1997), which cut DNA at precise, site-specific locations positioned next to gene segments to be recombined. This reaction produces double-stranded breaks in DNA that are blunt and 5′ -phosphorylated (Schlissel et al., 1993). The second major step recombines the DNA ends that have characteristic modifications, depending on whether they are coding joins, which form hairpins, or signal joins, which are precise and often produce recircularization of the cleaved byproduct (Schatz, 1997; Schatz et al., 1992). This NHEJ reaction requires several identified proteins: XRCC4 and ligase IV, which form a heterodimer (Critchlow, et al., 1997; Grawunder et al., 1997); Ku proteins (a heterodimer of Ku70 and Ku80); and DNA-dependent protein kinase. Studies from several laboratories, including our own, have analyzed genes involved with DNA rearrangement in studies of the nervous system or as an adjunct to immunological studies. Genes associated with the cleavage reaction— RAG1 and RAG2—do not produce obvious brain phenotypes, despite clear disruption of V(D)J, in null mutants (Mombaerts et al., 1992; Shinkai et al.,

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1992). Neural expression of RAG1 (but not RAG2) and indirect effects related to Ig light chain (Chun et al., 1991; Weiner and Chun, 1997a) or potential DNA transposition (Agrawal et al., 1998) leave open possible nonessential functions for RAG1. However, the actual function of RAG1 in the nervous system is not known. In dramatic contrast, deletion of genes involved with NHEJ, thought to be ubiquitous in expression, produce not only V(D)J disruption but also a remarkable phenotype within the CNS: early death of embryonic neurons (Barnes et al., 1998; Gao et al., 1998; Gu et al., 2000). The effect is obvious even using the same histological techniques that were incapable of accurately identifying PCD in the normal thymus or embryonic brain (noted above). More sensitive PCD detection techniques, such as ISEL+, have not been reported for these mutants. However, the presence of pyknotic cells within or just basal to the VZ indicates that their demise commenced within the VZ (Fig. 8), when the cells were neuroblasts (discussed in Chun and Schatz, 1999a,b). The actual percentage of neuroblasts that may be susceptible to NHEJ mutations is difficult to determine, since the most severe phenotypes—XRCC4 or ligase IV (null genotypes)—are also associated with embryonic lethality that ultimately results in the death of all cells. Because of this, the developmental point at which cell death is assessed in these null mutants will influence the extent of death observed. What is also clear in these studies is that NHEJ defects do not uniformly affect all developing neurons simultaneously (discussed in Chun and Schatz, 1999a,b) Indeed, based on results from Ku-deficient animals (Gu et al., 2000), it would appear that increased neuroblast loss (milder than XRCC4 or ligase IV, but still present) is compatible with a normal-appearing brain. This indicates that the lost cells were not needed for normal brain function, representing a dispensible subpopulation. In light of the absence of PCD effects in both the thymus and the majority of neuroblasts in caspase 3–deficient mice, the heterogeneity in NHEJ mutant neuroblast PCD is not unexpected. By contrast, T cells deficient for NHEJ genes still show uniform death that is accompanied by clear disruption of the actual NHEJ associated with V(D)J recombination (Frank et al., 1998; Gao et al., 1998; Gu et al., 2000). This raises the question of whether NHEJ is also disrupted in neuroblasts. There is no direct way to address this question in the absence of a known, endogenous target locus. However, a positive correlation exists between the extent of defective NHEJ and PCD in lymphocytes, and the severity of neuroblast PCD (Gao et al., 1998; Gu et al., 2000), which is consistent with involvement of NHEJ. On the other hand, mechanisms of NHEJ PCD in neuroblasts may not be cell autonomous or may be defective in growth control, as suggested for yeast deficient in lig4−/−(Barnes et al., 1998). Recent studies demonstrating that the absence of p53 can rescue neuronal cell death in a NHEJ-null genotype (XRCC4), but not death in T cells (Gao et al., 2000) further complicates

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CP

IZ

Zone of -/- dead cells

Zone of normally dying cells

VZ

V

V

V

FIG. 8. Distribution of dead cells in NHEJ-null mutant cerebral cortices, as compared to the normal distribution of dying cells identified by ISEL+ (in situ end-labeling plus). CP, Cortical plate; IZ, intermediate zone; V, ventricle; VZ, ventricular zone.

simple models of how neuroblasts are affected by the loss of NHEJ components (Fig. 9). T cells clearly have different requirements, compared to neuroblasts, for molecular death signals such as caspases. Moreover, even within a population of neuroblasts, there appear to be different requirements for both caspase death signals and NHEJ components. All these differences represent uncertainties that will no doubt be clarified in coming years. Initial postulates of nervous system DNA recombination in the 1960s (Dreyer et al., 1967) envisioned changes related to axonal connections during regrowth of axons into the tectum (midbrain) of goldfish. No locus for DNA rearrangement has yet been identified, but the best data to support such a mechanism to date, are the effects of NHEJ mutants on neuroblast PCD before the outgrowth of axons. Whether an actual DNA rearrangement is taking place is purely hypothetical but

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NHEJ+

?

