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ADVANCES IN

Immunology VOLUME 72

This Page Intentionally Left Blank

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 72

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1999 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-1999 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. 0065-2776/99 $30.00

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CONTENTS

ix

CONTRIBUTORS

The Function of Small GTPases in Signaling by Immune Recognition and Other Leukocyte Receptors

AMNONALTMANAND MARCELDECKERT

I. Introduction 11. The Function of Ras in IRR Signaling 111. The Function of Rho-Family GTPases in IRR Signaling IV. CD28 Signaling in T Cells: The Roles of Small GTPases V. The Function of Rab GTPases in Leukocytes VI. Small GTPases and Aberrant Leukoc e Functions VII. The Role of Small GTPases in Lymp ocyte Development References

i:

1 3 25 49 52 53 64 70

Function of the CD3 Subunits of the Pre-TCR and TCR Complexes during T Cell Development

BERNARD MALISSEN, LAURENCEARDOUIN,SHIH-YAO LIN,ANNEGILLET,AND MARIEMALISSEN I. Introduction

11. Mouse cup T Cell Development 111. The Pre-TCR Sensor

IV. The ap TCR Sensor V. Raison dEtre of the Pre-TCR VI. Are the Roles o f the Pre-TCR and TCR Complexes Limited to Ensure Cell Survival? VII. The Limits of Genetic Analysis: Redundancy and Adaptive Response to Certain Mutations VIII. Conclusions References V

103 106 109 128 131 131 138 140 141

vi

CONTENTS

Inhibitory Pathways Triggered by ITIM-ContainingReceptors

SILVIABOLLANDAND JEFFREY V. RAVETCH I. 11. 111. IV. V. VI. VII.

149 150 154 158 161 163 165 166

Introduction Inhibitory Receptors and Activatin Counterparts FcyRII-Mediated Inhibitory Signa Mechanism of Inhibition by SHIP Inhibition by KIR Receptors Lessons from SHIP, SHP-1, and SHP-2 Knockout Mice Conclusions References

P

ATM in Lymphoid Development and Tumorigenesis

YANGXu

I. Lymphoid Defects and Tumorigenesis in Ataxia-Telangiectasia Patients 11. The ATM Gene 111. Dissecting the Lymphoid Defects and Tumorigenesis in A-T Mouse Models IV. Future Perspectives References

179 180 181 185 186

Comparison of Intact Antibody Structures and the Implications for Effector Function

LISAJ. HARRIS, STEVEN B. LARSON, AND ALEXANDERMCPHERSON 191 192 193 196 198 199 203 205

I. Introduction 11. Hinge-Deleted Dob and Mcg 111. Partial Structure of Kol IV. Mab231 V. Mab61.1.3 VI. Biolo 'cal Implications and Effector Functions VII. Conc uding Remarks References

?i

Lymphocyte Trafficking and Regional Immunity

EUGENE c. BUTCHER, MARNA WILLIAMS, KENNETHYOUNGMAN, MICHAEL BRISKIN

LUSIJAH ROTI',

AND

I. Introduction 11. Interaction of Blood Lymphocytes with Endothelium Involves Multiple Steps That Control the Specificity of Lymphocyte Recruitment

209 211

CONTENTS

111. Molecules Involved in Lymphocyte Interactions with Intestinal Endothelium IV. Subset S ecificity and Mechanisms of Lymphocyte Targeting to IntestinaY Tissues in Vivo V. a4P7 and the Segregation of Intestinal from Systemic Memory VI. Clinical Sipficance and Therapeutic Opportunities References

vii

213 229 237 24 1 243

Dendritic Cells

DIANA BELL,JAMES W. YOUNG, AND JACQUES BANCHEREAU I. Introduction 11. Features of Dendritic Cells 111. Ontogeny of Dendritic Cells IV. Maturation of Dendritic Cells V. Interactions of Dendritic Cells with T Cells VI. Interactions of Dendritic Cells with B Lymphocytes VII. Dendritic Cells in Clinical Disease States VIII. Concluding Remarks References

255 257 274 279 28 1 285 29 1 305 305

lntegrins in the Immune System

Yojr SKIMIZU, DAWDM. ROSE,AND MARKH. GINSBERG Introduction Ligand-Bindin Sites in Integrins Integrin Ligan s in the Immune System Integrin Signaling Integrin Function and the Immune System Integrins in Lymphocyte Recirculation The Role of Integrins in Immune Responses and Inflammation: Two Case Studies VIII. Re ulation of Integrin Ligand Expression in Inflammation Re erences

325 325 330 335 337 344

INDEX CONTENTS OF RECENTVOLUMES

381 389

I. 11. III. IV. V. VI. VII.

5!

f

348 351 353


CONTRIBUTORS

Nunhers in parentheses indicate the pages on which the authors’ contributions begin

Amnon Altman (l),Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121 Laurence Ardouin (103), Centre d’Immunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France Jacques Banchereau (255), Baylor Institute for Immunology Research, Saminons Cancer Center, Dallas, Texas 75204 Dianas Bell (255), Baylor Institute for Immunology Research, Sammons Cancer Center, Dallas, Texas 75204 Silvia Bolland (149), The Rockefeller University, New York City, New York 10021 Michael Briskin (209),LeukoSite, Inc., Cambridge, Massachusetts 02142 Eugene C. Butcher (209), Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305; and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 Marcel Deckert (l),Unite INSERM 343, HGpital de I’Archet, 06202 Nice Cedex 3, France Anne G a e t (103),Centre d’Immunologie, INSERM-CNRS de MarseilleLuminy, 13288 Marseille Cedex 9, France Mark H. Ginsberg (325), Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037 Lisa J. Harris (191), Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900 Steven B. Larson (191), Department of Molecular Biology and Biochernistry, University of California, Inine, Irvine, California 92697-3900 Shih-YaoLin(103),Centre dInimunologie, INSERM-CNRS de MarseilleLuminy, 13288 Marseille Cedex 9, France Bernard M a h e n (103), Centre d’Immunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France ix

X

CONTRIBUTORS

Marie Malissen (103), Centre dImmunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France Alexander McPherson (191), Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900 Jeffrey V. Ravetch (149), The Rockefeller University, New York City, New York 10021 David M. Rose (325), Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037 Lusijah Rott (209), Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford university School of Medicine, Stanford, California 94305; and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 Yoji Shimizu (325), Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis, Minnesota 55455-0392 Mama Williams (209), Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305; and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 Yang Xu (179), Department of Biology, University of California, San Diego, La Jolla, California 92093-0322 JamesW. Young (255),Memorial Sloan Kettering Cancer Center, Cornell University Medical College, New York City, New York 10021 Kenneth Youngman (209), Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305; and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304

ADVANCES IN IMMUNOLOGY, VOL. 72

The Function of Small GTPases in Signaling by Immune Recognition and Other Leukocyte Receptors AMNON ALTMAN AND MARCEL DECKERT' Division of Cell Biology, Irr hllo Institub for A l l e w o d Immunology, San Diego, California 92 121; and 'Uniie INSEM 343, M p i d de I'Archet, 06202 Nice Cedex 3, France

1. Introduction

A. THEFUNCTION AND REGULATIONOF RASSUPERFAMILY GTPA~ES This article focuses on the role and regulation of small GTP-binding (G) proteins in leukocyte functions. The complex and rapidly evolving field of small G proteins has been extensively reviewed (Boguski and McCormick, 1993). Therefore, only a brief discussion of these proteins and their regulation is provided here in order to set the stage for the following review. The ras genes were first identified as the transforming principle of the Harvey and Kirsten strains of rat sarcoma viruses, and subsequently described as sites of somatic mutations in some 30% of human tumors. The Ras superfamily consists of -60 mammalian genes encoding small (20-29 kDa) membrane-associated G proteins. Their highly conserved structure in evolution, from yeast to humans, suggests basic and critical functions during development, growth, and differentiation. Despite their large number and ability to elicit diverse sets of responses in distinct cell types, the small GTPases all share two biochemical properties, i.e., guanine nucleotide binding and intrinsic GTPase activity. The products of this gene superfamily have been divided into several families. The major ones are the Ras, Rho, and Rab families, members of which play major roles in regulating cellular growth and differentiation, organization of the actin cytoskeleton, and intracellular membrane and vesicle trafficking, respectively. The other two families are Ran and Ad. GTPases cycle between an inactive, GDP-bound state and an active, GTPbound form. The activity of small GTPases is determined by the ratio of these two forms. In resting cells, GTPases are mostly inactive. When cells are stimulated via different receptors, including tyrosine kinase receptors, immune recognition receptors (see below), and cytokine receptors, bound GDPis exchanged for GTP, shiftingthe GTPaseto its active state. Oncogenic GTPases, the result of mutations at several residues, are constitutivelyin the GTP-bound state, because they are either not subject to negative regulation or undergo an accelerated rate of GDP dissociation that results in GTP binding. The activity of small GTPases is regulated by three functional classes of 1

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AMNON ALTMAN AND MARCEL DECKERT

proteins. First, hydrolysis of bound GTP is accelerated by GTPase-activating proteins (GAPS)that inactivate the GTPases. Conversely,exchange of bound GDP for GTP is mediated by guanine nucleotide exchange factors (GEFs), resultingin GTPase activation.A third group ofproteins, the guanine nucleotide dissociation inhibitors (GDIs),maintain some small GTPases in an inactive or active state by inhibitingthe dissociation of GDP or GTP, respectively, and/or the basal or GAP-stimulated GTPase activity. The most significant advances in the broad area of small GTPases and their role in signal transduction are probably represented by four sets of findings. First, small GTPases activate distinct serinelthreonine kinase cascades, known as mitogen-activated protein kinases (MAPKs), that ultimately lead to gene transcription (Treisman, 1996); second, “cross talk,” with important functional implications,occurs among Ras and Rho proteins (Khosravi Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995a,b, 1997); third, in addition to their “traditional” role as regulators of the cytoskeleton, Rho-family GTPases also regulate growth-signalingcascades that control cellular proliferation and/or programmed cell death (Hill and Treisman, 1995; Symons, 1995; Treisman, 1996); and, finally, small GTPases are each coupled to multiple effectors, thereby generating a large diversity in the potential outcomes of their activation (White et al., 1995; Joneson et al., 1996; Lamarche et al., 1996; Westwick et al., 1997). B. SIGNALTRANSDUCTION BY IMMUNE RECOGNITION RECEPTORS The family of immune recognition receptors ( IRRs) includes the antigenspecific T and B cell receptors (TCRs and BCRs, respectively) and receptors for the Fc fragment of immunoglobulin. IRRs evolved with unique strategies to transmit activation signals. They all constitute protein complexes consisting of 3-10 subunits. The tasks of ligand binding and signal transduction are divided among distinct subunits. In T cells, for example, the signaling subunits comprise the y , 6, and E chains of the CD3 complex and the TCR-associated 6 chain. The cytoplasmic tails of the signaling subunits in all IRRs share a motif, i.e., the immunoreceptor tyrosine-based activation motif (ITAM), critical for signal transduction. Based on current genetic and biochemical studies, it is believed that IRR-initiated hematopoietic cell activation results from the sequential activation of protein tyrosine kinases (PTKs) of the Src and Syk families, and their interaction with phosphorylated ITAMs. Src-family kinases (Lck and/or Fyn in T cells) are thought to induce the early tyrosine phosphorylation of the ITAMs, which leads to the recruitment and subsequent activation of Syk-family PTKs (Zap-70 and Syk) via their tandem Src-homology 2 (SH2) domains. The activation of receptor-coupled PTKs represents an obligatory and early event in IRR-mediated signaling cascades. The activated PTKs induce

GTPases IN IMMUNE RECOGNITION RECEPTOR SIGNALING

3

increased cellular tyrosine phosphorylation, thereby modulating the activity and/or cellular localization of various enzymes and adapter proteins, and activating several signaling pathways, including phospholipase Cy (PLCy), various small GTPases, phosphatidylinositol 3-kinase (PIS-K), and cytoplasmic serine/threonine kinases such as MAPKs, which are divided into three families: extracellular-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinases. This results in gene transcription, differentiation, and cellular proliferation. In T cells, induction of the interleukin2 (IL-2) gene serves as a hallmark and end point of cellular activation. The properties of IRRs and the pathways they use in order to transduce activation signals have been widely reviewed (Weiss and Littman, 1994; R a i n et al., 1995; Chan and Shaw, 1996; Wange and Samelson, 1996; Alberola-Ila et al., 1997; DeFranco, 1997; Kurosaki, 1997). To some extent, lymphocytes (and T cells in particular) occupy a special place in the extensively studied area of small GTPases and their biological functions. This is so because, historically, the first evidence for physiological Ras regulation came from the study of T cell activation by mitogenic antiCD3 antibodies or phorbol ester (Downward et al., 1990). Since then, stimulation of hematopoietic cells via other IRRs has also been found to activate Ras and other small GTPases, and their known downstream serine/ threonine kinases. The functions of Ras proteins (Cantrell, 1994; Cantrell et al., 1994; Izquierdo Pastor et al., 1995) and, to a lesser extent, of Rho and Rab proteins (Chavrier, 1993; Reif and Cantrell, 1998) in lymphocytes have been reviewed before. Nevertheless, both the rapid progress in this research field and the lack of comprehensive reviews that integrate distinct small GTPase families into a single scheme in the context of IRR-mediated signaling processes prompt us to revisit this fundamental problem. The aim of this review is, therefore, to summarize our current knowledge on the role of small GTPases, primarily of the Ras and Rho families, during lymphocyte activation and development. Although we focus on IRR signaling, the function of small GTPases in signal transduction by other leukocyte receptors is also reviewed, albeit in less detail. Because function and regulation of small GTPases have been analyzed more extensively in T lymphocytes than in any other cell type of the hematopoietic lineage, much of the information presented here is derived from the analysis of T cells. II. The Function of Ras in IRR Signaling

A. RAs ACTIVATIONBY IRRs As pointed out previously, analysis of TCR-stimulated T cells provided the first experimental evidence of physiological Ras activation (Downward et al., 1990). These studies demonstrated that stimulation of human leuke-

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AMNON ALTMAN A N D MARCEL DECKERT

mic (Jurkat) or peripheral blood T cells with an anti-CD3 monoclonal antibody induced a marked increase in the cellular fraction of active, GTPbound Ras. All three forms of Ras, i.e., H-Ras, K-Ras, and N-Ras, were activated under these conditions. The observation that phorbol ester, a well-known protein kinase C (PKC) activator, can also activate Ras in the same cells suggested a role for PKC, and additional experiments indicated that phorbol ester-mediated Ras activation was due, at least in part, to inhibition of ras-GAP activity (Downward et al., 1990).The reported activation of Ras by the T cell accessory receptor CD2 (Graves et al., 1991) may reflect an effect of TCWCD3 ligation because, first, CD2 usually cannot signal in the absence of a functional TCWCD3 complex in mature T cells (Altman et al., 1990) and, second, a physical association between CD2 and TCWCD3 has been demonstrated (Beyers et al., 1992).Subsequent studies confirmed the ability of anti-CD3 antibodies or phorbol ester to activate Ras in T cells (Franklin et aZ., 1994; Ohtsuka et al., 1996),and demonstrated that BCR ligation by specific antigen, anti-Ig antibodies, or phorbol ester similarly activates Ras in B lymphocytes (Hanvood and Carnbier, 1993; Lazarus et aZ., 1993; Tordai et aZ., 1994; Sarmay et al., 1996; Tridandapani et al., 1997a).FcERI ligation on rat basophilic leukemia (RBL) cells ( Jabril Cuenod et aZ., 1996) or FcyRIII (CD16) cross-linking on natural killer (NK) cells (Galandrini et al., 1996)was also found to stimulate Ras activity. The physiological relevance of TCR-induced Ras activation was established by demonstrating that, first, constitutively active (CA) Ras mutants can synergize with Ca2+ionophore to activate the IL-2 gene in T cells (Baldari et al., 1992b; Rayter et al., 1992; Ohtsuka et aZ., 1996) or the IL3 and granulocyte/macrophage colony-stimulatingfactor (GM-CSF) genes in a mast cell line (Hahn et al., 1991) and, second, transient expression of a dominant-negative (DN) Ras mutant (N17Ras)inhibited TCR- or phorbol ester-induced IL-2 induction (Rayter et al., 1992; Baldari et al., 1993; Ohtsuka et al., 1996). Similarly, introduction of a neutralizing Ras-specific antibody into Jurkat T cells interfered with T cell activation (Werge et al., 1994). These studies indicate that Ras activation is necessary but not sufficient for stimulation of IL-2 production. Later studies have characterized the IL-2 promoter-associated transcription factors that are regulated by Ras in T cells (Section 11,D). The involvement of Ras in cytokine production is also evident from the finding that transformation of a mast cell line by an oncogenic mutant of ras is associated with constitutive IL3 production and an autocrine IL-3 response (Nair et al., 1992). However, it is clear that the IL-2 or other cytokine genes are not the only targets for Ras in hematopoietic cells. Thus, Ras function is also important for up-regulation of activation antigens such as CD69 (D’Ambrosioet al., 1994) and the IL-2 receptor a chain (Sirinian et al., 1993), and for transcription of

CTPases IN IMMUNE RECOGNITION RECEPTOR SIGNALING

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the human TCR-P gene (Wotton et al., 1993) in T cells. The targets of Ras are discussed in detail below. Figure 1presents a scheme of the IRRcoupled Ras signaling pathway, using the TCWCD3 complex as the model. A report by Boussiotis et al. (1997) indicated that Rapl, a member of the Ras family that is known to antagonize the activation of Ras (Hata et aZ., 1990; Kitayama et al., 1990), may play an important role in maintaining T cell anergy (Fig. 1). Thus, whereas antigen stimulation of control T cells led to a transient stimulation of Rapl, activated (GTP-bound) Rapl was constitutively present at a high level in anergic T cells, and this level was not affected by antigen stimulation (Boussiotis et al., 1997). The aberrant function of the Ras signaling pathway in anergic lymphocytes is discussed in more detail in Section VI,A. OF RASACTIVATION AND COUPLING TO IRRs B. REGULATION 1. The Role of PTKs versus PKC in Ras Activation The precise mechanism(s) by which IRRs activate Ras remains unclear (Fig. 1).Nevertheless, IRRs follow, at least in part, the paradigm established for growth factor receptors that possess intrinsic tyrosine kinase activity. Combined genetic, biochemical, and pharmacological approaches have established that Ras is activated by these receptors in a PTKdependent manner and that activated Ras couples receptor PTKs to downstream signaling cascades, leading to cellular growth and differentiation (Egan and Weinberg, 1993). Similarly, Ras activation by IRRs depends on intact PTK activity. This is evident from the findings that specific tyrosine ldnase inhibitors can prevent Ras activation induced by ligation of the TCR (Izquierdo et al., 1992a; Ohtsuka et al., 1996) or the BCR (Lazarus et aZ., 1993; Kawauchi et al., 1996). The contribution of PTKs to activation of the Ras pathway in lymphocytes is implicated by several other findings: first, the expression of CA Lck in thymoma cell lines derived from Lcktransgenic mice is associated with constitutive activation of the Ras/Raf1/ERK pathway (Lin et al., 1995); second, transient overexpression of Syk in an Lck-negative variant of Jurkat T cells (JCaM1) enabled the TCW CD3 complex to induce activation of the ERK pathway and the transcription factor nuclear factor of activated T cells (NFAT), which binds to the IL-2 gene promoter. This effect of Syk required its catalyhc activity, and it was blocked by a DN Ras mutant (Williams et al., 1997). As discussed below, adapter proteins, which either function as PTK substrates or bind PTK substrates via their SH2 domain, most likely represent the link between IRR-coupled activated PTKs and Ras. It is clear, however, that, in addition to the ubiquitous tyrosine kinasedependent Ras activation pathway shared by different receptors and cell

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FIG.1. TCR-induced Ras signaling pathways and their interactions with other GTPases. TCR-coupled PTKs include members of the Src (Lck, Fyn)and Syk (Zap-70, Syk) families. The scheme depicts two potential scenarios for Ras activation, i.e., via a LAT/Crb2/Sos complex (right) or via a ShdGrbWSos complex (left). The potential role of a Cbl/Crk/Rapl complex in T cell anergy (Section VI,A) is indicated. Rac is required (but not sufficient) either downstream of, or parallel to, Ras in a pathway leading to NFAT activation (Genot d al., 1996). Rac, calcineurin, and PKCO are involved in JNK activation in T cells (Werlen et aL, 1998; Ghaffari-Tabrizi et al., 1998), but the exact relationship between the three signals is unclear. Vavmac-mediated cytoskeletal reorganization is linked to TCR-derived growth signals (Holsinger et al., 1998). Known PTK substrates in T cells are lightly shaded, small GTPases are darkly shaded, and transcription factors are in black. DAG, Diacylglycerol; IP, inositol phosphates.

types, T and B cells display a Ras activation mechanism that is mediated by PKC. This is evident from the ability of PKC activators such as phorbol myristate acetate (PMA) to activate Ras in T (Downward et al., 1990;


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Franklin et al., 1994; Ohtsuka et al., 1996) or B (Harwood and Cambier, 1993; Tordai et al., 1994) cells. PKC-mediated Ras activation is a lymphocyte-specific response not found in myeloid or mast cells (Downward et al., 1992; Izquierdo et al., 1992a), but this notion may need to be reexamined in view of the very recent finding that PKC can also lead to Ras activation in COS cells (Marais et al., 1998). The nature of the PKC-dependent Ras activation mechanism and its relationship to the IRR-mediated pathway of Ras activation have been addressed in a number of studies, and are only partially understood. As mentioned previously, activated PKC may stimulate Ras by inhibiting the activity of ras-GAP (Downward et al., 1990; Izquierdo et al., 1992a). Because PMA-mediated Ras activation is sensitive to specific PKC inhibitors but is not affected by selective PTK inhibitors (Hanvood and Cambier, 1993; Ohtsuka et al., 1996),PKC-dependent Ras activation does not appear to require tyrosine kinase activity. The use of a selective PKC inhibitor (Ro 31-8425) and transient expression of a CA Ras mutant indicated that PKC does not act downstream of Ras in the induction of NFAT and AP1 transcriptional activity and in the expression of IL-2 in Jurkat T cells (Williamset al., 1995).This conclusion is consistent with two other findings: first, an intracellularly introduced neutralizing anti-Ras antibody inhibited TCR-induced NFAT activation, but was much less efficient in inhibiting NFAT activation induced by direct PKC stimulation (i.e.,by PMA treatment) (Werge et al., 1994);second, Ro 31-8425 prevented ERK2 activation by PMA, but not by CA Ras (Izquierdo et al., 1994b). These data also suggest that PKC may have other effectors in addition to Ras. The contribution of PKC to TCR-mediated Ras activation was tested in permeabilized T cells under conditions in which TCR-induced PKC activation was blocked by using either a PKC pseudosubstrate peptide inhibitor or ionic conditions that prevent inositol phospholipid hydrolysis and, hence, diacylglycerol production and PKC stimulation (Izquierdo et al., 1992a). In the absence of PKC stimulation, TCR ligation still induced Ras activation, which correlated with ras-GAP inactivation. The selective PTK inhibitor, herbimycin A, prevented the non-PKC-mediated, TCRinduced stimulation of RAS (Izquierdo et nl., 1992a). These data suggest that the TCR is coupIed to Ras via a PKC-independent pathway that involves tyrosine kinases. However, another study reported that calphostin C, a specific PKC inhibitor, blocked not only PMA-induced but also TCRmediated accumulation of Ras-GTP and, furthermore, that PKC downregulation by prolonged PMA treatment severely impaired the activation of Ras in response to TCR stimulation (Ohtsuka et al., 1996). These results suggest that the activation of PKC is important for TCR-mediated Ras activation in Jurkat cells. The apparent discrepancy between these two

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conclusions may reflect the use of distinct T cells, which may depend to varying degrees on the PKC-mediated Ras activation pathway, i.e., human peripheral blood lymphoblasts (Izquierdo et al., 1992a) versus leukemic Jurkat cells (Ohtsuka et al., 1996). Thus, the functional relevance of PKC in coupling the TCR to Ras activation remains unclear. One potential problem with earlier studies that assessed the contribution of PKC to Ras activation in T cells stems from the fact that PMA was used to activate cellular PKC. Phorbol esters nonselectively stimulate the conventional, Ca2'-dependent (a,/3, y ) and novel, Ca'+-independent (6, E , 7,6, p ) PKC enzymes. Therefore, interpretation of the effects of PMA on Ras activation is compounded because PKC isoforms manifest specificity in terms of their ability to translocate to different cellular compartments, differentiallyphosphorylate cellular substrates, and regulate distinct signaling pathways. Thus, at present it is unknown which, if any, of the T cellexpressed PKC isoforms can activate Ras.