Postmitotic neuron

p53-

DSBs NHEJ-

Dead neuron Postmitotic neuron

FIG. 9. Summary of neuroblast effects in NHEJ-null mutants (e.g., XRCC4 or ligase IV) with or without p53. The DNA double-stranded breaks (DSBs) are somehow produced during neurogenesis. In the absence of NHEJ components, marked increases in cell death, detectable even by standard histology, are observed. In addition, many apparently unaffected neurons also remain in these mutants. If the NHEJ-null mutant (at least for XRCC4) is crossed into a p53-null background, the neuroblast death phenotype is rescued. The explanation for differential effects on neuroblast subpopulations is not known.

would likely be distinct compared to V(D)J recombination, based on all available evidence. It must be noted, however, that the complexity of the nervous system combined with cellular and molecular differences noted here leaves open the possibility that DNA rearrangements may yet be identified. VII. Conclusion

Comparison of the developing immune and nervous systems reveals significant similarities but also significant differences. Many analogous functions exist, as do shared molecules. The closest similarities appear to be between the ongoing cellular interactions of double-positive T cells and those of embryonic neuroblasts during the generation of postmitotic neurons. The operation of PCD in both systems demonstrates varying susceptibilities to the functions of caspases, NHEJ molecules, and p53 as well as efforts to understand the mechanisms behind both similarities and differences provide a fertile area for current research. The issue of DNA rearrangement in neurons remains a viable possibility, especially considering the involvement of NHEJ in subpopulations of developing neuroblasts and neurons. However, if this occurs, it will be distinct from V(D)J recombination, and the only suitable proof will be the identification of a rearranged genomic locus within a normal neuron. ACKNOWLEDGMENTS I thank Drs. Ken Shortman and Roland Scollay for their comments, Dr. Marcy Kingsbury for reading the manuscript, and Casey Cox for preparation of the figures. This work was supported by the National Institutes of Health.

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INDEX

A Acetylhydroxylases, PAF, 163 Actin, changes in, linked to ion channel control, 31–32 Actin-binding proteins, regulation of actin cytoskeleton, 25–28 Actin cytoskeleton actin-binding proteins, 25–28 in cross-talk with raft membrane domains, 75–76 exogenous agents modifying actin, 28–30 lipid rafts and, 21–23 microfilament reorientation, 14–15 polymerization regulation, 24–25 rapid remodeling, 30 structure, 23–24 and T cell signaling, 2 in T cell signal transduction, 33–34 Actin-depolymerizing factor, see Cofilin Actin-related protein 2/3 complex, 12–13, 27 Activation basophils IgE dependent, 101–104 IgE independent, 104–106 Btk, 129–137 PLC␥ , 145–148 PTK, 7 serum response factor, 32 T cells, architecture of, 77–79 Activation-induced cell death, 11 Activation markers, basophil, 96–98 Active immunization models current protocols, 273 immunization protocol, 267–273 introduction of allergen to lungs, 264–265 323

modulation of, 276 selection of antigen, 265–267 Acylations, multiple, Src kinases membrane anchored by, 55–56 Adapter molecules Gads, interaction with SLP-76, 147–148 recruitment, 4–8 ADCC, see Antibody-dependent cellular cytotoxicity Adenovirus, neutralization, 210–211 Adhesion molecules, basophil, 94–95 Adjuvant, in active immunization models, 270 Airway hyperresponsiveness in vivo, 266–267 mouse models of RSV, 285–286 and mouse strains, 271–273 passive transfer models and, 277–282 to spasmogenic stimuli, 263 Allergen challenge late-phase response to, 112–113 transient gene transfer models, 284–285 Allergic disease, basophils and, 112–114 Alphaviruses, antibody protection against, 232–233 Antibodies, see also Monoclonal antibodies antireceptor, infection inhibition by, 223–225 antiviral activity, 196 in established infection, 241–244 coating of virion particles, 214–219 cytotoxicity mediated by, 226 in vivo protection by, 227–240 neutralizing, 196 nonneutralizing, 199 in passive transfer models, 277 phagocytosis mediated by, 226 Antibody-dependent cellular cytotoxicity, HSV and, 236–237

324

INDEX

Antibody neutralization adenovirus, 210–211 based on coating of virion particles, 214–219 and binding to virus, 198–200 early studies, 200–202 HIV-1, 211–213 host cell-derived proteins incorporated into virions, 222 influenza virus, 220–222 picornaviruses, 202–205 rabies virus, 211 single-hit vs. multi-hit kinetics, 205–210 and vaccine design, 247–248 Anticoagulation, sPLA2-IIA and, 176 Antigen, selection for active immunization models, 265–267 Antigen-presenting cells, conjugate with T cells, 77–79 Antimicrobial activity, sPLA2-IIA, 175–176 Apoptosis, in JNK pathway, 139–140 Arachidonic acid, metabolism, 170–175 Architecture poliovirus and HRV, 204–205 T cell activation, 77–79 Arenaviruses antibody protection against, 233 passive protection, 248 Asthma industrial model of, chemical compounds in, 267 RSV association with, 285–286 Atherosclerosis, sPLA2-IIA role, 177

B cell receptor pre-BCR checkpoint, 126–127 signal pathway, 123 translocation into DRMs, 69 B cells development BLNK and, 148–149 Btk and, 124–129 PLC␥ 2 activation in, 138 BCR, see B cell receptor BLNK B cell development and, 148–149 and Btk, in early B cell development, 149–150 Btk inducibly associated with, 134 integration of Btk and Syk activities, 142–148 Bone marrow, XLA, pre-B cells in, 125–127 Bovine papillomavirus, antibody neutralization, 214 Bruton’s tyrosine kinase activation, 129–137 B cell development and, 124–129 and BLNK, in early B cell development, 149–150 calcium signaling downstream of, 137–138 SH3 domain, 135–136 and Syk, integration via BLNK, 142–148 tyrosine phosphorylation, 129–132 WASP and TFII-I pathways downstream of, 141–142 Btk, see Bruton’s tyrosine kinase

C B Basophils activation IgE dependent, 101–104 IgE independent, 104–106 allergic disease and, 112–114 cell surface markers, 94–98 comparison with mast cells, 93 growth and maturation, 94 inflammatory mediators, 98–101 signal transduction and control of secretion, 106–112

Calcineurin, NF-AT and, 110 Calcium cytosolic, role in basophil pro-secretory events, 109–110 signaling BCR-induced, 144 downstream of Btk, 137–138 Calcium channels, L-type, actin-regulated, 32 Calponin homology domains, 10 Cancer, sPLA2-IIA role, 177–178 Carbohydrate recognition domain, sPLA2 receptor, 182

325

INDEX

Caspase 3, deletion, 312–313 Caveolin lipid rafts associated with, 18–19 major constituent of DRMs, 50–51 CC chemokines basophils primed by, 105 CCR5, in HIV-1, 224–225 CD4 and CD8: coreceptors for MHC, 74–75 in HIV-1 infection, 223–224 CD21, in enhancement of HIV-1 infection, 209 CD40, basophils expressing, 98 CD45 Lck activity and, 58 segregated from IRR patches, 73 CD59 delayed partitioning into DRMs, 51 GPI-linked, 22 CD3 ␨ , p21 form, 34 Cell surface markers, basophil, 94–98 Central nervous system, embryonic derivation, 300–303 Cerebral cortex embryonic development, 303–309 initial process outgrowth, 308–309 neurogenesis, 306–307 Challenge, allergen late-phase response to, 112–113 transient gene transfer models, 284–285 Challenge dose, effect on antibody activity, 240 Chemical compounds, in model of industrial asthma, 267 Chlamydia trachomatis, neutralization, 217–218, 244 Cholesterol extraction by M␤CD, 71–72 interaction with sphingomyelin, 47 lateral assembly, 45 c-jun N-terminal kinase pathway apoptosis and, 139–140 in Vav/Rac pathway, 10–11 Clathrin-coated vesicle pathway, 18 Cofilin inactivation by LIM kinase, 32 nuclear translocation, 26–27 Complement coating with, and virion neutralization, 210 neutralization of sensitized virus with, 207 virolysis mediated by, 225–226

Coronaviruses, antibody protection against, 233–234 Cortical plate, cerebral cortex, 304–305 Cross-linking GPI-anchored proteins, 64–65 lateral, plasma membrane proteins, 53–54 Cross-talk actin cytoskeleton with raft membrane domains, 75–76 sPLA2-IB to other enzymes, 183 Csk-binding protein, association with PAG, 58, 60 Cyclooxygenase isozymes, sPLA2-IIA and, 171–172 Cytochalasin D and FcεRI signaling, 76 modification of actin, 28–29 Cytokine receptors, basophil, 95–96 Cytokines basophil-releasing, 99–101, 113–114 induction of sPLA2-IIA expression, 170 Cytotoxic T lymphocytes, histamine effect on, 98–99