2. The Role of Shc and Grb2/Sos in Bas Activation The mechanisms that couple receptor PTKs to Ras activation have been a subject of intense research, and two coupling mechanisms have been established (Fig. 1).First, the adapter protein Grb2 (Lowenstein et al., 1992) directly binds, via its SH2 domain, to phosphorylated tyrosine residue(s) in the cytoplasmic tail of activated receptor PTKs (Buday and Downward, 1993; Egan et al., 1993; Li et al., 1993; Rozakis Adcock et al., 1993). Second, Grb2 can indirectly associate with growth factor receptors via an intermediate adapter protein, Shc (Pelicci et al., 1992). Shc contains an SH2 domain that binds directly to the phosphorylated receptor, and is itself a substrate of receptor and nonreceptor PTKs. Phosphorylated tyrosine residue(s) of Shc bind the SH2 domain of Grb2 (Rozakis Adcock et al., 1992; Skolnik et al., 1993a,b; de Vries Smits et al., 1995; Pronk et al., 1994). In either case, the recruitment of Grb2 to the activated receptor allows Ras activation, because Grb2 is constitutively associated with the ubiquitous Ras GEF, Sos (Chardin et al., 1993), thereby localizing Sos to the membrane where its target (i.e.,Ras) is localized. The finding in one (Graziadei et al., 1990),but not another (Boyeret al., 1994),study that BCR cross-linking induces cocapping of Ras with the BCR indirectly supports the existence of such a recruitment mechanism in lymphocytes. The notion that targeting of Sos to the plasma membrane in the vicinity of Ras is a primary mechanism leading to Ras activation is supported by the finding that enforced localization of Sos to the plasma membrane via the addition of membrane-targeting sequences is sufficient for activating the Ras signaling pathway (Aronheim et at., 1994; Quilliam et al., 1994).


9

Potential mechanisms that couple IRRs to Ras could include the stimulation and/or membrane recruitment of a Ras GEF, and/or the inhibition of GAP activity. As noted previously, Ras activation by its ubiquitous GEF, Sos, does not reflect an increase in the specific activity of Sos but, rather, recruitment of Sos by Grb2 and/or Shc to the vicinity of Ras in the membrane. Nevertleless, one group reported that TCR stimulation of T hybridoina cells increased the guanine nucleotide exchange activity of Sos immunoprecipitates and induced Fyn-Shc, Shc-Grb2, and Grb2-mSOS complex formation (B. Li et al., 1996). Although the physiological GEF that may activate Ras in lymphocytes has not been identified, a role for Sos is implicated by the finding that membrane localization of Sos mimicked activated Ras and synergized with a Ca'+-dependent signal to induce NFAT activation in T cells; furthermore, this effect of Sos was strongly inhibited by DN Ras (Holsinger et al., 1995). Because physiological Sos recruitment to the vicinity of Ras in the membrane (albeit not its exchange activity per se) requires association with Grb2 and/or formation of a trimolecular phospho-ShdGrbWSos complex, a number of studies have attempted to elucidate the mechanisms through which Shc and/or Grb2 can be localized to the membrane following the triggering of IRRs (reviewed in Koretzky, 1997) (Fig. 1).One clue came from a study demonstrating that TCR stiinulation induces tyrosine phosphorylation of Shc and subsequent formation of a Shc/GrbWSos complex; moreover, the SH2 domain of Shc directly interacted with the phosphorylated f chain of the TCWCDS complex (Ravichandran et al., 1993). Additional studies by the same group demonstrated that TCR ligation increased Sos-Grb2 association. Shc was implicated as a regulator of this association because a Shc-based phosphopeptide that displaces Shc from Grb2 abolished the enhanced association between Grb2 and Sos, and addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with Sos (Ravichandran et al., 1995).These findings potentially account for the apparent increase in the exchange activity of Sos observed in T cells overexpressing activated Fyn (B. Li et al., 1996). The TCR-induced, Shc-mediated up-regulation of the Grb2/Sos complex in T cells is different from the situation found in fibroblasts, wherein Grb2 and Sos are constitutively associated and the level of the Grb&/Soscomplex is not affected by growth factor stimulation. Other studies also demonstrated that Shc is a substrate for TCR-stimulated PTKs (Baldari et al., 1995a; B. Li et al., 1996; Lin and Abraham, 1997),where it forms a complex with Grb2 (Lin and Abraham, 1997). Similarly, BCR, FcERI, FcyRI, or FcyRIII (CD16) ligation on B cells, mast cells, monocytes, and NK cells, respectively, also induces tyrosine phosphorylation of Shc, its association with phospho-ITAMs, and formation

10


of complexes containing phospho-Shc, Grb2, Sos, and additional tyrosinephosphorylated proteins (Saxton et al., 1994; Smit et al., 1994,1996; Kumar et al., 1995; Galandrini et al., 1996, 1997b; Jabril Cuenod et al., 1996; Kimura et al., 1996b; Park et al., 1996; Teramoto et al., 1997; Tridandapani et al., 1997a). This response was dependent on intact Syk kinase activity (Jabril Cuenod et al., 1996). Taken together, these findings suggest that Shc, on becoming phosphorylated by receptor-associated PTKs, couples IRR activation to the Ras signaling pathway. However, this conclusion has been called into question by other studies suggesting that Shc does not play an important role in Ras activation or in GrbWSos recruitment to the membrane in T cells. First, TCR or FceRI stimulation failed to induce an increase in the constitutive tyrosine phosphorylation of Shc in T cells (Gupta et al., 1994) or in mast cells (Turner et al., 1995), respectively, or formation of an Shc/GrbWSos complex in Jurkat T cells (Buday et al., 1994; Gupta et al., 1994), despite the fact that Grb2, together with additional tyrosine-phosphorylated proteins, translocated from the cytosol to the membrane under similar conditions of TCR stimulation (Nel et al., 1995). Second, the interaction of Shc with phosphohas a much lower dfinity compared to 6 interaction with the Zap-70 or Fyn tyrosine kinases (Osman et al., 1995).Finally, transfection of the Grb2 SH2 domain (Northrop et al., 1996),but not the Shc SH2 domain (Baldari et al., 1995b; Northrop et al., 1996), inhibited T cell activation. These findings, along with other reports documenting the membrane localization of Grb2 in TCR-stimulated T cells (Buday et al., 1994; Sieh et al., 1994; Nel et al., 1995), suggest that although Grb2 plays an important role in coupling Ras activation to stimulated IRRs, Shc is not critical for this process. A potential resolution to the controversial role of Shc in coupling TCR signals to Ras activation comes from studies that addressed the relative contribution of TCWCD3 versus CD4 signals to the activation of NFAT, a known Ras-dependent event in T cells (Rao et al., 1997). Whereas DN Ras inhibited NFAT activation induced by either CD3 or CD4 crosslinking, a DN Shc mutant consisting of its isolated SH2 domain only inhibited the CD4-mediated NFAT activation (Baldari et al., 1995b).Moreover, Lck and tyrosine kinase activity coimmunoprecipitated with Shc following CD4, but not CD3, triggering (Baldari et al., 1995a), and CD4 cross-linking by monoclonal antibodies recognizing distinct CD4 epitopes invariably led to increased tyrosine phosphorylation of Shc (Baldari et al., 1995a). However, only some of these antibodies caused NFAT activation, consistent with the notion that tyrosine phosphorylation of Shc is necessary, but not sufficient, for NFAT (and, hence, Ras) activation. These findings raise the possibility that the primary role of Shc is to couple the CD4 (and

r


11

CD8?) coreceptor to Ras activation, whereas the TCR is coupled to the Ras pathway via an alternative mechanism. This model and the controversial findings regarding the role of Shc in TCR-mediated Ras activation are consistent with the fact that the relative requirement of CD4-dependent signals for optimal T cell activation varies among different T cells, and depends on the strength of the TCR signal (Viola et al., 1997). 3. Linker for Activation of T Cells Evidence reviewed above suggests that a Shc-independent mechanism may mediate the recruitment of the Grb2/Sos complex to activated IRRs. A Grb2-associated tyrosine-phosphorylated protein of 36-38 kDa is a prime candidate for mediating this coupling in T cells. p36-38 is the major PTK substrate to associate with Grb2 in activated T cells, and has been found to associate with several other well-known signaling proteins, including Sos, PLCyl, and PI3-K (Buday et al., 1994; Motto et al., 1994; Reif et al., 1994; Sieh et al., 1994; Lahesmaa et al., 1995; Nel et al., 1995; Ingham et aE., 1996). The inducible association of Grb2 with p36-38 (Buday et aE., 1994; Nunes et al., 1994; Reif et al., 1994; Sieh et al., 1994), the finding that formation of a p36-38/Grb2/Sos complex occurs rapidly and correlates with Ras activation (Nunes et al., 1994), and the exclusive localization of p36-38 in the T cell plasma membrane (Buday et al., 1994; Sieh et at., 1994) are consistent with a putative role as a coupling element between the activated TCR complex and Ras. The association of p36-38 with Grb2 is mediated by the SH2 domain of Grb2 (Koretzky, 1997) and, in this regard, it is of interest that although various signaling proteins coimmunoprecipitate with p36-38, Shc is not one of them. This would perhaps be expected if phosphorylated p36-38 and Shc compete for binding to the Grb2 SH2 domain. The importance of p36-38 in TCR signaling is suggested by the finding that expression of a chimeric receptor with a cytoplasmic tail consisting of the Grb2 SH2 domain (which binds p36-38) and the catalytic domain of CD45 selectively reduced the TCR-induced tyrosine phosphorylation of p36-38, and this correlated with inhibition of inositol phosphate production and Ca2+mobilization;surprisingly,however, Ras and ERK2 activation remained intact under the same conditions (Motto et al., 1996a). The finding that p36-38 associates with the SH2 domain of Grb2 more stably than Shc (Osman et al., 1995) also supports the notion that a p36-38/Grb2/Sos complex (rather than a Shc/Grb2/Sos complex) is likely to be more relevant in TCR-induced Ras activation. Considerable effort has been invested in identifylng p36-38. Initially, this effort led to the cloning of Lnk, a 38-kDa protein that, like p36-38, becomes phosphorylated on tyrosine in activated T cells and associates with the SH2 domains of Grb2, PLCyl, and PI3-K (Huang et al., 1995).

12


However, subsequent analysis of transgenic mice expressing a Ink transgene in the thymus suggested that Lnk does not play an important role in TCR signaling. Thus, although TCR stimulation induced tyrosine phosphorylation of Lnk in the transgenic T cells, Lnk did not play a limiting role in TCR signaling and, furthermore, it could be biochemically distinguished from the more prominent tyrosine-phosphorylated p36 in activated T cells (Takaki et al., 1997). It has been reported that p36-38 almost certainly corresponds to a novel protein, linker for gctivation of T cells (LAT), which was isolated by a combination of biochemical purification, partial peptide sequencing, and cDNA cloning (Zhang et al., 1998). The deduced amino acid sequence of LAT identified an integral membrane protein containing multiple potential tyrosine phosphorylation sites. LAT was phosphorylated by Zap-7O/Syk and associated with Grb2, PLCyl, the regulatory subunit (p85)of PI3-K, and several other tyrosine-phosphorylated proteins in activated T cells. The functional relevance of LAT in T cell activation and its potential connection to the Ras signaling pathway are indicated by two findings: first, mutation of two tyrosine residues in LAT abolished its association with Grb2 and p85, and greatly reduced PLCyl binding, in activated T cells; second, transient transfection of Jurkat cells with the tyrosine-mutated (but not wild-type) LAT inhibited the anti-CD3-induced transcriptional activity of AP-1 and NFAT (Zhang et al., 1998), which is known to require intact Ras function (Foletta et al., 1998; Rao et al., 1997; Su et al., 1994). Based on these findings, LAT appears to be a critical membrane linker that couples activated Zap-7O/Syk to the stimulation of the Ras, PLCyl, and PI3-K signaling pathways in T cells (Fig. 1).The isolation of LAT will certainly lead to intense efforts aimed at characterizing its function and regulation in T cells. Furthermore, because LAT is selectively expressed in T cells, mast cells, and NK cells (Zhang et al., 1998), it would be interesting to determine whether a structurally and/or functionally related linker is expressed in other hematopoietic cells, e.g., B lymphocytes and monocytes. 4. The Role of Other Grb2-Associated Proteins in Ras Activation

The Ras-specific GEF, Sos, is the most obvious link between Grb2 and Ras activation. However, a number of Grb2-associated proteins that could potentially affect upstream regulators or downstream targets of Ras have been isolated, and their role in TCR-mediated T cell activation has been studied in detail (reviewed in Koretzky, 1997).Although a complete survey of Grb2-associated proteins is beyond the scope of this review, three of these proteins, i.e., SLP-76, Cbl, andVav, have been implicated in signaling pathways mediated by Ras- or Rho-family GTPases.

GTF'ases IN IMMUNE RECOGNITION RECEPTOR SIGNALING

13

An SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) was originally described by several groups as a 76-kDa tyrosine-phosphorylated protein associated with Grb2 in activated T cells (Buday and Downward, 1993; Motto et al., 1994; Reif et al., 1994; Sieh et al., 1994). Affinity purification of the protein using antiphosphotyrosine antibodies and a glutathione S-transferase (GST)-Grb2 fusion protein and peptide microsequencing led to cloning of the corresponding cDNA (Jackman et nl., 1995). Expression of SLP-76 mRNA is restricted to T cells, B cells, and monocytes. The protein contains an N-terminal acidic region with several tyrosine phosphorylation sites that are involved in binding the SH2 domain of Vav (Wu et al., 1996), a central proline-rich region that binds Grb2 (Jackman et nl., 1995), and a C-terminal SH2 domain that binds two tyrosine-phosphorylated protein of -62 and -130 kDa (Motto et al., 1996b). Although the exact function of SLP-76 in TCR signaling is unknown, several findings indicate an important regulatory role with potential connection to the Ras pathway. First, transient overexpression of SLP-76 enhanced TCR-mediated NFAT, AP-1, and IL-2 promoter activities, and this activity required intact phosphorylation of SLP-76 by Zap-70 (Fang et al., 1996; Motto et al., 199613;Wardenburget al., 1996; Musci et al., 1997). Second, SLP-76 interacts with Vav, and the two synergize to augment IL2 promoter activity (Wu et al., 1996), but this interaction per se is not essential for TCR-induced IL-2 production in all T cells (Raab et al., 1997). Finally, transient SLP-76 overexpression does not affect calcium signaling, but augments TCR stimulation of ERK (Musci et al., 1997). Because AP1, NFAT, and ERK activation by the TCR depends, at least in part, on Ras function, a link between SLP-76 and the Ras pathway is implicated. Cbl, a 120-kDa protein that was originally isolated in its oncogenic form, is a prominent PTK substrate in activated T cells (Donovan et al., 1994). Cbl consists of an amino-terminal transforming region, a zinc ring finger, multiple proline-rich stretches that mediate constitutive association with Grb2 (Donovan et al., 1994; Fukazawa et al., 1995; Meisner et al., 1995), and several tyrosine phosphorylation sites. It is rapidly phosphorylated on tyrosine and serine residues in response to stimulation of many cell surface receptors and, as a result, becomes inducibly associated with a number of intracellular signaling proteins such as PTKs, PIS-K, Crk, and 14-3-3 through different protein-interacting modules, leading to the formation of multimolecular signaling complexes (reviewed in Liu and Altman, 1998). The exact biological function of this adapter protein remains largely unknown. Nevertheless, Cbl and its transforming mutants have been shown to display both negative and positive regulatory activities, including in PTK- and Ras-mediated signaling pathways.