D Degranulation, sPLA2-IIA and, 176–177 Detergent insolubility cell membranes, 48–49 lipid rafts, 17 Detergent-resistant membrane association with FcεRI, 67–69 PIP2, 65–66 BCR translocated into, 69 LAT targeting into, 61 lipidated proteins associated with, 49–51 Lo-like membrane phase, 48–49 TCR recruited into, 70–71 Dimyristoyl phosphatidylcholine, in raft domains, 53 DNA rearrangement, nonhomologous end-joining and, 313–316 Dose antigen, in active immunization models, 271 neutralizing antibody, 204 virus challenge, 240 DRM, see Detergent-resistant membrane

326

INDEX

E Early studies, antibody neutralization, 200–202 Effector cells, neurons, 297–299 Eicosanoid biosynthesis, sPLA2-IIA, 170–175 COX-2 role, 171–172 heparan sulfate proteoglycan, 172–173 membrane asymmetry, 173–174 oxidation of cPLA2-derived products, 174–175 Embryonic development, cerebral cortex, 303–309 Embryonic divisions, nervous system, 299–303 Energy transfer, GPI-linked proteins, 51–52 Envelope spikes antibody binding, 198–199 heterogeneous, 246 HIV-1, 213 neutralization-irrelevant, 215 Enzymatic properties, sPLA2s, 167–168 Epitopes, neutralizing, 197–198 Extracellular signal-regulated kinase, see also Mitogen-activated protein kinase cPLA2 and, 108 Ezrin/radixin/moesin proteins, 27–28

F Fab fragments neutralization of viral particles, 218–219 in RSV, 242 Fc␣R signaling, raft role, 68 FcεRI cross-linked, 23 signaling and control of secretion, 106–112 cytochalasin D and, 76 raft role, 66–69 FcεRI␣, and IgE levels, 97 Fc␥ RIIA, 63, 68 Filament assembly/disassembly, 25–28 nucleation by cytochalasin D, 29 Filoviruses, antibody protection against, 234–235 Flaviviruses, antibody protection against, 235–236 Fluid mosaic model, plasma membrane structure, 45

F-Met-Leu-Phe, histamine induced by, 104 Friend-MuLV, antibody protection against, 230–231 Fungi, for active immunization models, 265 Fyn kinase enrichment, 78 N-terminal region mutations, 57 raft-associated, 55–56

G G-actin ATP-bound form, 24 pool, buffering by thymosins, 27 Gads, interaction with SLP-76, 147–148 Gelsolin, actin filament-binding, 27 Genes NHEJ, 314–315 pro-cell death, 311–312 sPLA2-X, 166 Genetics, mouse strains used in active immunization models, 271–273 Gene transfer systems tissue-specific expression systems, 283–285 transgenic models, 282–283 Glia, as noneffector cells of nervous system, 299 Glycerophospholipids, esterification, 46 Glycolipids, signaling via, 63–65 Glycosphingolipid-enriched membrane microdomains, see Lipid rafts Glycosphingolipids, ceramide-based, 46–47 GPI-anchored proteins energy transfer measurements, 51–52 enriched in DRMs, 49 signaling via, 63–65 GPI-linked folate receptor, 18 G proteins, heterotrimeric, ␤␥ dimer, 133 GTPase-activating protein, 9 Guanine nucleotide exchange factor, 7–11

H Hemagglutinin, associated with raft-like lipid assembly, 52 Heparanoids, sPLA2-binding, 168 Heparan sulfate proteoglycan, sPLA2-IIA and, 172–173

327

INDEX

Herpes simplex virus antibody protection against, 236–237 neutralization, 219 Histamine, synthesized by basophils, 98–99 Histamine-releasing factor, 104–106 HIV-1 antibody neutralization and binding to, 198–199 CD4 in, 223–224 enhancement of infection, 208–209 importance of binding site occupancy, 211–213 and LDV: comparing antibody activities, 239–240 H-ras detection in DRMs, 50 membrane-anchored, 72 HSV, see Herpes simplex virus Human cytomegalovirus, neutralization, 218 Human immunodeficiency virus type 1, see HIV-1 Human rhinovirus antibody neutralization, 214 neutralization, 203–205

I IgE, basophil activation and, 101–106 IgE receptors interreceptor spacing, 73 numbers on basophils, 96–97 patches, 67 Immune receptor-tyrosine-based activation motif, 3, 5, 34, 66–70 Immune recognition receptor signaling, 54 raft perturbation effects, 71–73 Immunity, sterile, 246–247 Immunization protocol, for active immunization models, 267–273 Infection antibody-mediated enhancement, 199–200, 208–209 antibody role, 196 established, antiviral antibody activity in, 241–244 inhibition by antireceptor antibodies, 223–225 retardation by antibody, 208 viral, mouse models, 285–286

Infectious virus, persistent fraction, 206–207 Inflammation allergic, 263 sPLA2-IIA role, 177 Influenza virus antibody neutralization, 220–222 antibody protection against, 237 Integrins, on basophils, 95 Interferon ␥ receptor, 281 Interleukin 3, basophils primed by, 105–106, 111 Interleukin 4 basophils as major source, 99–101 tissue-specific expression systems, 283 Interleukin 11, effector functions, 284 Interleukin 13 generation, 111 Ig isotype switch induced by, 99–100 tissue-specific expression systems, 284 Intermediate zone, cerebral cortex, 304–305 In situ end-labeling plus (ISEL+), 310–312 IRRs, see Immune recognition receptors Ischemia, sPLA2-IIA role, 177 Isoelectric point, changes after neutralizing antibody binding, 203 ITAM, see Immune receptor-tyrosine-based activation motif

J Jasplakinolide, modification of actin, 30 JNK, see c-jun N-terminal kinase pathway

K Kinetics models neutralization, 197, 204 single-hit vs. multi-hit, 205–210

L Lactate dehydrogenase-elevating virus, neuropathogenic, 239–240 lacZ gene, Btk gene replaced with, 128 Lag time, virus neutralization, 201, 205–206 LAT, see Linker of activated T cells Late-phase response, to allergen challenge, 112–113 Latrunculin, modification of actin, 29–30

328

INDEX

Lck enrichment, 78 mutant: deregulated activity, 57–58, 60 raft-associated, 55–56 LDV, see Lactate dehydrogenase-elevating virus Leukocyte protein, SLP-76, 7–8 Leukotriene C4 basophil-generated, 99 generation and release, 102 LIM kinase actin polymerization and, 26 inactivation of cofilin, 32 Linker of activated T cells complexes with signaling proteins, 60–61 mutants defective in DRM association, 61 palmitoyl acceptor sites, 49–50 in T cell signal transduction, 6–7 Lipid domains formation, 47–48 ordered-phase, stabilization, 53–54 Lipid metabolism, PI3K and, 9 Lipid rafts and actin cytoskeleton, 21–23 domains immunoreceptor signaling and, 54–79 structure in plasma membrane, 51–53 functions, 18–23 microscopic analysis, 17–18 in signal transduction, 19–21 structure, 16–17 12/15-LOX, 174–175 Lymphocytes, as effector cells of immune system, 298–299

M mAbs, see Monoclonal antibodies Major histocompatibility complex CD4 and CD8 coreceptors, 74–75 CD4 engaged by, 5 peptide-pulsed, 15–16 MAPK, see Mitogen-activated protein kinase Mast cells, comparison with basophils, 93 Maturation, basophils, 94 M␤CD, extraction of cholesterol from membranes, 71–72 Membranes, see also Plasma membrane cell

asymmetry: sPLA2-IIA and, 173–174 detergent-insoluble, 48–49 phase behavior, lipid domain formation and, 47–48 targeting of Btk, 132–134 Messenger RNA, IL-4, 102–103 MHC, see Major histocompatibility complex Microscopic analysis, lipid rafts, 17–18 Microtubule-organizing center, reorientation, 14 Mitogen-activated protein kinase activation: correlation with apoptosis, 139 p38, Cdc42-regulated, 10 Ras/MAPK pathway, 8 Models active immunization, 264–273 late-phase response to allergen challenge, 112–113 molecular, picornavirus neutralization, 219–220 mouse, limitations, 286–287 passive transfer, 277–282 rafts serving as signaling platforms, 20–21 Monoclonal antibodies anti-nucleoprotein, 234 anti-TCR, 12 antiviral, 229 neutralizing, 243 nonneutralizing, 207 Monocyte chemotactic proteins, 96 Mouse mammary tumor virus, nonneutralizing mAbs against, 207 Mouse strains, genetic background, 271–273 Murine hepatitis viruses, antibody protection against, 233–234 Murine leukemia virus, antibody protection against, 230–231 Mutations, Fyn N-terminal region mutations, 57

N Nervous system effector cells: neurons, 297–299 embryonic divisions, 299–303 noneffector cells: glia, 299 Neuroblasts, programmed cell death: ventricular zone, 309–313 Neurogenesis, cerebral cortex, 306–307