14


Several observations suggest that Cbl negatively regulates the Ras signaling pathway. First, genetic studies on vulval development in the nematode Caenorhabditis elegans revealed activating mutations in sli-1 (the C. elegans homolog of c-Cbl ) that restore normal development in nematodes expressing inactive Ras (LET-60) mutants (Yoon et al., 1995); second, a transforming mutant of Cbl, 70Z3, activates NFAT in a Ras-dependent manner in T cells (Liu et al., 1997);third, as discussed in more detail in Section VI,A, hyperphosphorylation of Cbl on tyrosine may indirectly lead to activation of a Ras-related protein, Rap-1, which functions as a suppressor of Ras and may play an important role in the induction of T cell anergy (Boussiotis et al., 1997);finally, the association of Cblwith Zap-70, Syk, or Fyn (Lupher et al., 1996; Deckert et al., 1998), and its ability to suppress Syk activation and mast cell degradation on FcERI triggering (Ota and Samelson, 1997), suggest that it may indirectly affect Ras activation by modulating the activity of IRR-coupled PTKs required for Ras activation. However, another study showed that Cbl overexpressionor inhibition of Cbl expression by antisense oligonucleotidesdid not have any detectable effect on EGF receptor signals leading to activation of the Ras pathway, although these manipulations affected a signaling pathway involving the Janus kinases ( JAKs) and signal transducers and activators of transcription (STATs) (Ueno et al., 1997). Because Cbl interacts with many signaling proteins, it probably displays versatile regulatory activities, the exact nature of which is dictated by the specific cellular context and the spectrum of the proteins that associate with it. Thus, the details of the functional interactions between Cbl and the Ras signalingpathway in lymphocytes and other cells remain to be elucidated. The role of Vav and its Grb2 association in regulating the activity of small GTPases is discussed in detail in Section III,D,l. 5. GAPS GAPSinhibit the activity of small GTPases by accelerating their intrinsic GTPase activity, thereby promoting the hydrolysis of GTP to GDP and, hence, inactivation of the GTPase. Two mammalian ras-GAPS, pl20-GAP and neurofibromin (NFl), have been isolated (reviewed in Boguski and McCormick, 1993), and both are expressed in lymphocytes (Boyer et al., 1994; Downward et al., 1992). pl20-GAP becomes phosphorylated on tyrosine in response to stimulation of various receptors, including IRRs (Gold et al., 1993; Lazarus et al., 1993; H. L. Li et aI., 1997), and it represents a binding partner and a substrate of the T cell-expressed kinase Lck (Amrein et al., 1992). However, the functional relevance of this phosphorylation event remains unclear. In addition, pl20-GAP was found to be associated with two tyrosine-phosphorylated proteins of -62 and


15

-190 kDa in activated B cells in one (Gold et al., 1993),but not in another (Lazarus et al., 1993), study. These two proteins correspond to ~ 6 (Carpino et al., 1997; Yamanashi and Baltimore, 1997) and a rho-GAP (Settleman et al., 1992a,b), respectively. Inhibition of GAP activity by IRR-, CD2-, or PKC-mediated signals has been described in both T (Downward et al., 1990; Graves et al., 1991; Izquierdo et al., 1992a) and B (Lazarus et al., 1993) cells, but its precise mechanism is unknown. In addition to PKC, PTK activity has been implicated in regulating ras-GAP activity in T cells (Izquierdo et al., 1992a). However, the inhibited GAP activity in B cells does not appear to be regulated by tyrosine phosphorylation because GAP activity following BCR triggering remained inhibited at a time when the inducible tyrosine phosphorylation of GAP was no longer detectable (Lazarus et al., 1993). It is not known which of the two ras-GAPS is inhibited by antigen receptor triggering or PMA treatment. The most compelling evidence that p12O-GAP plays an important role in T cell activation comes from a study demonstrating that overexpression of this protein in Jurkat cells inhibited TCR-mediated NFAT activation. The inhibition was overcome by expressing a CA Ras mutant (Baldari et al., 1994). In addition, pervanadate stimulation of T hybridoma cells overexpressing an active form of Lck was reported to induce the formation of a complex containing CD45, Lck, p12O-GAP, Grb2, and Sos (Lee et al., 1996).A study by Boyer et al., (1994) suggested a potential role of rasGAP in regulating BCR signaling. Thus, analysis of the distribution of p120GAP and NF1 in splenic B lymphocytes by immunofluorescent staining indicated that BCR cross-linking induced the redistribution of NF1, but not p12O-GAP. NF1 colocalized both spatially and temporally with the BCR, and this translocation was inhibited by cytoskeletal disrupting agents. However, cocapping of N F 1 with the BCR was independent of the Ras redistribution (Boyer et al., 1994). These findings indicate that NF1 and pl20-GAP can be differentially regulated in B cells and suggest that NF1 is a component of the signaling pathway initiated by BCR cross-linking (Boyer et al., 1994). C. RASEFFECTORS IN IRR SIGNALING Activated (GTP-bound) Ras regulates independent signaling cascades (White et al., 1995) via its ability to bind different partners. The highly conserved effector domain of Ras mediates this binding, and a number of yeast, C . elegans, Drosophila, and mammalian proteins, collectively termed Ras effectors, have been found to bind activated Ras directly (Marshall, 1996). Some of the known mammalian Ras effectors include GAPS (p120GAP and NF1); Raf kinases (Raf-1, A-Raf, and B-RaO, which function as

2

~

16


MAPK kinase kinases in the ERK pathway; the lipid kinase PI3-K; RalGDS, a GEF for the Ras-related protein Ral; PKCG and MEKK-1, a MAPK kinase kinase in the JNK pathway. These effectors initiate signaling pathways consisting primarily of serinehhreonine kinase cascades, the stimulation of which ultimately leads to the activation of transcription factors in a receptor- and cell type-specific manner. The Raf-1 kinase and its downstream serinekhreonine kinase cascade represent the most extensively characterized Ras effector pathway (Fig. 1).In this cascade, membrane-localized activated Ras directly binds Raf1,leading to its enzymatic activation. Activated Raf-1 stimulates two MAPK kinases, MEK-1/2, which in turn activate the MAPKs ERK1/2. A number of studies have demonstrated that IRR stimulation or IRR-coupled PTKs of the Src and Syk families induce the association of active Ras with Raf1 (Finney et al., 1993), and stimulate all members of this cascade, i.e., Raf-1, MEKs, ERKs, or the ribosomal S6 kinase p90rskin T cells (Nel et al., 1990;Siegel et al., 1990, 1993; Izquierdo et al., 1993, 1994a,b; Franklin et al., 1994; Gupta et al., 1994; Lin and Abraham, 1997; Williams et al., 1997; Dumont et al., 1998), B cells (Tordai et al., 1994; Kumar et al., 1995; Kawauchi et al., 1996; H. L. Li et al., 1997; Tridandapani et al., 1997a), mast cells (Fukamachi et al., 1993; Hirasawa et al., 1995a,b; Rider et al., 1996; Ishizuka et al., 1997; Teramoto et al., 1997; Turner and CantreU, 1997; Zhang et al., 1997), monocytes (Durden et al., 1995; Taylor et al., 1997), and neutrophils (Alonso et al., 1996). CA Raf mimics the effect of Ras and synergizes with Ca2' signals to induce the IL-2 gene (Owaki et al., 1993)or activate ERK2 (Izquierdo et al., 1994a), and stable overexpression of ERKl in Jurkat T cells enhances expression of IL-2, IL-3, and GM-CSF mRNA (Park and Levitt, 1993). Finally, a signaling complex containing Vav (Section III,D,l), Grb2, Raf-1, and ERK2 was found to associate with FceRI in RBL cells (Song et al., 1996). The importance of Ras/Raf-1 in coupling IRRs to downstream activation events is indicated by the findings that DN Ras (Izquierdo et al., 1993; Williams et al., 1997), DN Raf-1 (Owaki et al., 1993; Wotton et al., 1993; Izquierdo et al., 1994a), or PD90859, a specific pharmacological inhibitor of MEK-1 (Dumont et al., 1998; Zhang et al., 1997),were found to inhibit IRR-mediated activation of the downstream serinehhreonine kinase cascade or cytokine production. Cytokine genes are not the only targets for the Raf-l/MEK/ERK cascade. Thus, by using DN Raf-1 mutants, it has been shown that Raf-1 is also required for the TCR-induced up-regulation of CD69 (Taylor Fishwick and Siegel, 1995) and the TCR-/3 chain (Wotton et al., 1993). The mechanisms leading to Raf-1 or ERK activation in T cells have been addressed in a number of studies. Generally, Raf-1 is activated as a


17

result of its binding to Ras-GTP (Zhang et al., 1993) and its subsequent membrane localization (Leevers et al., 1994). The reported association of Raf-1 with the 6 and y chains of the CD3 complex (Loh et al., 1994) may facilitate this localization in T cells. However, full Raf-1 activation by at least some receptors, including the IL-2 receptor (Tuner et al., 1991, 1993), appears to also require its tyrosine phosphorylation by Src-family PTKs (Morrison et al., 1989; Marais et al., 1995; Jelinek et al., 1996; Stokoe and McCormick, 1997). In addition, PKC can also directly activate Raf-1 (Sozeri et al., 1992; Kolch et al., 1993). Similarly, PKC-dependent ERK activation pathways have also been described, including in T cells (Izquierdo et nl., 1993, 199413; Gupta et al., 1994). The finding that, in comparison to phorbol ester-induced ERKZ activation, ERK2 activation induced by TCR stimulation was considerably more sensitive to inhibition by DN Ras (Izquierdo et al., 1993) led to the conclusion that activation of the Raf-1IERK2 cascade by PKC is, at least in part, Ras independent. However, this notion has to be reexamined in view of findings that have shed new light on the role of Ras in PKC-mediated Raf-1 activation (Marais et al., 1998). Thus, although DN Ras did not block PMA (i.e., PKC)mediated ERK and Raf-1 activation in COS cells (Howe et al., 1992; Marais et al., 1998), PMA stimulation caused Ras activation and formation of Ras/ Raf-1 complexes containing active Raf-1; moreover, a Raf-1 mutation that prevented its ability to associate with active Ras, or microinjection of a neutralizing anti-Ras antibody (Y13-259),blocked ERK or Raf-1 activation by PMA stimulation (Marais et al., 1998).These findings demonstrate that the absence of an effect of DN Ras does not necessarily indicate that Ras is not part of a given signaling pathway, and that Ras activation is, in fact, a component of PKC signaling. In T cells, either TCR or PMA stimulation activates Raf-1 (Siegel et nl., 1990, 1993). TCR-activated Raf-1 was found to be phosphorylated on serine but not on tyrosine and, furthermore, PKC depletion by prolonged PMA treatment abrogated TCR-induced Raf-1 activation (Siegel et at., 1990) suggesting that tyrosine phosphorylation is, at best, a minor part of the mechanism utilized by the TCR to activate Raf-1. Rather, TCRstimulated PKC plays a predominant, if not exclusive, role in Raf-1 activation in T cells. Nevertheless, a role for T cell-expressed PTKs in Raf-1 activation cannot be excluded. It has recently become clear that in addition to their role in regulating growth factor-induced cell proliferation, Ras and Raf-1 are also activated during mitosis (Laird et al., 1995; Taylor and Shalloway, 1996; Downward, 1997). Activation of Raf-1 during mitosis was also described in T cells, and its dependence on Lck activity suggests that in this case, Raf-1 is regulated by tyrosine phosphorylation (Pathan et al., 1996). In addition, binding of purified HIV-1 or its envelope glycoprotein,

18

A M N O N ALTMAN A N D M A R C E L D E C K E R T

gpl20, to CD4 on T cells resulted in association, tyrosine phosphorylation, and activation of Lck and Raf-1 (Popik and Pitha, 1996). Under the same conditions, Ras activation or its association with Raf-1 were undetectable, suggesting that Lck activates Raf-1 via a CD4-mediated, Ras-independent activation pathway. Studies conducted in T cells have made it clear that, as in other cell types, IRR-induced Ras activation leading to the induction of cytokine genes and other targets does not operate exclusively through the Raf-l/ MEUERK cascade, but also stimulates additional Ras effectors and their downstream signaling pathways (Fig. 2), which coordinately act to stimulate gene transcription. This is supported by several findings: first, although a DN MEK transgene can mimic Ras and inhibit positive selection of thymocytes (Section VII,A), it does not inhibit the TCR-induced, Ras-dependent proliferation of thymocytes (Alberola-Ila et al., 1995); second, CA MEK1 fully activates ERK2, but does not substitute for activated Ras and synergize with Ca2+signals to induce NFAT in T cells (Genot et al., 1996); third, functional Rac, which is clearly in a pathway distinct from the Raf1/MEWERK pathway, is required for TCR-induced, Ras-dependent T cell activation (Genot et al., 1996); finally, the use of CA or DN mutants of Ras or MEKK-1, an activator of the JNK pathway, demonstrated that Ras regulates TCR-induced activation of the MEKK-UJNK pathway (Fans et al., 1996). Analysis of the role of Ras effectors other than Raf-1 in IRR signaling is still in its infancy, and is likely to be a productive area of future research. D. TRANSCRIPTION FACTORS AS TARGETS OF THE RAS SIGNALING PATHWAY Induction of the IL-2 gene and production of its protein product represent the hallmark of T cell activation by the combination of TCR-derived

FIG.2. Ras effectors and their known functions (Marshall, 1996). Effectors that have been found to be involved in leukocyte functions are boxed.


19

and costimulatory signals. The promoter region of the IL-2 gene has been extensively characterized, and is now known to contain a collection of response elements that bind die transcription factors AP-1, NF-KB, Oct1,and NFAT (Crabtree and Clipstone, 1994).Efficient IL-2 gene transcription depends on higher order assembly and synergistic interactions among the various transcription factor complexes that bind to the promoter (Garrity et al., 1994). The finding that the combination of phorbol ester and Ca" ionophore can mimic the TCR signal and lead to full T cell activation, including IL-2 production and concomitant proliferation (Truneh et al., 1985), established a fundamental paradigm for TCR-mediated signaling. Subsequent studies attempted to define the physiological signals that are mimicked by these two pharmacophores, and the transcription factors that represent the targets of these signals and regulate expression of the IL-2 gene. It is now well established that Ras and the Ca"/calmodulin-dependent protein phosphatase 2B (calcineurin) are the targets of the phorbol ester and Ca" signals, respectively (Crabtree and Clipstone, 1994; Weiss and Littman, 1994; Su and Karin, 1996; Alberola-Ila et al., 1997; Rao et at., 1997). Thus, CA Ras can synergize with Ca2+ionophore or CA calcineurin to activate the IL-2 gene or its NFAT element (Baldari et al., 1992b; Rayteretal., 1992;Woodrowet al., 1993a;Ohtsukaetal., 1996).Conversely, DN Ras blocks the induction of these transcriptional activation events by the TCR (Rayter et al., 1992; Baldari et nl., 1993; Ohtsuka et al., 1996). As a corollary of the requirement for Ras, MEK-1 and ERKs (which are downstream targets of Ras) are also required for the stimulation of IL-2 gene transcription in T cells (Whitehurst and Geppert, 1996). At the transcriptional level, the AP-1 (Foletta et al., 1998) and NFAT (Rao et al., 1997) transcription factors are the targets for the Ras- and calcineurin-mediated signals, respectively. Ca" ionophore represents a sufficient signal for NFAT activation as measured by NFAT dephosphorylation, nuclear translocation, and the increase in its DNA binding affinity (Rao et al., 1997). Similarly, phorbol ester or CA Ras alone activates AP1, including in T cells (Rayter et al., 1992; Williams et al., 1995), and regulates both of its components, Jun and Fos, at the transcriptional and posttranscriptional levels (Foletta et al., 1998). Nevertheless, it is well established that optimal NFAT activation requires a Ras signal in addition to the Ca2+/calcineurinsignal. For example, CA Ras synergizes with Ca2+ ionophore or with CA calcineurin to activate NFAT (Woodrow et al., 1993a) and, conversely, DN Ras inhibits TCR-induced NFAT activation (Woodrow et al., 199313). The requirement of Ras for optimal NFAT activation reflects the fact that NFAT proteins interact with the AP-1 complex and bind cooperatively to the composite NFAT/AP-1 site in the promoter of the IL-2 and other cytokine genes (Raoet al., 1997).Therefore,

20


the contribution of Ras to NFAT activation reflects its role in AP-1 regulation. A similar picture emerges from the characterization of NFAT complexes in B, NK, or mast cells. Stimulation via the BCR or CD40 on B cells (Yaseen et al., 1993, 1994; Choi et al., 1994; Venkataraman et al., 1994), FcyRIIIA (CD16) on NK cells (Aramburu et al., 1995), or FcERI on mast cells (Baumruker et al., 1997; Hutchinson and McCloskey, 1995; Prieschl et al., 1995; Turner and Cantrell, 1997; Weiss et al., 1996) induces Ca2'dependent, cyclosporin A-sensitive NFAT activity. Furthermore, like T cells, these complexes contain JudFos heterodimers, i.e., active AP-1. The presence of AP-1 in the NFAT complexes observed in B, NK, and mast cells implicates a role for Ras in their activation. However, in only two of these studies was the role of Ras directly addressed. Thus, Ras function was required for the FceRI-mediated activation of the NFAT complex that binds to the NFAT response element in the IL-4 gene promoter (Turner and Cantrell, 1997), or to the promoter region of the genes encoding IL-5 and the chemokine MARC (Prieschl et al., 1995);however, NFAT action on the MARC promoter occurred in the absence of AP-1, suggesting that NFAT cooperates in this case with another, yet to be characterized factor (Prieschl et al., 1995).The finding in both of these studies that PKC was not required for this transcriptional activation event (Prieschl et al., 1995; Turner and Cantrell, 1997) suggests that Ras activation was PKC independent, consistent with the report that PMA does not activate Ras in mast cells (Izquierdo et al., 1992b). PKC almost certainly represents the link between the phorbol ester signal and Ras activation leading to AP-1 induction in T cells. It acts either upstream of, or in parallel to, Ras, but it is not a Ras effector in T cells (Williams et al., 1995). As discussed earlier, the functional relationship between PKC and Ras activation in T cells has been a subject of intense study, and has not been completely resolved. Although it is reasonably clear that PKC can activate Ras in T (Downward et al., 1990; Franklin et al., 1994; Ohtsuka et al., 1996), B (Harwood and Cambier, 1993; Tordai et al., 1994), and COS (Marais et al., 1998) cells, the mechanism of this activation is unknown. Direct assessment of the role of PKC in Ras activation will require genetic approaches, e.g., by testing the ability of CA mutants of distinct PKC isoforms to activate Ras. This approach has not been implemented to date, although PKC mutants were used to address indirectly the relationship between Ras and PKC in signaling pathways leading to transcriptional activation of the IL-2 gene and its isolated response elements. In one study (Baier Bitterlich et al., 1996), transient overexpression of wild-type PKC 6, but not PKCa, in murine EL4 leukemic T cells increased


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the PMA-induced transcriptional activation of AP-1. Expression of a CA PKCB (but not PKCa) mutant was sufficient to activate AP-1 in the absence of PMA stimulation. Conversely, a catalytically inactive PKCO (but not PKCa) mutant abrogated PMA-mediated AP-1 activation. The ability of a DN Ras mutant to block the PKCB-induced AP-1 activation indicates that intact Ras function was required for the PKCB effect (Baier Bitterlich et al., 1996). However, it is not clear whether PKCB acts upstream of, or in parallel to, Ras in the signaling cascade leading to AP-1 activation. Another study (Genot et aZ., 1995) demonstrated that PKCE and, to a lesser extent, PKCa (but not PKCC) can activate the transcription factors AP-1 and NFAT-1. PKCE mimicked the stimulatory effect of CA Ras in this regard. Unlike Ras, however, none of the activated PKC mutants upregulated CD69 expression, a known Ras-dependent event (D’Ambrosio et al., 1994). Another indication of the potential importance of Ras in PKC-dependent signaling events is provided by the finding that, at sufficiently high levels of overexpression, DN Ras can inhibit ERK2 activation mediated not only by TCR, but also by phorbol ester stimulation (Izquierdo et al., 1993). It has been found that transient transfection of Jurkat T cells with CA PKCB, but not with other PKC isoforms (aor E ) , cooperated with CA calcineurin to activate the IL-2 promoter (Werlen et al., 1998).This combination was as effective as, or even more potent than, the combination of PMA and Ca2+ionophore, which generally stimulates maximal induction of the IL-2 promoter and is routinely used as a positive control in IL-2 gene induction studies. The PKC specificity of this induction operates at the level of JNWc-Jun activation because PKC 8 also specifically synergized with CA calcineurin to stimulate JNK and c-Jun transcriptional activity via a Rac-dependent pathway; in contrast, ERK was activated nonselectively by several PKC isoforms (Werlen et al., 1998). Finally, expression of DN PKCB, but not PKCa, inhibited the activation of JNK by PMA plus ionomycin. These findings are consistent with the selective ability of PKCB to activate AP-1 (Baier Bitterlich et al., 1996) and stimulate JNK (GhaffariTabrizi et aZ., 1998) in T cells, and with the demonstration that the specific MEK-l/JNK activator, MEKK-1, regulates the IL-2 promoter and is regulated, in turn, by Ras (Fans et al., 1996). Thus, PKCB, which is selectively expressed in T cells (Baier et al., 1993), and specifically colocalizes with the TCR to the contact area between antigen-specific T cells and antigenpresenting cells (APCs) (Monks et al., 1997), becomes an attractive candidate to mediate the PMA effect on Ras activation and IL-2 gene induction in T cells. It will be equally important to determine whether, in addition to its potential ability to activate Ras, PKCB also regulates other effectors involved in JNK andlor IL-2 gene activation.

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It is not clear whether Ras regulates transcription factors other than AP1 and NFAT in the IL-2 promoter. Reporter gene experiments demonstrated that NF-KB transcriptional activity is not affected by expression of activated Ras (Williams et al., 1995). However, TCR-induced NF-KB activation was found to require intact Raf-1 kinase function and a Ca2'/ calcineurin signal, suggesting a functional synergy between Raf-1 and calcineurin (Kanno and Siebenlist, 1996). In the face of the apparent lack of role for Ras (Williams et al., 1995), the Raf-1 requirement for NF-KB activation may reflect activation of Raf-1 by a Ras-independent mechanism, e.g., by PTK-mediated phosphorylation similar to what has been observed in CD4-ligated T cells (Popik and Pitha, 1996). Other transcription factors whose activation appears to require intact Ras function are Elk-1 (Turner and Cantrell, 1997) and Egr-1 (McMahon and Monroe, 1995). Elk-1 is a member of the ternary complex factor (TCF) family, members of which form complexes with serum response factor (SRF). These complexes are important for activation of the serum response element (SRE), which regulates the transcription of immediateearly genes such as c-fos and egr-1 (Treisman, 1994). Elk-1 can be phosphorylated and activated by various members of the MAPK family, i.e., ERK, JNK, and p38 kinases (Treisman, 1994). FceRI ligation or PMA (but not ionomycin) stimulation of RBL cells induced activation of Elk-1. Elk-1 was also induced by CA mutants of Ras or Raf-1 and, conversely, its FceRI-, active Ras-, or active Raf-l-mediated activation was markedly inhibited by DN Ras or DN Raf-1, as well as by PD90859, a specific MEK1 inhibitor (Turner and Cantrell, 1997). These findings indicate that Elk1 is activated by the Ras/Raf-l/MEK/ERK cascade in response to FceRI ligation on mast cells. The primary response gene egr-1 encodes a sequence-specific transcription factor whose expression is necessary for antigen receptor-stimulated activation of B lymphocytes (Monroe et al., 1993). Expression of activated Ras resulted in egr-1 induction similar to that induced by BCR crosslinking. Conversely, DN mutants of Ras and Raf-1 inhibited BCR-induced egr-1 induction (McMahon and Monroe, 1995). Although Ras is generally considered as a positive regulator in IRRmediated signaling pathways, a recent study (Chen et al., 1996) indicated that it may also exert negative regulatory influences. Expression of activated Ras inhibited induction of the immediate-early genes egr-1, c-fos, and cjun by Ca2' ionophore in Jurkat T cells. This inhibition was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin. A later reflection of this inhibitory effect was down-regulation of AP-1 activity and subsequent coordinate reduction in IL-2 mRNA and protein expression (Chen et al., 1996).These results suggest that Ras is an essential


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mediator not only in positive but also in negative modulatory mechanisms controlling the competence of T cells in response to inductive stimuli (Chen et al., 1996). The ability of Ras to regulate negatively Ca'+/calcineurindependent immediate-early gene induction in T cells may be related to the late phase of NFAT deactivation found to be induced in T cells by TCR ligation (Loh et al., 1996). Because this deactivation was facilitated by PMA treatment (Loh et al., 1996), it may also involve PKC and/or Ras activation. The role of Ras in IRR signaling was analyzed almost exclusively in the context of the Raf-1IMEWERK effector pathway. However, with the recent realization that Ras activates different effector pathways (RodriguezViciana et al., 1994; White et al., 1995; Marshall, 1996), the role of other Ras effectors in NFAT activation has been addressed in T cells (Genot et al., 1996) and in mast cells (Turner and Cantrell, 1997). Although CA MEK-1 f d y activated ERK2, it did not substitute for activated Ras and synergize with Ca2+/calcineurinsignals to induce NFAT. Expression of DN Rac also prevented TCR- and Ras-mediated activation of NFAT, but did not inhibit ERK2 activation. Similarly, the induction of AP-1 by Ras also required Rac-1 function. Activated Rac-1 mimicked active Ras to induce AP-1, but not NFAT. Moreover, the combination of activated MEK1 and Rac-1 could not substitute for activated Ras and synergize with Ca" signals to induce NFAT. Thus, Ras regulation of NFAT in T cells requires the activity of multiple effector pathways, including those regulated by MEK-lERK2 and Rac-1 (Genot et al., 1996). This is consistent with the role of Rac in the PKCB- and calcineurin-mediated signaling pathway leading to JNK activation in T cells (Werlen et al., 1998). A similar conclusion was drawn from the analysis of NFAT activation (using an IL-4 promoter element) by FcERI ligation in mast cells (Turner and Cantrell, 1997). In this case it was found that Ras-dependent NFAT activation did not involve the Raf-lIMEWERK pathway but, rather, a Racassociated pathway. These studies indicate that, as in other cells, IRR triggering also stimulates multiple Ras effector pathways that coordinately act to regulate cytokine gene induction. Much remains to be learned in this important area. E. NEGATIVE SIGNALING AND THE RASPATHWAY The majority of studies on signal transduction pathways in various cell types have focused on receptors that, upon ligation, lead to cellular activation. However, it has recently become clear that many cell types express inhibitory receptors. Triggering of these receptors initiates a negative signal that intercepts activation cascades at defined points and most likely acts as a negative regulatory mechanism to terminate activation responses (Cam-

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bier, 1997). Examples of inhibitory receptors on lymphocytes are provided by the FcyRIIbl receptor on B lymphocytes (Tridandapani et al., 1997b), killer cell inhibitory receptors (KIRs) on NK cells (Leibson, 1995; Yokoyama, 1995),and KIRs or CTLA-4 (Bluestone,1997;Thompson and Allison, 1997) on T cells. Essentially all known inhibitory receptors share the property of an immunoreceptor tyrosine-based inhibitory motif ( ITIM) present in their cytoplasmic domain. Receptor ligation induces tyrosine phosphorylation of the ITIM motif, thereby creating a docking site for the SH2 domain of two types of enzymes, i.e., the structurally related SH2containing phosphotyrosine phophatases 1 and 2 (SHP-1 and SHP-2, respectively) and SH2-containing inositol polyphosphate 5-phosphatase (SHIP). Activation of SHP-112 or SHIP generates negative signals that attenuate tyrosine phosphorylation or elevations in inositol phosphate and intracellular Ca2+concentrations (Scharenberg and Kinet, 1996). Several recent reports indicate that the Ras signaling pathway may represent one target of inhibitory receptors. Co-cross-linking of the BCR and FcyRIIbl on B cells has been known to inhibit B cell proliferation and antibody production, and probably serves as physiological mechanism to prevent excess antibody production (Sinclair and Panoskaltsis, 1989). This inhibition is mediated by recruitment of SHIP to the phosphorylated ITIM in FcyRII (On0 et al., 1996).Biochemical analysis of signaling events induced by BCR cross-linking alone versus BCWFcyRII coligation in human (Sarmay et al., 1996)or murine (Tridandapani et al., 1997a) B cells established that, under coligation conditions, the activation of Ras, Raf-1, and ERK was inhibited. A clue for the mechanism of this inhibition comes from the findings that, first, Shc is hypophosphorylated on tyrosine and coimmunoprecipitateswith FcyRII in the coligated B cells (Sarmay et al., 1996) and, second, the BCR-induced association of Shc and Grb2 was abrogated under negative signaling conditions; instead, Shc associated with a tyrosine-phosphorylated 145-kDa protein (Tridandapani et al., 1997a), previously identified as SHIP (Damen et al., 1996), only under conditions of negative signaling. Based on these findings, it was suggested that SHIP mediates Fcy RII-induced inhibition of B cell activation by competing with Grb2 for binding phospho-Shc (Tridandapani et al., 1997b). The resulting dissociation of the ShdGrb2 complex would disrupt Ras activation because it would prevent recruitment of the GrbYSos complex to the membrane. This attractive hypothesis does not take into account, however, the role of the catalytic activity of SHIP in inhibiting cellular activation. It is clear that the enzymatic activity of SHIP is also important for its inhibitory action (Deuter-Reinhard et al., 1997; Bolland et al., 1998) and, therefore, it is possible that the adapter


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and catalpc functions of SHIP are both necessary for optimal inhibition under physiological conditions. The role of the Ras signaling pathway as a potential target for other inhibitory receptors has not been explored in detail. However, it was reported that KIR recognition of major histocompatibility complex (MHC) class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adapter protein, pp36 (which is most likely LAT), with PLCyl in NK cells; furthermore, tyrosine-phosphorylated pp36 was a substrate in vitro for the KIR-associated tyrosine phosphatase SHP-1 (Valiante et al., 1996). Thus, it is possible that in KIR-ligated NK cells, hypophosphorylated LAT is also deficient in its ability to bind the GrbUSos complex, a situation that would be expected to prevent or reduce Ras activation. F. RASIN CVTOKINE RECEPTOR SIGNALING Although this review focuses primarily on the function of small GTPases in IRR signaling, it is well established that Ras is also activated by most cytokine receptors (Satoh et al., 1991; Graves et al., 1992; Izquierdo et al., 1992b; Nakafuku et al., 1992; Izquierdo and Cantrell, 1993; Miura et al., 1994). In T cells, PTKs, but not PKC, have been found to couple the IL2 receptor to Ras activation (Izquierdo and CantreU, 1993). The Raf-1 kinase is also activated by IL-2 (Turner et al., 1991, 1993), IL-3, and GM-CSF (Carroll et al., 1990). The Ras signaling pathway is one of two independent cascades that cooperate to mediate cytokine receptor signaling, the other one involving JAK-family kinases and STAT transcription factors. It is generally accepted that the mechanism by which cytokine receptors induce Ras activation involves tyrosine phosphorylation and receptor recruitment of a ShdGrbUSos complex. As an example, stimulation of the IL-2 (Ravichandran and Burakoff, 1994; Ravichandran et al., 1996) or GM-CSF (Pratt et al., 1996) receptors induces tyrosine phosphorylation of Shc and its binding to the phosphorylated receptor. This scenario allows Shc to interact simultaneously with phosphorylated tyrosine residue(s) in the cytoplasmic domain of the activated receptor via its phosphotyrosinebinding (PTB) domain, and with the SH2 domain of Grb2 via its own phosphotyrosine(s). Other details of signal transduction by cytokine receptors and the role of Ras in these events are discussed in reviews by Ihle et al. (1995) and Karnitz and Abraham (1995). 111. The Function of Rho-Family GTPases in IRR Signaling

A. INTRODUCTION Small GTPases of the Rho family (RhoA, RhoB, RhoC, RhoE, RhoC, Racl, Rac2, Cdc42, and TC10) were until recently considered to be primar-

26


ily involved in the organization of the actin cytoskeleton. Cdc42, Rac, and Rho independently induce the formation of filopodia, lamellipodia and membrane ruffles, or stress fibers and focal adhesion complexes, respectively (Nobes and Hall, 1995).However, these GTPases are also linked in a linear cascade in which Cdc42 stimulates Rac, which in turn activates Rho (Nobes and Hall, 1995) (Fig. 3). The major discoveries regarding Rho GTPases were made through the study of their functions in fibroblasts. However, there is now compelling evidence for a similar role for these

FIG. 3. The cascade of Rho-family GTPases in fibroblasts and their role in cytoskeleton organization. Rho GTPases are regulated by GEFs, GAPS,or GDIs (Section 1,A).Extracellular stimuli activate Cdc42, Rac, and Rho, leading to the formation of filopodia, lamellipodia, and stress fibers, respectively. These GTPases are also linked in a linear functional cascade whereby Cdc42 activates Rac, which in turn activates Rho. Furthermore, the Ras GTPase can “talk to the Rho GTPases cascade by activating Rac. The mechanisms that connect these GTPases remain largely unclear. Moreover, the functional implications of such a cascade in lymphocyte activation have not been addressed. LPA, Lysophosphatidic acid.


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proteins in additional actin-dependent cell structures, e.g., tight junction regulation by Rho in polarized epithelia; control of axonal outgrowth by RacKdc42 in mammalian neuronal cells and Drosophila; and regulation of cell polarization by Cdc42 in the budding yeast. Through their ability to establish different types of adhesive structures including focal complexes, adherens junctions, and tight junctions, Rho-family GTPases provide a molecular basis for many of the morphological and motility changes that cells undergo during differentiation, migration, and mitosis (Chant and Stowers, 1995; Ridley, 1995; Symons, 1995; Tapon and Hall, 1997; van Aelst and D’Souza-Schorey, 1997; Hall, 1998). In the past 3 years or so it has become clear that Rho GTPases also have critical functions in the control of cellular proliferation initiated by multiple receptor signals or, under aberrant conditions, in malignant transformation. Significant progress has been made toward characterizing the signaling cascades that couple signals emanating from Rho-family GTPases to the nucleus. As a result, it is now clear that Rho-family GTPases activate members of the JNKs and p38 families of stress-activated protein kinases (SAPKs) (Minden et al., 1995; Coso et al., 1995; Olson et al., 1995; Vojtek and Cooper, 1995), and deliver signals that regulate transcriptional activation by SRF and induce cell cycle progression through the GI phase (Hill and Treisman, 1995; Hill et al., 1995; Olson et al., 1995; Symons, 1995; Treisman, 1996) (Fig. 4).The functions of Rho GTPases in regulating the cytoskeleton versus cell growth appear to be independent and to be mediated by distinct effectors (White et al., 1995;Joneson et al., 1996; Lamarche et al., 1996; van Aelst and D’Souza-Schorey, 1997; Westwick et al., 1997; Hall, 1998). Ras interacts with the Rho GTPases cooperatively to regulate these signaling events (Khosravi Far et al, 1995; Prendergast et al., 1995; Qiu et al., 1995a,b, 1997). Mostly indirect evidence has also accumulated implicating an important role for Rho-family GTPases in coupling IRRs and other receptors in the reorganization of the actin cytoskeleton in leukocytes. The use of genetic and biochemical approaches has also started to yield important clues on the role of these small G proteins in IRR-initiated signaling pathways leading to proliferation and cytokine production by T cells and mast cells. These studies are reviewed below.

B. RHO-FAMILY GTPASESAND THE CYTOSKELETON IN LEUKOCYTES The morphological changes occurring during lymphocyte polarization induced by chemokines, adhesion molecules, or antigenic stimulation were described by Haston et al. (1982). The migrating lymphocyte extends a cytoplasmic pseudopod-like protrusion called a uropod, which is involved in adhesion to endothelial or extracellular matrix (ECM) proteins, motility,

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FIG.4. Effectors of Rho-family GTPases and their known functions. Cdc42 or Rac effectors, which share a CddiWRac interactive binding (CRIB) motif, are shaded, and Rhofamily effectors which have been imphated in leukocyte functions are boxed. Rho kinase (p160) was shown to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (myosin PPase) and this, in turn, inhibits the phosphatase activity (Kimura et al., 1996a).

and activation. In addition, important regulatory and effector cell-cell contacts occur among cells of the immune system, accompanied by secretion of various soluble products. Polarized secretion is a common property of many cell types and lymphocytes are no exception. Two major T cell interactions involve vectorial secretion, which requires cytoskeleton reorganization and polarization: contact between T helper cells and APCs, which results in cytokine production; and cytotoxic T lymphocyte (CTL)-target cell interactions accompanied by cytotoxic granule release and target cell lysis (Kupfer et al., 1987; Po0 et al., 1988; Kupfer and Singer, 1989; Podack and Kupfer, 1991). For example, conjugate formation between cytotoxic


29

effector lymphocytes and target cells is accompanied by capping of lymphocyte function antigen-1 (LFA-1) to the contact area, followed by talin colocalization (Podack and Kupfer, 1991). It is therefore not surprising that small GTPases of the Rho family have been implicated in the regulation of lymphocyte adhesion, polarization, motility, and effector functions (Chavrier et al., 1993; Quinn, 1995; Dharmawardhane and Bokoch, 1997; Reif and Cantrell, 1998). Direct evidence for the activation of Rho-family GTPases by IRRs and other leukocyte receptors, or morphological evidence for their distinct roles in inducing defined cytoskeletal structures in leukocytes, is missing. The difficulty in demonstrating Rho-family GTPase activation is not unique to leukocytes. It reflects the fact that GTPases of this family display a much higher intrinsic GTPase activity compared to Ras, thus making it technically more demanding to isolate and detect their active (GTP-bound) form. Morphological analysis of actin cytoskeleton structures in leukocytes is very difficult because these cells either grow in suspension and/or contain relatively little cytoplasm. Nevertheless, indirect evidence based on morphological, pharmacological, biochemical, or genetic approaches implicates important roles for Rho-family GTPases in reorganization of the cytoskeleton in leukocytes (Fig. 5). Many studies have demonstrated that triggering of IRRs (TCR, BCR, FcsRI, FcyR) leads to actin cytoskeleton reorganization in T cells (DeBell et al., 1992; Donnadieu et al., 1992, 1994; Pardi et al., 1992; Parsey and Lewis, 1993; Phatak and Packman, 1994; Negulescu et al., 1996), B cells (Melamed et al., 1991a,b; Cox et al., 1996), and mast cells (Apgar, 1991; Norman et aZ., 1994, 1996; Pfeiffer and Oliver, 1994; Barker et al., 1995; Prepens et al., 1996; Guillemot et al., 1997). Reorganization of the cytoskeleton is manifested by an increase in the content of filamentous actin (Factin), formation of membrane ruffles and focal adhesions (most likely reflecting Rac and Rho activation, respectively), spreading and adhesion to the substrate, uropod extension and cell elongation or rounding, and changes in motility, pinocytosis, and phagocytosis. In activated T cells, the redistributed F-actin colocalized and was physically associated with the adhesion receptors LFA-1 (CDllaICD18) and VLA-4 (Pardi et al., 1992). These changes are accompanied by Ca2+oscillations (Donnadieu et al., 1992, 1994; Valitutti et al., 1995a; Negulescu et al., 1996). Pharmacological approaches based on the use of agents that disrupt the cytoskeleton, e.g., cytochalasins or phalloidin, demonstrated that reorganization of the actin cytoskeleton is tightly coupled to secretion in mast cells (Norman et al., 1994), T cell shape changes induced by contact with antigen-pulsed APCs (Donnadieu et al., 1992), TCR internalization (DeBell et al., 1992), and B cell proliferation (Melamed et al., 1991a).However,

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FIG.5. The role of Rho-family GTPases in leukocyte signaling. The TCFVCD3 complex is used as an example. Integrins and chemokine receptors activate Rho-family GTPases and can provide a costimulatory signal whose exact biochemical nature is unclear. Known PTK substrates in activated T cells are lightly shaded, and small GTPases are in black. For details, see Section 111.

these agents do not seem to inhibit degranulation in mast cells (Prepens et al., 1996).Similarly, overexpressionof transfected mutant small GTPases, which interefere with cytoskeletal reorganization, does not inhibit mast cell secretion (Norman et al., 1996)or IL-2 production by T cells (Stowers et al., 1995).Importantly, these findings do not exclude the likely possibility that reorganization of the cytoskeleton is essential for a physiologically relevant polarized secretion, e.g., localized lymphokine secretion at the site of contact between antigen-specific T cells and AF'Cs (Po0 et al., 1988; Stowers et al., 1995). The stimulus-coupled reorganization of the actin cytoskeleton is not only important for changes in cell shape, adhesion, and motility. Rather, this reorganization is also essential for the proper transmission of growth signals.


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This is not surprising because intact architecture of the cytoskeleton is most likely essential for recruitment of signaling complexes to the phosphorylated ITAM motifs in IRRs, including those that contribute to the activation of Ras, i.e., Shc, LAT, Grb2, and Sos. Studies on the role of the actin cytoskeleton in antigen-specific T cell activation demonstrated that agents that disrupt the actin cytoskeleton prevent not only the cell shape changes occurring on contact with APCs but, in addition, also interfere with Ca" mobilization, which is known to regulate gene transcription (Dolmetsch et al., 1997; Timmerman et al., 1997), and with interferon-? production (Valitutti et al., 1995a), as well as with TCFUCD28-induced proliferation and NFAT activation (Holsinger et al., 1998). These results indicate that the actin cytoskeleton drives TCR cross-linking and sustains signal transduction, and offer an explanation for the well-known phenomenon that T cells, despite being equipped with a low-affinity TCR, can be triggered by very few MHC/peptide complexes (Demotz et al., 1990) that serially engage many TCRs (Valitutti et al., 199513). Cytoskeletal changes induced in T cells by antigen/MHC binding, which are most likely mediated by Rho GTPases, may also be essential for the ligand-specific oligomerization of TCFUMHClpeptide ternary complexes on the surface of T cells (Reich et al., 1997). The quantity and/or quality of cytoskeleton assembly and the degree of oligomerization may, in turn, be important determinants in differential TCR signaling that can lead to distinct functional outcomes, i.e., activation and proliferation, anergy, or apoptosis (Alberola-Ila et al., 1997). The exact mechanisms through which cytoskeleton reorganization regulates PTK-mediated growth signals in T cells are unknown. However, several clues have emerged. First, T or B cell activation was found to induce increased tyrosine phosphorylation of a-tubulin (Ley et al., 1994; Mane Cardine et al., 1995; Peters et al., 1996), most likely mediated by Syk-family tyrosine kinases (Huby et al., 1995; Peters et al., 1996). The presence of phosphorylated a-tubulin in the unpolymerized soluble fraction of T (Ley et al., 1994) and B (Peters et al., 1996)cells, and its association with Zap-70 (Huby et al., 1995) or Syk (Peters et al., 1996), suggest that tyrosine phosphorylation of a-tubulin may inhibit its polymerization into microtubules (Ley et al., 1994) and/or serve to dissociate Syk from the BCR, thereby allowing it to phosphorylate cytosolic proteins (Peters et al., 1996). Phosphorylated a-tubulin was also found to associate with Vav in intact T cells (Huby et al., 1995), and with the SH2 domain of Fyn in vitro (Marie Cardine et al., 1995). The association of a-tubulin with Vav and Zap-70 in unactivated T cells (Huby et al., 1995) most likely reflects the constitutive tyrosine phosphorylation occurring in the transformed

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Jurkat cells. Indeed, a-tubulin was not phosphorylated on tyrosine in untransformed resting human T lymphocytes (Marie Cardine et al., 1995). TCR-induced reorganization of the cytoskeleton may also regulate growth signaling pathways by affecting a population of phospho-( chains (and their associated TCR complexes) residing in the cytoskeleton. Thus, two groups have reported that cell surface-expressed TCR-J is associated with the actin cytoskeleton (Caplan and Baniyash, 1995, 1996; Caplan et al., 1995; Rozdzial et al., 1995). A fraction of cell surface-expressed ( was found to be associated with the cytoskeleton in resting T cells and in transfected COS cells, and a unique 16-kDa tyrosine-phosphorylated species of [was detected only in the cytoskeletal fraction (Caplan et d., 1995). The phosphorylated species of ( in the cytosolic and cytoskeletal fractions were distinct in both nonactivated and activated lymphocytes, and assembly of the CD3 subunits with cytoskeleton-associated (was necessary for their maximal localization to the cytoskeleton (Caplan and Baniyash, 1996). It was further demonstrated that TCR ligation induced increased association of phospho-(with the cytoskeleton (Caplan et al., 1995), and that cytochalasin treatment disrupted it (Caplan et al., 1995; Rozdzial et al., 1995). The importance of this association in growth signaling is implicated by the finding that it correlated with IL-2 production by activated T cells, and was not detected in immature thymocytes (Rozdzialet al., 1995).Anchorage of cell surface-expressed ( chain to the cytoskeleton in T cells may, therefore, facilitate recycling of receptor complexes and/or allow the transduction of TCR signals into the cell. Studies have addressed the role of Ca” or different enzymes in cytoskeleton reorganization using either transfection of PTKs such as Syk (Cox et al., 1996), or selective pharmacological inhibitorshnducers that modulate Ca2+mobilization or the activities of PKC and P13-K. The conclusions drawn were that changes in the cytoskeleton are dependent on tyrosine kinase (Melamed et al., 1991b; Pfeiffer and Oliver, 1994; Cox et al., 1996) and PKC (Phatak et al., 1988; Apgar, 1991; Melamed et al., 1991b; Par& et al., 1992; Pfeiffer and Oliver, 1994) activity. Ca2+also modulates these events (Apgar, 1991; Donnadieu et ul., 1992,1994; Negulescu et al., 1996), apparently via a calcineurin-independent mechanism (Stowers et al., 1995; Negulescu et al., 1996), although an increase in intracellular Ca2+concentration is neither necessary nor sufficient for actin polymerization (Apgar, 1991). Selective inhibition of PIS-K activity in intact cells indicated that PI3-K is required for the polarization of antigen-specific T cells toward APCs, possibly via a Cdc42-mediated pathway (Stowers et al., 1995), and for membrane ruffling and fluid pinocytosis in FcsRI-stimulated RBL cells (Barker et al., 1995). However, PI3-K apparently was not required for actin


33

polymerization, receptor internalization, spreading, and adhesion plaque formation (Barker et al., 1995). Because Cdc42, Rac, and Rho are all involved to varying degrees in cytoskeleton reorganization and, furthermore, these GTPases as well as Ras are linked in a signaling cascade, it is impossible to conclude from studies assessing cell morphology or the effects of pharmacological agents which Rho-family GTPase regulates a particular aspect of cytoskeleton reorganization. This question was addressed more directly by pharmacological approaches based on the use of bacterial toxins that selectively inhibit defined GTPases, or genetic approaches relying on transfection with wildtype or mutated forms of Rho-family GTPases. This analysis allowed more precise conclusions. In mast cells, transient transfection with CA mutants of RhoA and Racl (V14RhoA and V12Rac1, respectively) demonstrated that although Rho was responsible for de novo actin polymerization occurring in the cell interior, presumably from a membrane-bound monomeric actin pool, Racl was required for entrapment of the released cortical filaments following activation by several stimuli (Norman et al., 1994); furthermore, both of these active GTPases enhanced the stimulated secretion by increasing the proportion of cells responding to the stimulus (Price et al., 1995) in a cytochalasin-resistant manner (Norman et al., 1996). Inhibition of Rac and Rho by a DN Rac mutant (N17Racl) or Clostridium botulinurn C3 exotoxin (which ADP-ribosylates and inactivates Rho), respectively, reduced the stimulus-induced secretory response of the cells (Price et al., 1995). Expression of DN mutants in RBL cells also had distinct effects on mast cells. DN Cdc42 decreased cell adhesion, interfered with FcsRI-induced actin plaque assembly, and reduced the recruitment of vinculin at the cell-substratum interface, whereas the inhibitory Racl mutant abolished FcsRI-mediated membrane ruffling. Both mutants significantly inhibited antigen-induced degranulation (Guillemot et al., 1997). Similar approaches were also used in T cells. Introduction of recombinant C3 exotoxin into electropermeabilized NK cells or CTLs led to a dosedependent inhibition of their cytolpc function; the only substrate efficiently ADP-ribosylated by C3 was RhoA, which was localized in the cytosol (Lang et al., 1992). A role for Rho in T cell activation is also suggested by the finding that lysophosphatidic acid (LPA), a selective Rho activator, induced Ca2+mobilization and increased IL-2 production in Jurkat T cells (Xu et al., 1995). Similarly, when a B lymphoblastoid cell line was treated with C3, only RhoA was ADP-ribosylated in situ in a time- and concentration-dependent manner, and this correlated with inhibition of PMAinduced LFA-lhntercehlar adhesion molecule (1CAM)-l-dependent cell aggregation (Tominaga d al., 1993). This result indicates that RhoA functions downstream of PKC to induce LFA-l-dependent cell aggregation.