INDEX

Neurons, as mature effector cells of nervous system, 297–299 Neutralization antibody, see Antibody neutralization intracellular, 227 in vitro, mechanisms, 195 picornavirus, molecular model, 219–220 single-hit vs. multi-hit kinetics, 205–210 stoichiometry, 214–219 virions, by coating with complement, 210 virus, kinetic studies, 197, 204 Newcastle disease virus, mAbs against, 209–210 NF-AT, see Nuclear factor of activated T cells Nonhomologous end-joining, and DNA rearrangement, 313–316 Nonneutralizing antibodies, 199 Nonreleaser phenotype, basophil, 107 Nuclear factor of activated T cells calcineurin and, 110 transcriptional activation, 6–7 translocation, 11 Nucleation, actin filaments, by cytochalasin D, 29

O Occupancy antibody, at neutralization, 217–219 binding site, importance in HIV-1, 211–213 neutralization in relation to, 244 Organization, cerebral cortex, 303–305 Orthomyxoviruses, antibody protection against, 237 Outgrowth, initial process of cerebral cortex, 308–309 Ovalbumin, for active immunization models, 266–267 Overexpression Btk and Syk, 146 sPLA2-IID, 180 Oxidation, cPLA2-derived products, 174–175

P PAF, see Platelet-activating factor PAG, association with Csk, 58, 60

329

Palmitoylation protein, effect of PUFAs, 72–73 Src kinase, 56–57 Pancreatitis, sPLA2-IB role, 169 Paramyxoviruses, antibody protection against, 238 Parasites, for active immunization models, 265 Passive transfer models, lymphocyte CD4+ T cells, 277–278 effector Th cells, 279 modulatory effect of effectors, 279–282 T cell clones, 278–279 Peptides, complex with TCR and MHC, 15–16 Peripheral nervous system, placodes, 300 Persistent fraction, infectious virus, 206–207 Phagocytosis antibody-mediated, 226 sPLA2-IIA and, 175 Phorbol myristate acetate, activator of basophils, 109 Phosphatidylcholine hydrolyzed by sPLA2-X, 181 sPLA2-binding, 167–168 Phosphatidylinositol 4,5-bisphosphate, in raft dynamics, 65–66 Phosphatidylinositol 3-kinase coexpression with Btk, 133–134 lipid metabolism and, 9 p85 subunit, 132 pYxxM binding, 5 Phospholipase A2 secretory enzymatic properties, 167–168 heparanoid binding, 168 sPLA2 receptors, 182–183 structures, 164–167 sPLA2-IB, 168–169 sPLA2-IIA anticoagulation, 176 antimicrobial activity, 175–176 degranulation, 176–177 eicosanoid biosynthesis, 170–175 expression, 169–170 pathology, 177–178 sPLA2-III, 181 sPLA2-V, expression and functions, 178–180 sPLA2-X, expression and functions, 181 Phospholipase C-␥ 2 activation in B cells, 138 Syk connected with, 144–146

330

INDEX

Phospholipase C-␥ 1 pathway, T cell signaling, 8 Phosphoprotein associated with glycolipid-enriched membranes, see PAG Phosphotyrosine binding domain, 4–5 Picornaviruses antibody protection against, 237–238 neutralization, 202–205 molecular model, 219–220 PI3K, see Phosphatidylinositol 3-kinase PIP2, see Phosphatidylinositol 4,5-bisphosphate PLA2, see Phospholipase A2 Placental alkaline phosphatase, GPI-linked, 18, 48, 54 Placodes, akin to neural crest, 300 Plasma membrane raft domain structure in, 51–53 structural models, 45 Platelet-activating factor, acetylhydroxylases, 163 Platforms, signaling, rafts serving as, 20–21 Pleckstrin homology domain Btk, 127, 132–134 PTK, 124 Poliovirus, neutralization, 202–205 Polymerization, actin cytoskeleton, regulation, 24–25 Polyunsaturated fatty acids, effect on protein palmitoylation, 72–73 Profilin, as ATP nucleotide exchange factor, 25–26 Programmed cell death neuroblast: ventricular zone, 309–313 nonhomologous end-joining and, 313–316 Protein antigens, for active immunization models, 266–267 Protein kinase C F-actin association, 31 isozymes in basophils, 108–109 PKC␪, 8, 11, 13, 33, 35 Proteins, lipidated, DRM-associated, 49–51 Protein tyrosine kinase activation, 7 Btk/Tec family, 123–124 recruitment, 4–8 Src family, link with Btk, 129–130 Pseudogene, sPLA2-IIC as, 180 PSTPIP, SH3 domain, 131 PTK, see Protein tyrosine kinase PUFAs, see Polyunsaturated fatty acids