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It is not clear, however, whether C3 inhibits aggregation by interfering with cell polarization or by preventing Rho-dependent adhesion of ICAM1to LFA-1, because LFA-1 is known to undergo changes in ligand affinity and to inetarct with the actin cytoskeleton on activation (Hynes, 1992). Expression of CA or DN Cdc42 in antigen-specific T cells inhibited the polarization of both actin and microtubules toward APCs (Stowers et al., 1995). Finally, Rho-family GTPases also regulate the invasiveness of malignant T cells, most likely by controlling cell shape, adhesion, and/or motility. Thus, transfection of Tiam-1, a Rac-specific GEF, or active Racl induced invasiveness in the murine T lymphoma line BW5147 (Michiels et al., 1995). Pretreatment of several T lymphoma cell lines with C3 exotoxin inhibited the characteristic shape changes resulting from extension and retraction of pseudopodia and, concomitantly, the invasion of the cells through a fibroblast monolayer. These effects correlated with the absence of F-actin in the pseudopodia of the treated (but not control) cells (Verschueren et al., 1997). The role of Rho in FcyR-mediated phagocytosis was investigated by microinjecting 3774 macrophages with C3 exotoxin (Hackam et al., 1997). C3 induced retraction of filopodia, disappearance of focal complexes, and a global decrease in the F-actin content. In addition, the cells exhibited increased spreading and formation of vacuolar structures. Importantly, inactivation of Rho completely abrogated phagocytosis of opsonized particles. The same effects were also observed in FcyRIIA-transfected COS cells. Moreover, Rho was essential for the accumulation of tyrosinephosphorylated proteins and F-actin around phagocybc cups and for F v R mediated Ca2+signaling. The effect of the toxin was specific, because clustering and internalization of transfenin receptors were unaffected by C3 microinjection. These data i d e n q a role for Rho in FcyR-mediated phagocytosis in leukocytes (Hackam et al., 1997). C. RHO-FAMILY GTPASESCONTROL GROWTH SIGNALS IN LYMPHOCYTES The role of Rho-family GTPases in controlling growth signaling pathways (Symons, 1995; Vojtek and Cooper, 1995; Treisman, 1996)has just begun to be explored in lymphocytes (Genot et al., 1996; Turner et al., 1997; Reif and Cantrell, 1998) (Fig. 5). The limited studies conducted to date indicated that expression of a DN Racl mutant (N17Racl) prevents TCRand Ras-mediated activation of NFAT and AP-1, but does not interfere with ERK2 activation; conversely, activated Racl mimicked active Ras to induce AP-1, but not NFAT (Genot et al., 1996). Similarly, Racl was necessary, but not sufficient, for FceRI-mediated NFAT activation (and, hence, cytokine expression), acting either in parallel to, or downstream of,


35

Ras (Turner and Cantrell, 1997). Research in this important area is likely to expand in the near future. OF RHO-FAMILY GTPASESIN HEMATOPOIETIC CELLS D. REGULATION 1. Vau-An Integrator of Rho-Family and Ras Signals in Hematopoietic Cells The Dbl family of proteins consists of some -15 mammalian members that share a so-called Dbl-homology (DH) domain followed by a pleckstrinhomology (PH) domain. Dbl-family proteins function as physiological activators of Rho family GTPases, and display GEF activity for these GTPases (Cerione and Zheng, 1996). Among members of the Dbl family, special attention has been given to Vav because of its unique structural characteristics and specific expression in hematopoietic cells. Vav was originally isolated as the product of a transforming gene in fibroblasts, the result of a deletion of the amino-terminal 67 residues from the protooncogene product (Katzav et al., 1989). Vav is a 95-kDa protein that contains, in addition to its DH and PH domain, a cysteine-rich zinc-binding domain similar to that found in members of the PKC family, and an SH2 domain flanked by two SH3 domains. These domains mediate physical interactions with many unrelated proteins and with lipid second messengers (Romero and Fischer, 1996; Collins et al., 1997). It is, therefore, evident that, aside from its intrinsic enzymatic (GEF) activity, Vav is likely to serve as an adapter or docking molecule to help convey signals from membrane-associated signaling complexes. Severalkey findings have demonstrated the importance of Vav to hematopoietic cell function: first, Vav is phosphorylated on tyrosine in response to stimulation of a large variety of hematopoietic cell receptors and interacts with PTKs of the Src, Syk, Tec, and JAK families, suggesting a broad involvement in cellular responses; second, Vav-deficient mice display defective T and B lymphocyte development and responsiveness to antigen receptor-mediated activation signals (Fischer et al., 1995; Tarakhovsky et al., 1995; Turner et al., 1997; Zhang et al., 1995); third, overexpression of protooncogenic Vav in T lymphocytes activates transcription factors involved in IL-2 production, including NFAT, and potentiates TCRmediated activation of the IL-2 gene (Holsinger d al., 1995; Wu et al., 1995; Deckert et al., 1996);finally, Vav-deficient T cells were very recently found to display severe defects in cytoskeletal reorganization and actin-cap formation in response to TCR stimulation ( Fischer et al., 1998;Holsinger et al., 1998).The severe developmental and activation defects in lymphocytes of Vav-deficient mice represent perhaps the most compelling indirect evidence for a critical function of Rho-family GTPases in IRR signaling.

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Although Vav was originally reported to be an activator of Ras (Gulbins et al., 1993), more recent studies have shown that Vav functions as a GEF for Rho GTPases (Crespo et al., 1997; Han et al., 1997). Tyrosine phosphorylation mediated by Lck (Crespo et al., 1997; Han et al., 1997) and, possibly, by Syk-family kinases (Deckert et al., 1996) stimulates the exchange activity of Vav. The B cell negative regulatory receptor, CD22, may be linked to a signaling pathway that selectively inhibits the tyrosine phosphorylation of Vav and/or accelerates its dephosphorylation, thereby potentially down-regulating its exchange activity (Sat0 et al., 1997). Substrates or products of PI3-K were more recently found also to regulate the activity of Vav in a negative or a positive manner, respectively (Han et al., 1998). Dual regulation of Vav by PTKs and PI3-K-related lipids appears to be similar to the dual regulatory mechanism of Bruton tyrosine kinase (BTK) (Z. Li et al., 1997). The specificity of Vav for members of the Rho family is not fully established. Although one study demonstrated specific (or at least preferential) exchange activity for Rac (Crespo et al., 1997), two other studies based on in vitro exchange assays (Han et al., 1997) or morphological analysis of Vav-transfected COS cells (Olson et al., 1996) suggested that Vav regulates Rac, Cdc42, and Rho. Similar to Rac and Cdc42 (Coso et al., 1995; Minden et al., 1995; Olson et at., 1995; Teramoto et al., 1996), Vav overexpression in COS cells (Crespo et al., 1996; Olson et al., 1996) or in mast cells (Teramoto et al., 1997) leads to JNK activation. However, most of these studies utilized the oncogenic form of Vav, which is inert in T cells with regard to inducing transcriptional activation of the IL-2 gene (Wu et al., 1995). Thus, an important question that remains unsettled is whether, in the context of IRR signaling in hematopoietic cells, Vav is also a relevant physiological activator of the JNK cascade. In fact, findings indicate that TCWCDZ8-induced JNK activation remains intact in Vav-deficient T cells (Fischer et al., 1998; Holsinger et al., 1998). The positioning of Vav in lymphocyte activation pathways makes it an attractive candidate for coupling Ras- and Rho-family signaling pathways in hematopoietic cells (Fig. 5). It has recently become clear that these two families of small GTPases “talk’ to each other (Khosravi Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995a,b, 1997). Vav could fulfill the important function of integrating both pathways in hematopoietic cells. Such a function would be analogous to that of another Dbl-family member isolated from yeast, Scdl (Chang et al., 1994). Biochemical and genetic analysis has demonstrated that Scdl forms a complex in a cooperative manner with two small GTPases of the Ras and Rho families, Rasl and Cdc42sp, respectively. Although the functional relationship between Scdl and Cdc42sp has not been elucidated, it was determined that an active


37

(i.e., GTP-bound) Rasl acts upstream of Scdl to stabilize its interaction with Cdc42sp, respectively (Chang et al., 1994). As reviewed above, the link of Vav to Rho-family GTPases is evident from the demonstration of its in witro GEF activity for these GTPases; its ability to activate JNK via a Rac-dependent pathway, including in mast cells (Teramoto et al., 1997); the cytoskeletal changes induced in Vavtransfected cells; and the cytoskeletal defects in Vav-deficient T cells. However, it is clear that Vav also interacts with the Ras signaling pathway. First, Vav-mediated transformation of fibroblasts (Katzav et al., 1995) or NFAT activation in T cells (Wu et al., 1995) can be blocked by DN Ras. Conversely, DN Vav mutants also block fibroblast transformation by oncogenic Ras (Katzav et al., 1995) or Ras-dependent NFAT/AP-1 activation in T cells (Deckert et al., manuscript in preparation). Second, integrin receptor stimulation of neutrophils activated Ras and induced the association of Ras with tyrosine-phosphorylated Vav; moreover, a selective tyrosine kinase inhibitor prevented in parallel Ras activation, tyrosine phosphorylation of Vav, and the association between these two proteins (Zheng et al., 1996). Third, in mast cells, FcERI was found to associate with an active signaling complex that included Vav, Grb2, Raf-1, and ERK2 (Song et al., 1996). Finally, a phosphorylated Vav/SLP-76/Grb2 complex that also contained 36- to 38-kDa (most likely LAT) and 116-kDa (Cbl?) tyrosinephosphorylated proteins was observed in TCWCD28-activated antigenspecific T cells (Tuosto et al., 1996). At least three potential mechanisms through which Vav may be linked to Ras activation can be considered. The first two are related to Grb2. A proline-rich sequence in the amino-terminal Vav SH3 domain binds to the carboxy-terminal SH3 domain of Grb2 (Ye and Baltimore, 1994; RamosMorales et al., 1995), providing two potential mechanisms for linking Vav to Shc and Grb2 and, thus, potentially to Sos, the ubiquitous Ras GEF. First, a Shc/Grb2 complex may bind to Vav SH3. This, however, is unlikely to mediate interaction with Sos, because the SH3 domains of Grb2 have been shown to bind cooperatively to Sos (Li et al., 1993) and, therefore, would compete for association with Vav. Indeed, in some studies it has been difficult to detect a Vav/Grb2 complex in lymphocytes (Nel et al., 1995).The observed Grb2Nav association in intact activated T cells (Tuosto et al., 1996) or in in vitro “pull-down” experiments using Grb2 fusion proteins (Ramos-Morales et al., 1994) may be indirect and mediated by an adapter protein that binds both simultaneously, e.g., SLP-76 (Tuosto et al., 1996; Koretzky, 1997). Second, in activated cells, Shc could simultaneously bind phosphorylated Vav and Grb2 SH2 via its own SH2 domain or phosphorylated tyrosine residue(s), respectively. In this complex, the Grb2 SH3 domains would mediate association with Sos. Formation of such

38


a Vav/Shc/Grb2/Sos complex could offer a potential mechanism for the integration of signals from the Ras pathway and a signaling pathway involving Rho-family small G proteins. A third potential coupling mechanism could be mediated by lipids that regulate Vav activity. Because the products of PI3-K up-regulate the exchange activity of Vav (Han et al., 1998), and PI3-K is an immediate effector of Ras (Rodriguez-Vicianaet al., 1994), a pathway could be envisaged in which IRR-stimulated Ras would activate PI3-K, and the lipids generated by activated PI3-K would, in turn,cooperate with tyrosine kinases to stimulate the exchange activity of Vav toward Rho-family GTPases. 2. Other Regulators Rho-family GTPases expressed in leukocytes are most likely regulated by the same kinds of regulators that have been characterized in other cell types, i.e., GEFs, GAPS, and GDIs. A comprehensive review of these regulators is beyond the scope of this article, but some pertinent studies are of interest. In general, the expression of small GTPases is not regulated at the transcriptional level. One exception is Race, which was found to be expressed in peripheral blood lymphocytes, purified B and T cells, thymus, and several B and T cell lines, but not in other tissues analyzed, including liver, brain, lung, heart, and kidney (Reibel et al., 1991). After 24 hr of in vitro stimulation with phytohemagglutinin A, 30- to 50-fold accumulation of Rac2 mRNA was observed in peripheral blood lymphocytes and in purified T lymphocytes. These findings suggest that Rac2 fulfills specific roles in leukocyte activation. One such role may be related to the NADPH oxidase system (Section III,F,4). This plasma membrane-associated multimolecular enzyme complex generates superoxide anions that serve as bactericidal agents in phagocytes. NADPH oxidase is known to be regulated by Rac (Bokoch, 1994). Despite the 92% homology between Racl and Rac2, the latter interacted 6-fold better with p67phox (Dorseuil et al., 1996). A posttranslational mechanism regulating the activity of Rho GTPases has been described in NK cells (Lang et al., 1996)and leukocytes (Laudanna et al., 1997). The phosphorylation of RhoA on a single serine residue (Ser108)by CAMP-dependentprotein kinase (PKA)increased the affinity of the active, GTP-loaded form of RhoA for its GDI, resulting in a translocation of RhoA from the membrane to the cytoplasm (Lang et al., 1996) and inhibition of its guanine nucleotide exchange (Laudanna et al., 1997). As a result, the cytotoxic activity of NK cells (Lang et al., 1996) or the chemoattractanttriggered integrin-dependent leukocyte adhesion of neutrophils or an IL8 receptor-transfected lymphoid cell line (Laudanna et al., 1997) were inhibited.


39

IN IRR SIGNALING E. RHO-FAMILY EFFECTORS The evolving interest in the role of Rho-family GTPases as regulators of growth signals has been accompanied by an intense search for effectors of these GTPases. This search has led to the isolation of various proteins that bind to the activated (GTP-bound) forms of Rac, Cdc42, or Rho (Amano et al., 1996; Ishizaki et al., 1996; Joneson et al., 1996; Kolluri et al., 1996; Quilliam et al., 1996; Symons et al., 1996; Van Aelst et al., 1996; Watanabe et al., 1996; 1997; Aspenstrom, 1997; reviewed in Narumiya et al., 1997; van Aelst and D'Souza-Schorey, 1997; Hall, 1998) (Fig. 4). A detailed analysis of nearly all of these immediate effectors, with the exception of one (Section VI,C), in leukocyte function has not been conducted. However, studies have addressed the role of stress-activated protein kinases of the JNK and p38 families, which are downstream targets of Rac and Cdc42 (Coso et al., 1995; Minden et al., 1995; Olson et al., 1995), in IRR signaling in T cells, B cells, and mast cells. The pathway leading from Rad Cdc42 to JNWp38 activation in nonhematopoietic cells involves the p21activated protein kinase (PAK) family as an intermediate (Bagrodia et al., 1995; S. Zhang et al., 1995; Frost et al., 1996), but the role of PAKs in lymphocyte activation is less clear. TCR-mediated activation of T cells stimulates JNK (Su d al., 1994), its upstream activating kinase MEKK-1 (Faris et al., 1996), and its immediate activating kinases MKK4 (SEK1) and MKK7 (Matsuda et al., 1998), as well as activation of p38 (Salmon et al., 1997; Matsuda d al., 1998), its immediate activator MKK6 (Matsuda et al., 1998), and its downstream target MAPKAP kinase-2 (Salmon et al., 1997) (Fig. 5). Unlike ERK activation, which can be induced by TCR or phorbol ester stimulation alone (Su et al., 1994; Matsuda et al., 1998), MEKK-l/MKKUMKK7/ JNK or MKK6/p38 activation required CD28 costimulation because it was undetectable or very weak in the absence of CD28 (Su et al., 1994; Faris et al., 1996; Salmon et al., 1997; Matsuda et al., 1998). The two-signal requirement for JNKlp38 activation is also evident from the finding that it can be induced by a combination of phorbol ester and Ca" ionophore, but not by either of these signals alone (Su et al., 1994; Matsuda et al., 1998). The requirement for a Ca" signal reflects the important role of calcineurin, because activation of JNK and p38, as well as their upstream kinases, was sensitive to cyclosporin A (Su et nl., 1994; Matsuda et a]., 1998). Indeed, CA calcineurin was found to synergize with a PKC signal to activate JNK and the IL-2 promoter in T cells via a Rac-dependent pathway (Werlen et al., 1998). The important role of JNK and p38 in induction of IL-2 production is implicated by the finding that blocking of p38 by a specific pharmacological inhibitor (SB203580)or by a DN MKK6,

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as well as inhibition of JNK by expression of DN MKK7, abrogated transcriptional activation of the IL-2 promoter or NFAT (Matsudaet al., 1998). Further clues regarding the signals leading to JNK activation in T cells come from studies demonstrating that combinations of a CA form of Rac plus Syk tyrosine kinase (Jacinto et aL, 1998), or Vav plus CA calcineurin (Villalba-Gonzales and Altman, 1998), cooperate to activate JNK. In contrast to Syk, Lck did not cooperate with Rac to activate JNK. It has been suggested that Syk functions in a pathway parallel to Rac, which regulates PKC and calcineurin (Jacinto et al., 1998). The activation of various MAPKs in the murine immature B cell lymphoma, WEHI-231, was also analyzed in some studies. These cells undergo apoptosis in response to BCR ligation, which is prevented by coligation of the B cell costimulatory receptor CD40. BCR or CD40 cross-linking on freshly isolated or LPS-activated splenic B cells, or on WEHI-231 cells, resulted in activation of p38 and MAPKAP kinase-2 (Salmon et al., 1997). Inhibition of p38 activity by pretreating intact WEHI-231 cells with SB203580 had no effect on either BCR-induced apoptosis or anti-CD40mediated suppression of apoptosis. In another study, BCR ligation caused strong activation of ERK2, but only a weak or modest activation of p38 or JNK, respectively, in WEHI-231 cells; however, CD40 was a potent activator of JNK, p38, and MAPKAP kinase-2 in these B cells (Sutherland et al., 1996). These results suggest that, in this system, ERK2 activation correlates with B cell apoptosis, but that p38/MAPKAP kinase-2 activation is not required for BCR-mediated apoptosis. However, a different conclusion regarding the role of p38 in B cell apoptosis was reached in another study using a human B cell lymphoma line (B104). These cells undergo apoptosis in response to anti-IgM, but not anti-IgD, stimulation. In parallel, IgM, but not IgD, cross-linking led to activation of JNK and p38 in these cells. Similar activationwas induced by ionomycin, and ionomycin- or IgMinduced JNWp38 activation and apoptosis were inhibited in parallel by cyclosporin A (Graves et al., 1996).The difference between the two studies serves to emphasize the critical importance of the cellular context and developmental stage in determining the contribution of distinct signaling pathways to a particular biological outcome. This point is also evident from the finding that ligation of the incomplete BCR complex expressed on proB cells (which consists of the ITAM-containing IgdIgP signaling subunits bzlacks surface IgM), activates ERK2, but not JNK or p38 (Nagata et al., 1997). Several studies analyzed the activation of JNWp38 in mast cells. FcsRI ligation was found to lead to activation of MEKK-1 (Ishizuka et al., 1996, 1997), JNK (Ishizuka et. al., 1996, 1997; Kaga et al., 1997; Kawakami et al., 1997; Teramoto et al., 1997), and p38 (Ishizuka et al., 1997; Kawakami


41

et al., 1997; Zhang et al., 1997). The activation of JNK in mast cells was dependent on intact PI3-K activity (Ishizuka et al., 1996, 1997), and was mediated by a Vav/Rac-dependent pathway (Teramoto et al., 1997),consistent with the requirement for Rac in FceRI induction of NFAT in the same cells (Turner and Cantrell, 1997). Analysis of cultured bone marrowderived mast cells from Btk tyrosine kinase-deficient mice indicated that the FceRI-mediated activation of JNK and, to a lesser extent, of p38, but not ERK, was compromised, suggesting that Btk positively regulates JNK (and p38) activation (Kawakami et al., 1997). Different conclusions were reached in two studies with regard to the role of distinct MAP kinases in FceRI-stimulated TNF-a production by mast cells. Based on the use of specific pharmacological inhibitors of MEWERK (PD98059) or p38 (SB203580),or MEKK-1/JNK mutants, one study concluded that JNK, but not ERK, regulated TNF-m production by stimulated mast cells (Ishizuka et a/., 1997). In contrast, the other study (Zhang et al., 1997) demonstrated that ERK (but not p38) contributed to the FceRI-stimulated production of TNF-a and the release of arachidonic acid in these cells. Neither kinase, however, was essential for FceRI-mediated degranulation or constitutive production of TNF-0. The latter study also revealed that p38 negatively regulated activation of ERK and the responses mediated by this kinase (Zhang et al., 1997). Activation of neutrophils by FcyR cross-linkingor by trimeric G proteincoupled receptors was found to induce activation of PAK, a known downstream target of RacKdc42. However, only the FcyR-induced activation was dependent on cytoskeleton reorganization and intact PI3-K activity as revealed by the use of pharmacological inhibitors (Jones et al., 1998).

FUNCTIONS F. RHOGTPASESAND OTHERLEUKOCYTE Rho-family GTPases are coupled to leukocyte receptors other than IRRs, and regulate additional leukocyte functions such as cell adhesion and rolling mediated by integrins and selectins (reviewed in Springer, 1990, 1994; Hynes, 1992, 1996; Dunon et al., 1996), responses to chemoattractants and chemokines initiated by seven transmembrane-spanning and other receptors (reviewed in Bokoch, 1995; Downey et al., 1995), and the phagocyte NADPH oxidase system (reviewed in Bokoch and Knaus, 1994; Bokoch, 1995; Quinn, 1995; Dharmawardhane and Bokoch, 1997). In addition, these GTPases also regulate the process of programmed cell death, or apoptosis. These functions are briefly reviewed below. 1. Integrins and Cell Adhesion

Integrins are heterodimeric transmembrane receptors that couple components of the ECM to the actin cytoskeleton. Each integrin consists of

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an a and a p subunit, and, in mammals, 16 distinct a subunits combine with 8 p subunits to form over 22 receptors with distinct cellular distribution and biological functions. Binding of integrins to ECM proteins causes formation of complex protein structures, termed focal adhesions, which anchor the actin cytoskeleton to the plasma membrane. Integrin-mediated signals regulate various processes during embryogenesis and development, cell growth, motility, and survival and, in malignant cells, uncontrolled growth and metastasis. The best characterized signaling pathways triggered by integrins include activation of PTKs and Rho-family GTPases (reviewed in Clark et al., 1994; Schwartz et al., 1995; Dedhar and Hannigan, 1996; Hotchin and Hall, 1996; Parsons, 1996; Yamada and Geiger, 1997) (Fig. 6). Focal adhesion kinase (FAK) represents the major tyrosine kinase associated with focal adhesions (Zachary and Rozengurt, 1992; Parsons, 1996). FAK can couple ECM signals to the Ras signaling pathway via its association with the GrbWSos complex (Schlaepfer et al., 1994), leading to activation of Ras-dependent ERK kinases (Schlaepfer et al., 1994; Renshaw et al., 1996; Fig. 6). Although the integrin-mediated signaling pathways in lymphocytes remain largely unclear, integrin ligation by natural ligands can stimulate the Ras and Rho pathways in lymphocytes (Kapron-

FIG.6. The involvement of small GTPases in signaling by integrins and chemoattractant receptors. For details see Sections II,F,l and III,F,3.


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Bras et al., 1993; Schwartz et al., 1995). Integrin-dependent adhesion has been shown to generate additional signals in adherent cells, including turnover of phosphoinositides and Rho-dependent activation of its effector, phosphatidylinositol 4-phosphate 5-kinase (PIP5-K) (Chong et al., 1994). is a One major product of PIPS-K, phosphatidylinositol4,5-bisphosphate, substrate for both PLCy and PISK, two enzymes involved in the generation of critical lipid second messengers in IRR-stimulated leukocytes. Integrin-mediated adhesion to the extracellular matrix or APCs plays an important role in regulating lymphocyte homing and recruitment to inflammatory sites. Integrin ligation also generates a costimulatory signal for T lymphocyte proliferation (Van Seventer et al., 1991;Croft and Dubey, 1997). As an example, integrin a 4 p l and the TCR synergized to increase tyrosine phosphorylation of PLCy and calcium mobilization in T cells (Kanner et al., 1993). Conversely, IRR stimulation leads to an increase in the avidity of integrins for their ligands via an “inside-out” signalingpathway (springer, 1990; Schwartz,et al., 1995; Dedhar and Hannigan, 1996).Thus, FcyR cross-linking on human neutrophils was found to increase the avidity of a M P 2 integrin for its ligand via a pathway that required PI3-K activity and an intact cytoskeleton (Jones et al., 1998). The signals delivered by integrins are integrated with those originating from antigen or inflammatory cytokine receptors in order to activate reorganization of the cytoskeleton, gene expression, secretion, differentiation, and proliferation. The integration of the integrin-associated costimulatory signal with IRR signals may involve the association of the tyrosine-phosphorylated 6 subunit with polymerized actin (Caplan and Baniyash, 1995, 1996; Caplan et al., 1995; Rozdzial et al., 1995; Caplan and Baniyash, 1996) or the ITAM-associated Syklzap-70 kinases, because, for example, Syk was found to be required for BCR-dependent actin assembly in B lymphocytes (Coxet al., 1996).Two additional elements that could represent important points of integration between IRRs and integrins are Vav, which can undergo tyrosine phosphorylation (and, presumably, enzymatic activation) in response to ligation of both IRRs and integrins (Cichowski et al., 1996; Zheng et al., 1996; Gotoh et al., 1997), and FAK, which is phosphorylated and activated by integrin, including in leukocytes (T. H. Lin et al., 1995), or IRR (e.g., Haimovich et al., 1996; Berg and Ostergaard, 1997) stimulation. T helper cell-APC and CTL-target ceIl initial contact involves T cell-expressed /32 integrins (Springer, 1994), and PZ integrin (LFA-1)-dependent aggregation of lymphocytes (via ICAM-1 binding) is blocked by the Rho-inactivating C3 transferase exotoxin (Tominaga et al., 1993). 2. Selectins Regulation of T lymphocyte cell shape has to be considered as a critical process because the circulating T cell adheres to cellular and extracellular

44


matrices, migrates through blood vessel endothelia, and interacts with APCs in the crowded environment of the lymph nodes. Lymphocytes express another family of adhesion receptors, the selectins, which are involved in the rolling process, the initial step of leukocyte adhesion to endothelial cells (Springer, 1994; Dunon et al., 1996; Rosen et al., 1997). This process may play an important role in the recruitment of T cells to inflammatory sites, as indicated by the finding that P- and E-selectin mediate recruitment of T helper-1 but not T helper-2 cells into inflamed tissues (Austrup et al., 1997). T cell adhesion to P-selectin was found to induce tyrosine phosphorylation of FAK and other substrates (Haller et al., 1997).L-Selectin ligation has been shown to trigger a signaling cascade involving activation of the Src-kinase Lck and association of GrbWSos with L-selectin in Jurkat T cells; this correlated with Ras, ERK, and Rac2 activation, and with a transient increase of reactive oxygen intermediates (Brenner et al., 1996). L-Selectin also induced cytoskeleton reorganization in the cells as demonstrated by a marked increase in F-actin content, which was dependent on intact Ras and Rac2 function (Brenner et al., 1997). 3. Chemoattractant Responses In addition to selectins, integrin-mediated cell arrest and adhesion can be triggered when seven transmembrane-spanning, trimeric G proteincoupled receptors are stimulated by their ligands, e.g., during stimulation of neutrophils by formyl peptides, leukotriene B4, or IL-8 (Fig. 6). These chemoattractants are released by endothelid cells during inflammation and generate signals that activate leukocytes, induce their integrin-mediated adhesion to the endothelial surface, and, finally, stimulate transmigration. In addition, cytokines coupled to tyrosine kinase signaling pathways, e.g., GM-CSF in neutrophils (Coffer et al., 1998), or colony-stimulating factor1(CSF-1) in macrophages (Allen et al., 1997),also act as chemoattractants. The precise chemoattractant signaling mechanisms are not known. However, the “conventional” model that implicated trimeric G proteins as central players has been updated to include the important contribution of tyrosine kinases. For example, formyl peptide stimulation of human neutrophils was found to stimulate tyrosine phosphorylation and activation of the Lyn kinase and its association with tyrosine-phosphorylated Shc and PI3-K (Ptasznik et al., 1995). Phosphorylated Shc and activated PIS-K might lead, in turn, to recruitment of GrbWSos and subsequent Ras activation (see Section II,B,2) or activation of Rac, respectively. In addition, activated Ras may stimulate PI3-K activity (Rodriguez-Vicianaet al., 1994) and other effectors. Indeed, chemotactic peptides activate Ras, Raf-1, BRaf, MEK-1, and ERKs in human neutrophils (Buhl et al., 1994; Fialkow et al., 1994; Grinstein et al., 1994; Worthen et al., 1994). In addition, a


45

tyrosine kinase (Lyn?)-dependent pathway is required for chemoattractantstimulated PI3-K activation in human neutrophils (Ptasznik et al., 1996). Numerous studies have shown that small GTPases mediate important signaling functions in neutrophil chemoattractant signaling (reviewed in Bokoch, 1995; Downey et al., 1995) (Fig. 6). Phospholipase At (PLA,), which is activated by phosphorylation and mediates the production of lipid second messengers (arachidonic acid and, subsequently, eicosanoids and leukotrienes), appears to represent a major target for the chemoattractantstimulated Ras signaling pathway. One important target for Rho GTPases is PIP5-K, which, on activation, generates PIP2. This is required for the halting of lymphocyte rolling and a firm integrin-mediated adhesion (Campbell et al., 1996). Therefore, as also discussed above, lipid metabolism enzymes and intermediates appear to serve important regulatory functions in Ras and Rho GTPase-mediated cytoskeletal reorganization (Hartwig et al., 1995) and lymphocyte adhesion, and this property is shared by integrins, selectins, and chemoattractants. Agonist stimulation was reported to stimulate within seconds guanine nucleotide exchange (i.e., activation) on RhoA in lymphoid cells transfected with formyl peptide or IL-8 receptors. Inactivation of Rho by C3 transferase exoenzyme blocked agonist-induced lymphocyte a 4 p l integrin adhesion to vascular cell adhesion molecule-1 (VCAM-1)and neutrophil P2 integrin adhesion to fibrinogen. These findings suggest that Rho participates in signals from chemoattractant receptors, which trigger rapid adhesion in leukocytes (Laudanna et al., 1996). Selective inhibitors of the MEWERK pathway (PD98059) or P13-K (wortmannin and LY294002) were used to investigate the roles of these kinases in the regulation of neutrophil effector functions. GM-CSF, platelet-activating factor (PAF), or a formyl peptide were capable of activating ERK1/2 and PI3-K in human neutrophils. ERK activation correlated with the stimulation of Ras by both tyrosine kinase and G protein-coupled receptors. PI3-K inhibition interfered, to various degrees, with superoxide generation, neutrophil migration, and PAF release. PD098059 treatment, however, inhibited only the PAF release stimulated by serum-treated zymosan. This demonstrates that, although MEUERK kinases are not involved in the activation of respiratory burst or neutrophil migration, they have a potential role in the activation of cytosolic phospholipase A2. PI3-K, however, seems to have a much wider role in regulating neutrophil function (Coffer et al., 1998). The requirement for Rho-family members in cytoskeletal events mediated by structurally diverse chemoattractant receptors was examined in RAW 264.7 monocytic cells transfected with the human chemotactic peptide receptor and stimulated with formyl peptide, CSF-1, IgG-coated parti-

46


cles, or PMA. Expression of DN mutants of Racl or Cdc42 inhibited cytoskeletal responses to FMLP and CSF-1, blocked phagocytosis, and partially inhibited accumulation of F-actin-rich phagocytic cups. The finding that PMA-induced ruffling was not inhibited by expression of DN Racl, but was blocked by DN Cdc42, indicated that these GTPases acted via nonoverlapping pathways (Cox et al., 1997). The role of Rho-family GTPases in the chemotactic response of a CSFl-dependent murine macrophage cell line (Bac1.2F5) to CSF-1 was also analyzed by expression of DN or CA Racl, Cdc42, or RhoA mutants (Allen et at., 1997). In addition to stimulating the proliferation and motility of these cells, CSF-1 acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bacl cells, evidenced by formation of filopodia, lamellipodia, and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables inside the cell. Microinjection of CA Racl stimulated lamellipodia formation and membrane ruffling and, conversely, a DN Rac mutant inhibited CSF-l-stimulated lamellipodia formation and induced cell rounding. CA Cdc42 induced the formation of long filopodia, whereas the DN Cdc42 mutant prevented CSF-l-induced formation of filopodia but not lamellipodia. Finally, CA RhoA stimulated actin cable assembly and cell contraction, but the Rho inhibitor, C3 transferase, caused a loss of these cables (Allen et al., 1997). Thus, Cdc42, Rac, and Rho regulate the formation of distinct actin filament-based structures during the chemotactic response of Bacl macrophages to CSF-1. 4. NADPH Oxidase

The chemotactic response of leukocytes at inflammatory sites is tightly coupled to activation of the NADPH oxidase system in phagocytic leukocytes. These cells use this specialized enzyme to generate reactive oxygen metabolites that kill microorganisms engulfed by the phagocyte (reviewed in Bokoch, 1994; Bokoch and Knaus, 1994). The active membraneassociated NADPH oxidase consists of four components: flavocytochrome b558, SH3-containing p47ph""and p67ph", and Rac. Rac is absolutely necessary for reconstitution of NADPH oxidase activity in purified protein preparations (Abo et al., 1991; Knaus et al., 1991). In resting neutrophils, Rac is associated in the cytosol with a Rho-GDI, which negatively regulates its activity by inhibiting guanine nucleotide exchange and membrane translocation. NADPH activation is accompanied by translocation of a Raclp47ph"/ p67ph" complex, in which Rac-GTP directly binds p67p'" (Diekmann et al., 1994), from the cytoplasm to the membrane. The Ras-related small GTPase Rapl is also associated with the complex, but is not necessary for activity. Rapl may negatively regulate the activity of NADPH oxidase


47

because PKA-elevating agents (e.g., CAMP) inhibit oxidant production, and Rap1 was found to be a substrate for PKA (Quilliam et al., 1991). 5. Phospholipase D The phospholipase D (PLD) family includes several isoenzymes that hydrolyze phospholipids to generate phosphatidic acid (PA).PLD enzymes are stimulated by both tyrosine kinase receptors and G protein-coupled receptors. As an example, T cell activation by either anti-CD3 antibodies (Mollinedo et al., 1994; Reid et al., 1997) or chemokines (Bacon et al., 1998) stimulates PLD activity. The hydrolysis product of PLD, PA, acts as a second messenger in various growth and differentiation signaling pathways, and it can be metabolized to form other intercellular and intracelMar lipid messengers (reviewed in Boarder, 1994; English, 1996; Olson arid Lambeth, 1996; Spiegel et al., 1996). Selective PLD stimulation has been found to induce actin stress fiber formation (Cross et al., 1996; Colley et al., 1997). In the past few years it has been established that, in addition to its regulation by tyrosine kinases (Natarajan et ul., 1996), PLD activity is also regulated by two families of small GTPases, i.e., the Rho and ADPribosylation factor (Arf)families (reviewed in Frohman and Morris, 1996; Kanaho et al., 1996; Olson and Lambeth, 1996; Wakelam et al., 1997). Members of the Arf family, which are located in the Golgi apparatus and represent major components of non-clathrin-coated vesicles, regulate intracellular protein transport (Nuoffer and Balch, 1994; Boman and Kahn, 1995). 6. Apoptosis

The process of programmed cell death, or apoptosis, mediated by interaction of Fas (CD95) with its ligand (FasL), is also influenced by small GTPases. Although a comprehensive review of this subject cannot be provided here, a few examples are noteworthy. The association of Ras with positive signals leading to cell growth would intuitively suggest that Ras does not participate in apoptosis. However, experimental evidence indicates that Ras can contribute to apoptosis in T cells, and that distinct signaling pathways cooperating with Ras determine whether the outcome of Ras stimulation will be growth or apoptosis. Thus, Fas ligation activated Ras in one (Gulbins et al., 1995), but not another (Wilson et al., 1996), study, and a DN Ras mutant or introduction of neutralizing anti-Ras antibodies into intact cells by electroporation inhibited Fas-mediated apoptosis in T cells (Gulbins et ul., 1995).Because Fas ligation activates the sphingomyelinase pathway that results in ceramide production, but does not cause PKC activation (Gulbins et al., 1995), it appears that one outcome of the cooperation between the Ras and sphingomyelinase signaling pathways is

48


apoptosis; conversely, Ras cooperates with PKC to induce TCR-stimulated cell growth (Downward et al., 1990; Izquierdo et al., 1992a). This view is consistent with the findings that, under conditions wherein cellular PKC activity is inhibited, the expression of an activated Ras mutant caused apoptosis in T cells, which was antagonized by Bcl-2 (Chen and Faller, 1995, 1996). Along a similar line, PKC activation by PMA treatment was found to attenuate early signaling events induced by Fas ligation, i.e., cleavage of the CPP32 protease and its substrate poly(ADP-ribose) polymerase (Ruiz-Ruiz et al., 1997). Fas-mediated apoptosis is associated with the activation of JNK (Toyoshima et al., 1997; Wilson et al., 1996) and its activating kinase MKK7 (Toyoshimaet al., 1997),as well as activation of the MKKGIp38IMAPKAP kinase-2 pathway (Salmon et al., 1997; Toyoshima et al., 1997); the activation of these kinase pathways did not require CPP32-like proteases (Toyoshima et al., 1997). However, whether JNK or p38 activation is required for apoptosis is an unresolved question. In fact, another group reported that Fas-induced JNK activation occurred late, and that expression of a DN mutant of a JNK-activating kinase, MKK4, blocked Fas-induced JNK activation but had no effect on apoptosis. In addition, "-1 was not activated under these conditions (Lenczowskiet al., 1997). Similarly, pharmacological inhibition of p38IMAPKAP kinase-2 by SB203580 did not affect Fas-mediated apoptosis, indicating that p38 activation is probably not required for this event (Salmon et al., 1997). The Ras and JNK pathways can also affect apoptosis in T cells via regulation of FasL expression. Using a luciferase reporter construct containing elements of the FasL promoter in transient transfection assays, TCR-stimulated activation of the Ras signaling pathway was found to be required for optimal induction of the FasL (Latinis et at., 1997). In another study, inducible expression of CA MEKK1 (which activates the JNK pathway) resulted in apoptosis of Jurkat T cells in conjunction with prolonged JNK activation and induction of FasL expression (Faris et al., 1997). Ras is also involved in the apoptosis that cytokine-dependent cell lines undergo when deprived of the respective growth factor. By expressing a DN Ras mutant controlled by a tetracycline promoter in an IL-2AL-4dependent T cell line, it was found that Ras has a crucial role in both proliferation and prevention of apoptosis through the IL-2 receptor, whereas IL-4 promoted proliferation and inhibited apoptosis by Ras-independent signals (Gomez et al., 1996). Furthermore, Ras activation under conditions of IL-2 deprivation led to apoptotic cell death; in contrast, Ras enhanced proliferation in the presence of IL-2 (Gomez et al., 1997). Other studies have also implicated a role for Cdc42 or Rho in apoptosis. First, an infectious recombinant Sindbis virus expressing C3 exoenzyme


49

was used to infect EL4 T lymphoma cells. This resulted in modification and inactivation of virtually all endogenous Rho and, in parallel, in formation of multinucleate cells (likely by inhibiting the actin microfilament-dependent step of cytokinesis) and apoptosis (Moorman et al., 1996). Apoptosis occurred even when multinucleate cell formation was blocked by 5-fluorouracil, which induces cell cycle arrest. Second, an activated form of Cdc42 induced apoptosis in Jurkat T cells. This response was mediated by activation of a protein kinase cascade leading to JNK stimulation, and it was inhibited by expressing DN components of the JNK cascade or by caspase inhibitors (Chuang et al., 1997). The same group later demonstrated that Fas-mediated JNK activation and apoptosis were blocked by expression of a DN PAK mutant and, furthermore, that expression of the catalytically active C-terminal region of PAK in Jurkat cells, which is generated in situ during Fas-mediated apoptosis (Rudel and Bokoch, 1997), induced apoptosis (Rudel et al., 1998). IV. CD28 Signaling in T Cells: The Roles of Small GTPases

A. RASAND CD28 COSTIMULATION In addition to TCWCDS-derived signals, efficient T cell activation requires a second signal, which can be provided by a variety of costimulatory receptors expressed on T cells (reviewed in Van Seventer et al., 1991; Croft and Dubey, 1997). Among these, CD28 plays a critical role in IL2 production, autoimmunity, tumor immunity, and anergy (reviewed in Lenschow et al., 1996; Rudd, 1996; Ward, 1996; Chambers and Allison, 1997). The mechanisms of CD28 signaling are still incompletely understood, but the findings that CD28 becomes phosphorylated on tyrosine, and is associated with three defined signaling proteins, i.e., PI3-K (August et al., 1994; Prasad et al., 1994; Truitt et al., 1994), Grb2 (Schneider et al., 1995b; Kim et al., 1998), and the Itk/Emt tyrosine kinase (August et al., 1994; Marengere et al., 1997), represent important advances in this area (Fig. 7 ) . Although CD28 and the TCWCDS complex share some signaling properties, e.g., the ability to activate PIS-K, the two receptors clearly differ in several regards. In contrast to the TCR, CD28 cross-linking by its physiological ligand, CD80 (B7-1), fa& to stimulate Ras, Raf-1, and ERK2, or to induce tyrosine phosphorylation of LAT and SLP-76 (however, anti-CD28 monoclonal antibodies do induce these events); under the same conditions, a potent and prolonged phosphorylation of Vav on tyrosine is induced (Nunes et al., 1994). Conversely, CD28 but not TCWCDS crosslinking induces tyrosine phosphorylation of a 62-kDa protein (Nunes et al., 1996), which is most likely identical to ~ 6 (Carpino 2 ~ et ~al., 1997; Yamanashi and Baltimore, 1997).