R Rabies virus, neutralization, 211 RAG-2, knockout mice, 150 RANTES, 96 Ras/MAPK pathway, T cell signaling, 8 Regulatory tyrosine residues, Y551 and Y223, 130–132 Reoviruses antibody protection against, 238–239 neurally spreading, protection against, 243 Respiratory syncytial virus association with asthma, 285–286 effect of neutralizing antibody, 242 Retroviruses, antibody protection in vivo, 229–231 Rhabdoviruses, antibody protection against, 231–232 Rho family members, differential effects on cell morphology, 9–10 Route of immunization, in active immunization models, 267, 270 RSV, see Respiratory syncytial virus

S Schistosoma mansoni, intratracheal delivery, 265–266 Sensitized virus, neutralization with anti-antibody, 207 Serum response factor, activation, 32 SH2, see Src homology 2 domain SH3BP5, phosphorylation by Btk, 136 Signaling calcium, downstream of Btk, 137–138 immunoreceptor, 54 raft membrane domains in, 66–73 IRR, raft perturbation effects, 71–73 T cell, see T cell signaling via GPI-anchored proteins and glycolipids, 63–65 Signaling cascades, as linear flow charts, 1–2 Signaling molecules, raft-associated, 55–66 Signal transduction actin dynamics in, 30–34 and control of secretion, 106–112 lipid rafts in, 19–21 T cell, actin cytoskeleton in, 33–34

INDEX

SLP-76 interaction with Gads, 147–148 SH2 containing, 7–8 SMAC, see Supramolecular activation complex Sphingolipids amide and hydroxyl groups, 46 lateral assembly, 45 Sphingomyelin, interaction with cholesterol, 47 Src homology 2 domain, containing SLP-76, 7–8 Src homology 3 domain, Btk, 131–132, 135–136 Src kinases enzymatic activity regulation, 57–60 inner leaflet-anchored, 64 membrane anchored by multiple acylations, 55–56 palmitoylation, 56–57 Structure actin cytoskeleton, 23–24 lipid raft domains, in plasma membrane, 51–53 lipid rafts, 16–17 plasma membrane, fluid mosaic model, 45 sPLA2s, 164–167 Superantigens, 103–104 Supramolecular activation complex central and peripheral, 78–79 formation, 13–16 PKC␪ recruitment to, 33, 35 at T cell–APC contact, 21–22 Surface area, virus, antibody-coated, 215, 217 Surrogate light chain, expression in XLA, 125–126 Syk, and Btk, integration via BLNK, 142–148

T Targeting Btk to membrane, 132–134 LAT into DRM, 61 T cell line-adapted HIV-1 isolates, 212–213 T cell receptor complex, expressed by CD4+ T helper lymphocytes, 3–4

331

engagement, 34–35 recruitment into DRMs, 70–71 TCR–peptide–MHC complex, 15–16 T cells activation, architecture of, 77–79 CD4+, in passive transfer models, 277–278 clones, transfer of, 278–279 costimulation through CD28, 22–23 signal transduction, actin cytoskeleton in, 33–34 T cell signaling actin cytoskeleton and, 2 formation of SMAC, 13–16 PI3K and lipid metabolism, 9 PLC-␥ 1 pathway, 8 Ras/MAPK pathway, 8 recruitment of PTKs and adapter molecules, 4–8 TCR complex, 3–4 Vav/Rac pathway, 9–13 TCR, see T cell receptor Tdt-mediated dUTP nick end-labeling (TUNEL), PCD and, 309–310 Tec homology domain, 124 Btk, 136 TFII-I pathway, downstream of Btk, 141–142 T helper cells effector, transfer of, 279 Th2, inflammation mediated by, 280–282 Th1 cytokines, proinflammatory effects, 279–280 Th2-like cytokines, basophils and, 100 Thymosins, buffering of G-actin pool, 27 Tick-borne encephalitis, antibody protection against, 235–236 Tissue expression sPLA2-IIA, 169–170 sPLA2-IIE, 180 Tissue-specific expression systems, gene transfer, 283–285 Transferrin receptor, cross-linked, 22 Transgenic models, gene transfer systems, 282–283 Transient gene transfer models, allergen challenge, 284–285 Tyrosine phosphorylation Btk, 129–132 PLC␥ , 144–145

332

INDEX

V

W

Vaccine design, and neutralizing antibodies, 247–248 Vav/Rac pathway, T cell signaling, 9–13 V(D)J recombination, 313–314 Venezuelan equine encephalomyelitis virus, 232–233 Ventricular zone cerebral cortex, 303–304 neuroblast programmed cell death, 309–313 Vesicular stomatitis virus antibody protection against, 231–232 neutralization, 209 Virions HIV-1, 213 host cell-derived proteins incorporated into, 222 neutralization, by coating with complement, 210 particles, antibody coating of, 214–219 Virolysis, complement-mediated, 225–226