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FIG.7 . The integration of CD28 costimulatory signals and TCR signah leading to T cell activation via small GTPases. Although the key elements integrating these signals remain largely unclear, they appear to operate on at least two levels. First, productive activation of PTKs and optimal phosphorylation of their substrates require the two sets of signals. Second, both the TCWCDS complex and CD28 are linked to Ras and Rho-family GTPases, and to their regulatory proteins (e.g., Sos, Vav, GAPS).Lack of the CD28 costimulatory signal results in TCR-induced anergy. Known PTK substrates in activated T cells are lightly shaded, and small GTPases are in black. In addition to the TCR, CD28 cross-linking was also found to induce cytoskeleton reorganization in T cells (Kaga et al., 1997).For details, see Sections TV and VI,A.

The association of CD28 with the GrbYSos complex (Schneider et al., 1995b; Kim et al., 1998) provides a potential link to Ras activation (Fig. 7 ) .However, it is more likely that CD28 ligation by its physiological ligand does not activate Ras in itself (Nunes et al., 1994), but rather potentiates Ras activation by TCWCDS triggering. This view is consistent with the finding that CD28 costimulation converges with TCWCDS signals to activate JNKs, AP-1, and the IL-2 promoter in T cells (Su et al., 1994; Fans et al., 1996). The CD28 costimulatory signal is essential because TCW CD3 ligation alone does not activate JNK (Su et al., 1994) or its upstream activating kinase, MEKK-1 (Gupta et al., 1994), in T cells (although it can activate Raf-1 and ERK). The ability of DN Ras to block JNK activation by TCR plus CD28 cross-linking (Fans et al., 1996) indicates that Ras regulates JNK activation by these two receptors.


51

The inducible association of CD28 with PI3-K provides an additional potential mechanism through which CD28 could regulate Ras activation (Fig. 7), because PI3-K can activate the Ras signaling pathway in NIH 3T3 fibroblasts and Xenopus laevis oocytes (Hu et al., 1995), or function as an immediate Ras effector (Rodriguez-Vicianaet al., 1994). However, no definite information exists regarding a function of this putative mechanism in T cells. Although Grb2 and PI3-K (p85) bind to the same site in the phosphorylated cytoplasmic tail of CD28 (and, therefore, should compete for CD28 binding), both signalingproteins coimmunoprecipitate with CD28 from activated T cells. Thus, the relative contribution of PI3-K versus Grb2 association to CD28-mediated costimulatory signals remains to be determined. B. CD28 COSTIMUJATION Is LINKED TO RHO-FAMILY GTPASES Recent studies have connected CD28 signaling events to small GTPases of the Rho family (Fig. 7). First, CD28 cross-linking by CD80 induces a rapid and sustained increase in the tyrosine phosphorylation of Vav in the absence of TCR ligation (Nunes et al., 1994). Because the exchange activity of Vav is stimulated by tyrosine phosphorylation (Crespo et al., 1997; Han et al., 1997), this finding suggests that CD28 ligation would stimulate the guanine nucleotide exchange activity of Vav toward member(s) of the Rho family. This is consistent with the finding that stimulation of T cells with the CD28 ligand B7-2 promoted formation of focal adhesion-like plaques where Rho-family small G proteins accumulated (Kaga et al., 1997). However, it remains to be formally demonstrated that CD28-induced tyrosine phosphorylation of Vav enhances its exchange activity. Second, as mentioned earlier, CD28 costimulation is required for activation of JNK (Su et al., 1994) and MEKK-1 (Faris et al., 1996) in T cells. Because JNK activation is coupled to Rac and Cdc42 in fibroblasts (Coso et al., 1995; Minden et al., 1995; Olson et al., 1995) and in T cells (Villalba-Gonzales and Altman, 1998), and the oncogenic form of Vav was also found to induce JNK activation in COS cells (Crespo et al., 1996; Olson et al., 1996) and in mast cells (Teramoto et al., 1997), this finding suggests that CD28dependent MEKK-UJNK activation is mediated by a Vav/Rac (and/or Vav/ Cdc42) pathway. However, it is not known whether wild-type Vav alone activatesJNK in hematopoietic cells. Another T cell costimulatory receptor, CD5, was also found to stimulate a PI3-K-dependent signaling pathway regulated by Vav and Rac (Gringhuis et al., 1998).Similar to CD28 (Nunes et al., 1994), the B cell costimulatory receptor CD19 was found to induce potent tyrosine phosphorylation of Vav in the absence of BCR ligation (Sat0 et al., 1997).Thus, in terms of the underlying signaling events, CD19 coupling to Vav and PI3-K (Weng et al., 1994) and, potentially, to Rho-

52


family GTPases, may represent an analogous costimulatory pathway to the one mediated by CD28 in T cells. Finally, CD28 (but not TCR) ligation leads to tyrosine phosphorylation of a 62-kDa adapter protein (Nunes et al., 1996), now known as ~ 6 (Carpino et al., 1997; Yamanashi and Baltimore, 1997). This PH domaincontaining protein forms a complex with p120 ras-GAP and p190 rho-GAP (Fig. 7). Tyrosine phosphorylation of ~ 6 is 2thought ~ to ~ modulate the cellular distribution of the associated GAPSand sequester them away from their activated Rac and Ras targets, thereby prolonging their activated to regulate state (Reif and Cantrell, 1998). Thus, ~ 6 has2 the~potential ~ both Ras and Rho GTPases. An additional possibility is that via its coupling to CD28 (but also to TCR), PI3-K could couple immune receptors to Rad Rho signaling pathways. This notion is based on the finding that PI3-K products can stimulate Rac- and Rho-mediated cytoskeletal responses in fibroblasts (Reif et al., 1996). V. The Function of Rab GTPases in Leukocytes

The Rab family of small GTPases includes more than 40 mammalian members that localize in a highly restricted manner in distinct membrane compartments and regulate different vesicular transport steps along the endocytic and secretory pathways. Cycles of GTP binding and hydrolysis by Rab proteins are linked to the recruitment of specific effector molecules on cellular membranes (Nuoffer and Balch, 1994; Olkkonen and Stenmark, 1997). Because hematopoietic cell activation triggers production and secretion of different soluble mediators (cytokines, chemokines, antibodies, cytotoxic granules, inflammatory mediators, etc.), and leukocytes undergo phagocytosis, it is reasonable to expect that Rab proteins regulate some of these processes. In addition, the functional dichotomy between MHC class I and class I1 molecules is reflected by the distinct intracellular processing pathways of MHC molecules and antigens in APCs. Therefore, Rab proteins, by controlling distinct transport steps along these routes, are likely to play important roles in antigen processing. Because different Rab proteins are associated with specific subcellular compartments, they could potentially serve as ideal markers to identif) distinct compartments associated with antigen processing (Chavrier et al., 1993). However, this potentially productive area has not been addressed. Levels of several Rab proteins were shown to increase during the differentiation of precursor cell lines into macrophage- or neutrophil-like cells (Bokoch, 1995). A few studies have addressed the function of Rab proteins in exocytic membrane fusion events leading to mast cell degranulation. Intracellular perfusion of mast cells with a nonhydrolyzable GTP analog, GTPyS (which functions as a nonspecific activator of trimeric and small

2

~

~


53

GTPases), is sufficient to trigger complete mast cell degranulation. This process could be mimicked by synthetic peptides corresponding to the effector domain of Rab3 but not other Rab proteins (Law et al., 1993; Oberhauser et al., 1992).A more direct approach for evaluating the role of Rab proteins in FcERI-stimulated mast cells was undertaken by transfecting RBL cells with wild-type or mutant forms of Rab3 proteins (Roa et al., 1997; Smith et aE., 1997). One of these studies led to the conclusion that active Rab3a functions as a negative regulator of degranulation by inhibiting an early stage of granule targeting to the membrane, but it does not regulate granule fusion with the plasma membrane (Smith et al., 1997). However, another study reported that RabSd, but not RabSa, negatively regulates mast cell exocytosis (Roaet al., 1997).Taken together, these studies indicate that Rab3 proteins selectively regulate mast cell secretion. One group reported that peripheral blood mononuclear cells from patients with SBzary syndrome and other lymphoid and myeloid malignancies overexpress Rab2 at the mRNA and protein level (Culine et al., 1992). This overexpression was restricted to nonmalignant CD2+ peripheral lymphocytes (Culine et al., 1993) and its significance is unclear. Another member of the Rab family, Rab5, which has been implicated in the regulation of early steps in the endocytic pathway, appears to play a role in the down-regulation of the TCWCD3 complex induced by TCR ligation (Andre et al., 1997). This modulation, which is mediated by receptor endocytosis, probably serves to attenuate T cell activation by depleting antigen-binding receptors from the cell surface, thereby regulating the magnitude and/or duration of the activation response. Analysis of transgenic mice expressing a DN form of Rab5 in their T cells revealed that mature thymocytes developed normally, but the absolute number of CD4+CD8+ double-positive thymocytes was reduced. Fluid-phase endocytosis was severely impaired in the transgenic thymocytes. In peripheral T cells, the kinetics and rate of ligand-induced TCR down-modulation were delayed and reduced. These effects were correlated with enhanced early and late signaling responses (Andre et al., 1997). These findings suggest that TCR endocytosis is an important regulatory component of TCR signahng and that defects in this regulation can result in prolonged signaling and altered thymic development. The effects of the Rab5 transgene lend potential physiologic relevance to the finding that PMA stimulation of peripheral blood neutrophils induced translocation of Rab5a from the cytosol to the membrane (Vita et al., 1996). W. Small GTPases and Aberrant Leukocyb Functions

A. RAs SIGNALING AND T CELLANERGY Clonal anergy is a well-established mechanism for the maintenance of peripheral T or B cell tolerance, which plays a key role in preventing

54


reactivity to self antigens in the immune system (Goodnow, 1997; Schwartz, 1997). T cell anergy is usually induced when antigen receptor stimulation occurs in the absence of a costimulatory signal. The hallmark of anergic T cells is their inability to produce IL-2 or to proliferate in response to antigen stimulation. Ample evidence indicates that clonal anergy does not simply reflect a global failure of antigen receptor signal transduction but, rather, selective and aberrant triggering of a s’ubset of the signaling pathways that are normally induced by functional antigen receptor agonists (Alberola-Ilaet al., 1997), as reflected, for example, by the ability of anergic T cells to activate NFAT-1 and generate a normal increase in their intracelMar calcium concentration following antigenic stimulation (W. Li et al., 1996; Mondino et al., 1996; Schwartz, 1997). The obvious clinical implications of antigen-specific anergy have generated considerable interest in understanding the molecular basis of the aberrant signaling responses associated with this phenomenon. Several studies have implicated Ras (and, hence, its downstream elements) as a key target for the induction of T cell anergy (Fig. 1). The first clue that a defect in Ras activation may underlie T cell anergy came from a study in which it was demonstrated that activation of an IL2 promoter-reporter gene construct and, more specifically, activation of an AP-1 reporter and its DNA-binding activity were severely reduced in anergic T cells (Kang et al., 1992). A similar defect has since been documented by others (Mondino et al., 1996; Sundstedt et al., 1996). The defective AP-1 activation in anergic T cells results from the reduced nuclear expression of several inducible members of the Fos and Jun families of transcription factors that together combine to form AP-1 heterodimers (Foletta et al., 1998), i.e., c-Fos, FosB, and JunB (Mondino et al., 1996). The anergic state was not associated, however, with a global impairment of IL-2 gene induction based on the fact that other elements in the IL-2 gene promoter, i.e., NFAT-, NF-KB-, or Oct-binding sites, were not affected (Kang et al., 1992). Other studies reported, however, either a reduction in the transcriptional activation of reporter constructs containing multimerized NFAT-binding sites (Mondino et al., 1996) or a decrease in Fos/ Jun-containing NFAT complexes (Sundstedt et al., 1996). This reflects the fact that the AP-1 complex associates with NFAT and cooperatively binds to the NFAT site in the IL-2 promoter (Rao et al., 1997; Foletta et al., 1998), thereby stabilizing DNA association and transcriptional activation. Indeed, when NFAT activity is measured by more direct assays, i.e., dephosphorylation of NFAT and its nuclear translocation, it remains unaffected in anergic T cells (W. Li et al., 1996; Mondino et al., 1996),consistent with the intact TCR-induced Ca2+response in these cells (Mondino et al., 1996; Schwartz, 1997).


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Inasmuch as AP-1 activation in T cells depends on functional Ras, reflecting primarily the Ras-dependent phosphorylation of c-Jun and the transcriptional upregulation of the c-jun gene by JNK (Su et al., 1994; Su and Karin, 1996), the AP-1 defect in anergic T cells suggested that Ras activity may have been affected. This notion was directly confirmed by demonstrating that stimulation of anergic T cells with antigen-pulsed APCs failed to activate Ras as measured by the increased level of GTP-bound Ras (Fields et al., 1996a,b). In accordance with this finding, the activation of ERK1/2, which is known to require functional Ras (Izquierdo et al., 1993), was also suppressed in the anergic cells (Fields et al., 1996a,b). Deficient ERK activation was confirmed by other studies that additionally documented defective activation of JNK (which depends on a combination of Ras and calcineurin-dependent signals for optimal activation in T cells) (Su et al., 1994; Su and Karin, 1996), as well as the p38 stress-activated protein kinase, in anergic T cells (DeSilva et al., 1996, 1997; W. Li et al., 1996). Although the activation of p38 in T cells has not been formally shown to require Ras, the ability of PMA alone to activate p38 in T cells (DeSilva et al., 1997) suggests that this may be the case. The defect in Ras activation did not reflect an intrinsic inability of Ras to become activated because Ras activation was restored in anergic T cells treated with a combination of PMA and calcium ionophore (Fields et al., 1996a,b). This observation is most likely a reflection of the ability of PMA to bypass TCR-mediated signals and activate Ras via a PKC-dependent pathway in T cells (Downward et al., 1990, 1992; Izquierdo et al., 1992a). It is also in agreement with the findings that PMA partially or completely restores antigen-induced proliferation and IL-2 production in anergic T cells (Bhandoola et al., 1993; Fields et al., 1996a,b; W. Li d al., 1996). However, the restoration of IL-2 production or even ERK activation in anergic T cells by PMA was not observed in other studies (DeSilva et al., 1996; Schwartz, 1997), perhaps indicating that, in addition to Ras, other signaling pathways are also affected during T cell anergy. This is also implied by the findings that a defect in Ca2+mobilization occurs in at least some in vivo models of T cell anergy (e.g., Blackman et al., 1991). Some of the observed discrepancies may reflect differences in the protocols used to induce anergy, e.g., induction by antigen-pulsed fixed APCs, polyclonal mitogens, immobilized anti-CD3 antibodies, or superantigen using either in vitro or in vivo systems. Perhaps more important, however, is the possibility that these differences reflect the ability of distinct biochemical pathways to induce anergy. Whether defective Ras activation is common to all pathways leading to T cell anergy remains to be determined. Defects in the Ras signaling pathway, reflected by reduced levels of GTP-bound Ras and tyrosine-phosphorylated (and, thus, active) ERK, as

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well as defective PKC activity, were also reported in another model of T cell anergy, i.e., in nonobese diabetic (NOD) mice (Rapoport et al., 1993b). The mature thymic or peripheral T cells of these mice are hyporesponsive to TCR-initiated activation signals, as measured by proliferation and IL2 or IL-4 production, and the development of hyporesponsiveness correlates with the onset of insulitis. This fact, as well as the finding that hyporesponsiveness and diabetic disease can be reversed in parallel by IL4 both in vitro and in vivo (Rapoport et al., 1993a), suggests a causal relationship between T cell hyporesponsiveness and autoimmune disease development (Rapoport et al., 1993a). The relationship of this anergic model to more commonly studied models of T cell anergy (Schwartz, 1997) is not clear, particularly because IL-4 (rather than IL-2) is effective in reversing NOD T cell hyporesponsiveness (Rapoport et al., 1993a). Nevertheless, the similarity between this model and others in terms of inhibited Ras and ERK activation implies a defect in Ras activation as a common underlying mechanism in different types of T cell anergy. Interestingly, a similar T cell hyporesponsiveness has been documented in human type I diabetes (De Maria et al., 1994), where it is associated with defective PKC activation and CD6Qexpression. As in most other anergy models, however, the anergic human T cells display a normal increase in their intracellular Ca2’ concentration following anti-CD3 stimulation. Because PKC can lead to Ras activation in T cells (Downward et al., 1990, 1992; Izquierdo et al., 1992a), and CD69 expression in T cells has been found to depend on functional Ras (D’Ambrosioet al., 1994),the defect in CD69 up-regulation suggests that here, too, Ras activation is adversely affected. Ras is clearly a critical target in the induction of T cell anergy, but much less is known regarding the molecular basis for the defect in Ras activation. As discussed earlier, Ras activation in response to antigen receptor ligation is mediated by PTK-dependent or a PKC-dependent (but PTKindependent) pathways (Downward et al., 1990, 1992; Izquierdo et al., 1992a; Hanvood and Cambier, 1993).Thus, each of the intervening events that couple receptor ligation to Ras activation is potentially a candidate target for anergy induction. Several studies addressed the contribution of upstream elements in the Ras activation cascade to the anergic state. The tyrosine phosphorylation of Shc, as well as the expression of a phosphoShc/Grb2/Soscomplex, which represents a major mechanism for Ras activation by growth factor receptors (Rozakis Adcock et al., 1992; Skolnik et al., 1993a,b; Pronk et al., 1994; de Vries Smits et al., 1995), were found to be intact in anergic T cells (Fields et al., 1996a,b). However, this study did not determine whether this complex (or a LAT/GrbYSos complex) is properly recruited to the T cell plasma membrane, a step necessary for Sos-mediated Ras activation (Aronheim et al., 1994; Quilliam et al., 1994;


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Holsinger et al., 1995). Indeed, it has recently been reported that in the NOD mouse anergy model, TCR stimulation leads to deficient membrane translocation of Sos, and Sos (as well as PLCyl) was excluded from the phospho-4'-associated (and, hence, membrane-localized) G r b m T E a p 70 signaling complex (Salojin et al., 1997), implying that this complex is incompetent to activate Ras. Although one TCR-induced Ras activation pathway is mediated by PTKs (Izquierdo Pastor et al., 1995), it is unlikely that a global defect in the activation of TCR-coupled PTKs underlies the deficient Ras activation in anergic T cells, because, as mentioned earlier, the TCR-induced Ca2+ response, which also depends on TCR-coupled PTKs (June et nl., 1990; Mustelin et al., 1990),is usually intact in these cells (Mondino et al., 1996; Schwartz, 1997). Nevertheless, several studies reported abnormal tyrosine phosphorylation profiles in anergic T cells, including the lack of (Migita et al., 1995), or differential (Boussiotis et al., 1996), tyrosine phosphorylation of the TCR-associated f chain, deficient tyrosine phosphorylation of 38- and 74-kDa proteins (Bhandoola et al., 1993; Cho et al., 1993), and hyperphosphorylation of Fyn and/or concomitant increase in its catalyhc activity (Gajewski et al., 1995; Boussiotis et al., 1996; Salojin et al., 1997). In parallel, a reduction in the expression of Lck and, conversely, an increase in the level of Fyn (Quill et al., 1992), deficient recruitment of Zap-70 to 4' [which would be expected if f tyrosine phosphorylation was deficient (Migita et al., 1995; Salojin et al., 1997)], and decreased levels of Lck associated with the CD3/@ap-70 complex (Boussiotis et al., 1996) were also observed in anergic T cells. Consistent with the finding that, in the absence of CD28-mediated costimulation, TCR engagement alone leads to anergy (Schneider et al., 1995a; Schwartz, 1997),CD28 coimmunoprecipitated with the phospho-&/Lck/Zap-70complex in productively activated, but not in anergized, T cells (Boussiotiset al., 1996).Taken together, these findings suggest the following scenario: in anergic T cells, TCR engagement in the absence of CD28 costimulation leads to deficient Lck activation and hyperstimulation of Fyn. Consequently, the CD3 subunits and 4'are hypophosphorylated or abnormally phosphorylated in a manner that makes them incompetent to recruit Zap-70 via its tandem SH2 domains and activate it, a situation perhaps akin to the aberrant phosphorylation in T cells stimulated by altered peptide ligands (Sloan-Lancaster et al., 1994; Madrenas et al., 1995). As a result, Zap-70 and/or Lck cannot phosphorylate and recruit to the membrane adapters (or enzymes) that are critical for the activation of the Ras signaling pathway, such as Shc, LAT, Grb2, or Sos (Fig. 1).The deficient membrane translocation and Grb2 association of the Ras activator Sos (Salojinet al., 1997) are consistent with this scenario.