WASP, see Wiskott–Aldrich syndrome protein Western equine encephalitis virus, neutralization, 201 Wiskott–Aldrich syndrome protein, 11–13, 75, 130–131, 141 Worm models, allergic airway disease, 266

X XID mouse, B cell defects, 127–128 XLA, see X-linked agammaglobulinemia X-linked agammaglobulinemia bone marrow pre-B cells, 125–127 Btk defect causing, 124–125

Z ZAP-70, LAT and, 6–7

CONTENTS OF RECENT VOLUMES

Volume 73

Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy EITAN YEFENOF

Mechanisms of Exogenons Antigen Presentation by MHC Class I Molecules in Vitro and in Vivo: Implications for Generating CD8+ T Cell Responses to Infectious Agents, Tumors, Transplants, and Vaccines JONATHAN W. YEWDELL, CHRISTOPHER C. NORBURY, AND JACK R. BENNINK

INDEX

Signal Transduction Pathways That Regulate the Fate of B Lymphocytes ANDREW CHAXTON, KEVIN OTIPODY, ALMIN JIANC, AND EDWARD A. CLARK

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers KIMISHICE ISIHZAKA, YASUYUKI ISHII, TATSUMI NAKANO, AND KATSUJI SUGIK

Oral Tolerance: Mechanisms and Therapeutic Applications ANA FARIA AND HOWARD L. WEINER

Volume 74

The Role of Complement in B Cell Activation and Tolerance MICHAEL C. CARROLL

Caspases and Cytokines: Roles in Inflammation and Autoimmunity JOHN C. REED

Receptor Editing in B Cells DAVID NEMAZEE

T Cell Dynamics in HIV-1 Infection DAWN R. CLARK, BOB J. DE BOER, KATJA C. WOLTHERS, AND FRANK MIEDEMA

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection PIUS LOETSCHER, BERNHARD MOSER, AND MARCO BACCIOLINI

Bacterial CpG DNA Activates Immune Cells to Signal Infectious Danger HERMANN WAGNER Neutrophil-Derived Proteins: Selling Cytokines by the Pound MARCO ANTONIO CASSATELLA

Escape of Human Solid Tumors from T-Cell Recognition:Molecular Mechanisms and Functional Significance FRANCESCO M. MARINCOLA, ELIZABETH M. JAFFEE, DANIEL J. HICKLIN, AND SOLDANO FERRONE

333

334

CONTENTS OF RECENT VOLUMES

The Host Response to Leishmania Infection WERNER SOLBACII AND TAMAS LASKAY INDEX

Volume 75 Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria ¨ JURGEN HESS, ULRICH SCHAIBLE, ¨ RAUPACH, AND STEFAN H. E. BARBEL KAUFMANN The Cytoskeleton in Lymphocyte Signaling A. BAUCH, F. W. ALT, G. R. CRABTREE, AND S. B. SNAPPER TGF-␤ Signaling by Smad Proteins KOHEI MIYAZONO, PETER TEN DIJKE, AND CARL-HENRIK HELDIN MHC Class II-Restricted Antigen Processing and Presentation JEAN PIETERS T-Cell Receptor Crossreactivity and Autoimmune Disease HARVEY CANTOR Strategies for Immunotherapy of Cancer CORNELIS J. M. MELIEY, RENE E. M. TOES, JAN PAUL MEDEMA, SJOERD H. VAN DER BURG, FERRY OSSENDORP, AND RIENK OFFRINGA Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor ROBERT C. HSUEH AND RICHARD H SCHEUERMANN

The 3′ IgH Regulatory Region: A Complex Structure in a Search for a Function AHMED AMINE KHAMLICHI, ERIC PINAUD, CATHERINE DECOURT, CHRISTINE CHAUVEAU, AND MICHEL COGNE´ INDEX

Volume 76 MIC Genes: From Genetics to Biology SEIAMAK BAHRAM CD40-Mediated Regulation of Immune Responses by TRAF-Dependent and TRAF-Independent Signaling Mechanisms AMRIF C. GRAMMER AND PETER E. LIPSKY Cell Death Control in Lymphocytes KIM NEWTON AND ANDREAS STRASSEN Systemic Lupus Erythematosus, Complement Deficiency, and Apoptosis M. C. PICKERING, M. BOTTO, P. R. TAYLOR, P. J. LACHMANN, AND M. J. WALPORT Signal Transduction by the High-Affinity Immunoglobulin E Receptor FceRI: Coupling Form to Function MONICA J. S. NADLER, SHARON A. MATTHEWS, HELEN TUHNER, AND JEAN-PIERRE KINET INDEX