80%) of memory T cells are /31h'~h//37-/crE'. These memory T cells express high levels of a 4 p l and are particularly efficient at migrating into nonmucosal sites of inflammation through adhesion to endothelial VCAM-1. The remaining and a minor subset of these memory T cells also express memory T cells are /31'"w//37'"~'1, the a E subunit. High levels of /37 integrin expression allow for efficient trafficking of these memory T cells to gut-associated lymphoid tissue. The circulating aE+memory T cell subset may be involved in trafficking specifically to the intraepithelial compartment in the gut, although this has not been definitively established. Abbreviations: int, intermediate.

et al., 1998). These cells must exit the bloodstream at a wide variety of sites. The use of multiple exit sites may be facilitated by the marked heterogeneity in adhesion receptor phenotypes in these cells. Memory T cells (Fig. 4), in general, express higher levels of Pl and 02 integrins (Sanders et al., 1988; Shimizu et al., 199Ob).There is marked heterogeneity in expression of a4P7 (Schweighoffer et al., 1993; Rott et al., 1996; Abitorabi et al., 1996; Mackay et al., 1996) and these differences correlate with differences in adhesion to MAdCAM-1 (Rott et al., 1996). A small subpopulation of a407'& cells express aEP7 (Rott et al., 1996). As with naive T cells, these phenotypic characteristics are best described in the CD4 T cell lineage; limited studies of CD8' memory T cells show similar patterns, although a4P7 integrin expression is much more heterogeneous in CD8'CD45RAt naive T cells than in CD4+CD45RAf naive T cells (Rott et al., 1996).These differences in integrin expression between CD4+

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and CD8+ T cells likely reflect the different recirculation properties of these two major subsets of T cells, properties that are related to the different functions of CD4+ and CD8’ T cells (Kedl and Mescher, 1997), although definitive proof of this hypothesis is currently lacking. The gut-associated lymphoid tissue (GALT) consists of secondary lymphoid organs, such as Peyer’s patches, and effector sites, which consists of lamina propria and the intraepithelial lymphocyte compartment (Kraehenbuhl and Neutra, 1992). Homing of lymphocytes into the GALT is dependent on lymphocyte expression of the a4P7 integrin (Holzmann et al., 1989; Holzmann and Weissman, 1989).a4P7‘gh memory T cells and a4P7+ naive T cells directly extravasate into Peyer’s patches (Williams and Butcher, 1997). In contrast, a4P7-negative memory T ceIls do not home to Peyer’s patches (Williams and Butcher, 1997). Thus, there is a direct relationship between a4P7 expression and migration to Peyer’s patches in viva (Williams and Butcher, 1997). The adhesion of lymphocytes to Peyer’s patch HEVs involves L-selectin, a4P7, and aLP2 in a sequential fashion. However, a4P7 participates in both rolling and stable arrest (Bargatze et al., 1995). Like L-selectin, a4P7 is concentrated on the microvilli of lymphocytes, which mediate the initial contact with HEVs (Berlin et al., 1995). Furthermore, the activation state of the lymphocyte determines the relative role of a4P7 in mediating lymphocyte interactions with Peyer’s patch HEVs. Activated cells expressing high levels of a4P7 arrest on Peyer’s patch HEVs in an L-selectinindependent, a4P7-dependent, manner (Bargatze et al., 1995).Attachment of lymphocytes to lamina propria venules in viva is also dependent on a4/37, but independent of L-selectin (Berlin et al., 1995). Just as L-selectin-deficient mice exhibit dramatically decreased peripheral lymph nodes due to loss of influx of lymphocytes, p7 integrin-deficient mice exhibit a reduction in size and cellularity of Peyer’s patches (Wagner et al., 1996). The P7-deficient lymphocytes are unable to bind to Peyer’s patch HEVs in vitro and are unable to migrate into Peyer’s patches in viva Furthermore, loss of P7 profoundly reduced arrest and firm adhesion to Peyer’s patch HEVs (Wagner et al., 1996). Lymphocyte homing to Peyer’s patches is also defective in a4-negative chimeric mice (Arroyo et al., 1996), further supporting the preeminent role of a4P7 in mediating homing of lymphocytes to Peyer’s patches. C. THEaEP7 INTEGRINAND MUCOSAL IMMUNITY Intraepithelial lymphocytes ( IELs) comprise a unique T cell population that resides in the intestinal epithelium. Most of these T cells express the CD8 coreceptor, and can be subdivided based on expression of either an aP T cell receptor (TCR) or a y6 TCR (Goodman and Lefrancois, 1989).

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IEL development differs from other T cell development in that T cell receptor rearrangement in IELs occurs outside of the thymus (Lefrancois and Olson, 1994; Lin et nl., 1993). These cells also differ in having a reduced TCR repertoire (Sim, 1995),constitutive lyhc activity (Lefrancois and Goodman, 1989), and they localize preferentially to spaces between intestinal epithelial cells. aEP7 integrin is expressed at high levels on all IELs and dendritic cells found in the intestinal epithelium, as well as on many T cells in the lamina propria (Cerf-Bensussan et al., 1992; Kilshaw and Murant, 1990; Kilshaw, 1993). This is particular1 striking, given that only a very small subpopulation of peripheral a4/37'gh, Pl'c'wmemory T cells express aEP7 (Rott et al., 1996).Transforming growth factor P induces a E integrin expression. Because this cytokine is expressed in the small intestinal epithelium (Barnard et al., 1993), it may regulate aEP7 expression. E-Cadherin, the counterreceptor for aEP7, is expressed on intestinal epithelial cells (Roberts and Kilshaw, 1993; Cepek et al., 1993, 1994; Karecla et al., 1995; Higgins et al., 1998). Divalent cations and cellular activation (Higgins et al., 1998) can regulate aEP7-mediated adhesion to E-cadherin. aEP7 binding to E-cadherin on intestinal epithelial cells may retain IELs in the intestinal epithelium. A reduction in the number of IELs in mice deficient for the a E subunit (Higgins et al., 1998) is consistent with this model. However, others have noted an increase in p7-positive IELs in mice expressing a dominant negative form of N-cadherin, which disrupts E-cadherin expression in the intestine (Hermiston and Gordon, 1995). Furthermore, there is an influx of aEP7-negative CD8+ T cells into the intestinal epithelium in graft-versus-host disease (Kilshaw and Baker, 1988). The aEP7-mediated adhesion of IELs to E-cadherin on intestinal epithelial cells may also regulate IEL effector function (Kilshaw and Karecla, 1997), because aEP7 can provide signals that enhance TCRmediated activation of T cells (Sarnacld et al., 1992; Begue et al., 1995). Furthermore, the in vitro cytotoxic activity of some, but not all, y6+ tumorinfiltrating lymphocytes isolated from patients with colorectal cancer is partially inhibited by an anti-P7 antibody (Maeurer et al., 1996). P2 integrins are involved in IEL function. Mice deficient in expression of either P2 or ICAM-1 have defects in the activation and expansion of some lamina propria T cells and IELs (Huleatt and Lefrancois, 1996). Deficiency of 0 2 integrin or ICAM-1 results in almost complete loss of a subset of ap TCR+ T cells in both intestinal epithelium and lamina propria and in a dramatic reduction of Thy-l+ y6 TCR-positive IELs. Because these Thy-l+T cells exhibit constitutive lytic activity that is dependent on normal microbial flora, loss of P2 integrin or ICAM-1 expression may disrupt p2 integrin-dependent signals required for the development of

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Thy-l+ IELs. The inability of bone marrow from ICAM-1-deficient mice to restore the generation of some ap TCR IELs in radiation reconstitution experiments is consistent with this model (Huleatt and Lefrancois, 1996). An overall decrease in lymphocytes in Peyer’s patches was also noted in PZ-nuIl and ICAM-1-null mutant mice, but this phenotype may be due to diminished homing. In addition, antigen-induced stimulation of IELs in vivo leads to expression of the a m 2 integrin that may enhance IEL recognition of target cells during cytolyhc responses (Huleatt and Lefrancois, 1995). Other differences in integrin expression between ap TCR T cells and 76 TCR T cells have also been noted. High expression of a461 and a5pl on Val+ T cells correlates with increased adhesiveness to fibronectin (Nakajima et al., 1995). The increased adhesion may contribute to the accumulation and retention of V a l + T cells in intestinal epithelium (Halstensen et al., 1989).

D. DENDRITIC CELLS Dendritic cells capture antigen in the periphery and migrate to lymphoid organs, where they present the antigen to T and B cells (Banchereau and Steinman, 1998). Blood dendritic cells express pl and p2 integrins, and anti-fi2 and a n t i 4 antibodies inhibit the adhesion of blood dendritic cells to human umbilical vein endothelial cells (Brown et al., 1997). In addition, differential expression of the aXp2 integrin subdivides blood dendritic cells into two populations (O’Doherty et al., 1994) that may migrate to distinct areas of lymphoid organs (Banchereau and Steinman, 1998). The specific roles of integrins in dendritic cell migration into and within lymphoid organs, along with the factors that regulate the activity of integrins on dendritic cells, are important areas for future investigation. VII. The Role of Integrins in Immune Responses and Inflammation: Two Case Studies

As the foregoing discussion makes clear, integrins are critically involved in virtually all aspects of the development and patterning of the immune system. Consequently, manipulation of these receptors is a potential strategy for immunomodulation. A detailed review of all inflammatory diseases and immune reactions and the various integrins involved is beyond our scope. Consequently, we will discuss two representative “case studies”: the role of aLp2 in selected immune responses and the role of integrins on one autoimmune inflammatory disease, experimental autoimmune encephalomyelitis (EAE).

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A. aLp2 AND IMMUNE RESPONSES As described in the foregoing sections, aLp2 plays a pivotal role in lymphocyte trafficking. Moreover, its signaling functions can cooperate with specific immunologic receptors in many contexts (Mobley et al., 1993). Consequently, defects in immune responses of aLp2-deficient individuals are readily understandable. These include defects in rejection of allografts (Le Deist et al., 1989) and tumors (Schmits et al., 1996; Shier et nl., 1996). Antibody inhibition experiments confirm the role of this integrin in transplant rejection (Isobe et al., 1992). Furthermore, in vitro studies show that lymphocytes from aL-null mice do not aggregate and exhibit defects in proliferation in mixed lymphocyte reactions (MLRs) and in response to concanavalin A (Schrnits et al., 1996; Shier et al., 1996). Mice with null CD18 expression exhibit similar defects in T cell allogeneic responses (Scharffetter-Kochanek et al., 1998). In addition, inhibitory anti-aL antibodies (Odum et al., 1988; Kuijpers et al., 1990) or absence of ICAM-1 on antigen-presenting cells blocks MLRs (Sligh, et al., 1993). Thus, there is strong evidence that aLp2 plays an essential role in response to allografts. T cells from CD18-null mice also fail to proliferate in response to staphylococcal enterotoxin A (Scharffetter-Kochanek et al., 1998), consistent with earlier antibody blocking studies implicating aLp2 in superantigen-dependent T cell activation (Damle et al., 199313; Nickoloff et al., 1993; van Seventer et al., 1991a). In contrast, there are discrepant reports on the effects of a L gene ablation on the in vitru cytotoxic activity of natural killer cells toward YAC-1 target cells (Schmits et al., 1996; Shier et al., 1996). Responses of aL-deficient mice in delayed-type hypersensitivity (DTH) vary depending on the nature of the sensitizing agent. The response to dinitrofluorobenzene (DNFB) is reduced in aL-negative mice (Schmits et al., 1996) or in response to anti-aL antibodies (Kondo et al., 1994). In contrast, the response to sheep red blood cells (SRBCs) is comparable to aL-positive mice (Shier et al., 1996). Antigen-specific variance in T cell priming might explain these differences (Schmits et al., 1996; Shier et al., 1996). Some humans deficient in j32 manifest normal DTH responses in vivu and are not susceptible to severe viral infections (Anderson et al., 1985). In contrast, a patient with a selective defect in “activation” of aLp2 manifests reduced DTH responses due to an inability of antigen-specific T cells to infiltrate the skin (Kuijpers et nl., 1997).This individual suggests that activation of p 2 plays a role in human DTH. Possibly, the functional defect in aLp2 evokes less compensation than its complete loss, accounting for the apparent discrepancy between the quantitative and qualitative deficits in aLp2 function. In sum, the contribution of aLp2 to DTH is

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likely to be dependent on the type of antigen-presenting cell, the quantity of activating antigen, and the nature of the microenvironment in which the activation occurs. Early antibody inhibition studies implicated aLP2 in the killing of target cells by cytotoxic T lymphocytes (CTLs) (Davignonet al., 1981a,b; SanchezMadrid et al., 1982; Shaw et al., 1986). Remarkably, aL-negative mice manifest normal CTL effector function in response to certain viruses (Schmits et al., 1996); similar findings were reported in ICAM-l-deficient mice and in mice with a partial loss of P2 integrin expression (Christensen et al., 1996). Furthermore, P2-deficient humans do not seem predisposed to viral infections (Anderson et al., 1985), consistent with normal in vivo CTL function. CTL priming to these viruses occurs in the spleen (Schmits et al., 1996) and aL-null mice manifest splenomegaly due to markedly increased splenic T cells (Schmits et al., 1996). Consequently, increased aL-negative T cells in the spleen may compensate for the lack of aLP2 in the CTL response.

B. INTEGRINS AND LYMPHOCYTE-MEDIATED INFLAMMATORY DISEASE Lymphocyte entry into inflammatory sites utilizes the four-step adhesion cascade (Carlos and Harlan, 1994). The roles of 0 2 and a 4 integrins have received the most attention because their ligands, ICAM-1, VCAM-1, and MAdCAM-1, are dramatically up-regulated on inflamed endothelium. Indeed, these integrins are candidate therapeutic targets for a wide range of chronic inflammatory diseases, including inflammatory bowel disease (Hesterberg et al., 1996), asthma (Das et al., 1995), diabetes (Hanninen et al., 1996), and rheumatoid arthritis (Postigo et al., 1992; Diaz-Gonzalez and Ginsberg, 1996; Diaz-Gonzalez and Sanchez-Madrid, 1998). Perhaps the greatest amount of data has been obtained in a model of multiple sclerosis (Swanborg, 1995), experimental autoimmune encephalomyelitis. Studies in EAE vividly illustrate the role of integrins in lymphocytemediated inflammation and will therefore be the focus of our discussion. EAE is elicited by a T cell response to myelin or to certain of its components (Swanborg, 1995),leading to extensive lymphocyte infiltration of the central nervous system and damage to myelin sheaths. Integrins play a central role in the pathogenesis of EAE. ICAM-1 and VCAM-1 expression are dramatically increased on central nervous system venules in both multiple sclerosis and EAE (Irani and Griffin, 1996; Washington et al., 1994; Sasseville et al., 1992; Barten and Ruddle, 1994; Engelhardt, 1998). Furthermore, there is a direct relationship between a 4 integrin expression and the ability of autoreactive T cell clones to induce EAE in mice (Sasseville et al., 1992; Baron et al., 1993). Anti-a4 or anti-VCAM1 antibodies delay the onset and decrease the severity of EAE (Yednock

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et al., 1992; Baron et al., 1993; Soilu-Hanninen et al., 1997; Keszthelyi et al., 1996), possibly by preventing the influx of autoreactive T cells into the central nervous system. Autoreactive T cells that cause EAE do not express a407 (Engelhardt, 1998; Engelhardt et al., 1995). Consequently, a 4 p l is likely to be the relevant integrin. In addition to mediating adhesion, the a4plNCAM-1 interaction facilitates responses necessary for extravasation into the brain. These include metalloproteinase expression (Romanic and Madri, 1994), which may degrade the basement membrane, facilitating transmigration of T cells (Leppert et al., 1995). aLp2 does not induce metalloproteinase expression, perhaps accounting for the inability of antip2 or anti-ICAM-1 antibodies to ameliorate EAE consistently (Baron et al., 1993; Welsh et al., 1993). The integrin phenotype of the T cells changes once they enter the brain (Romanic et al., 1997).After transmigration, T cells down-regulate integrins involved in endothelial binding (e.g., a401, aLp2) and up-regulate those involved in matrix adhesion (e.g., a5p1, a 2 p l ) with resulting increased adhesion to collagen and fibronectin. These changes suggest mechanisms by which autoreactive T cells might be retained in the inflammatory site by firm adhesion to the extracellular matrix components found in tissue. Thus, a 4 p l and its ligands play a key role in the pathogenesis of EAE and are candidate therapeutic targets for multiple sclerosis. VIII. Regulation of lntegrin Ligand Expression in Inflammation

As noted above, changes in expression of integrin ligands play a role in the inflammatory response. The regulation of VCAM-1 and ICAM-1 has been most extensively studied, and many stimuli can influence their expression. Proinflammatory agents such as TNF-a, IL-lp, and LPS induce VCAM-1 as well as ICAM-1 expression on endothelium (Springer, 1995; Bevilacqua, 1993; Schleimer et al., 1992). This up-regulation appears to involve both transcriptional and posttranscriptional mechanisms. The 5' regions of the ICAM-1 and VCAM-1 genes contain several potential transcriptional regulatory elements, some of which are common to both genes. The activation of NF-KBtranscription factor and its interaction with multiple KB motifs on the VCAM-1 and ICAM-1 gene promoters is critical for TNF-a and IL-lp-induced expression (lademarco et al., 1995; Degitz et al., 1991). Other transcription factors can also modify this induction. For example, AP-1 potentiates TNF-a-induced VCAM-1 expression (Ahmad et al., 1998). Interestingly, this does not appear to involve AP-1 interaction with its cognate enhancer, but instead involves a direct interaction with NF-KB.

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Posttranscriptional mechanisms for regulating ICAM-1 and VCAM-1 expression have also been proposed. At least two regulatory regions have been defined for ICAM-1. The 3‘ untranslated regions (UTR) of ICAM1 contains multiple AUUUA motifs (Staunton et al., 1988). This motif is partially responsible for the short half-life of ICAM-1 mRNA as well as other gene products (Ohh and Takei, 1996). Removal of the AUUUA motifs increases the half-life of ICAM-1 mRNA and prevents the stabilization of mRNA induced by phorbol myristate acetate (PMA) stimulation (Ohh and Takei, 1996). Interestingly, ICAM-2 and ICAM-3, which are constitutively expressed at high levels, do not contain 3’ UTR AUUUA motifs, and thus this may partially explain the constitutive high expression. Inclusion of the 3’ UTR of ICAM-1 on the ICAM-2 gene shortens the mRNA half-life (Ohh and Takei, 1994). A second regulatory element located in the region encoding the cytoplasmic domain of ICAM-1 has also been implicated in the regulation of mRNA stability (Ohh and Takei, 1996). This region is responsible for the increased mRNA stability induced by IFN-.)I stimulation. Similarly, VCAM-1 may also be regulated by posttranscriptional mechanisms. For example, IL-4, in combination with TNF-a, seems to be a selective inducer of VCAM-1 expression on endothelial cells. IL-4 stimulation alone is a weak inducer of VCAM-1 expression; however, it acts synergistically with TNF-a to increase VCAM-1 expression dramatically (Schleimer et al., 1992; Thornhill et al., 1991; lademarco et al., 1992). This synergistic effect of IL-4 appears to occur largely through an increase in VCAM-1 mRNA stability (Iademarco et al., 1992). Other conditions can down-regulate VCAM-1 and ICAM-1 expression. Laminar shear stress on the vessel wall suppresses endothelial VCAM-1 expression (Ohtsuka et al., 1993; Korenaga et al., 1997). This appears to involve two AP-1 cis elements acting as negative regulators of transcription (Korenaga et al., 1997). Paradoxically, shear stress stimulates ICAM-1 expression on vascular endothelium (Nagel et al., 1994). This appears to involve a shear stress response element located on the promoter of ICAM1 but not on the VCAM-1 gene. Agents that elevate intracellular CAMP and activate protein kinase A, such as prostaglandins, have also been implicated in the suppression of ICAM-1 and VCAM-1 expression in both endothelial and smooth muscle cells (Pober et al., 1993; Panettieri et al., 1995; Braun et al., 1997).This suppression is at the level of gene transcription, but the precise mechanism is unclear. An additional potential mechanism for the down-regulation of ICAM1 and VCAM-1 expression involves shedding from the cell surface and release of soluble fragments. Soluble forms of ICAM-1 and VCAM-1 have been detected in the culture supernatant of human endothelial cells and


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in human plasma (Pigott et al., 1992; Leca et al., 1995; Rothlein et al., 1991). It is speculated that the cleavage of surface VCAM-1 involves a zinc-dependent metalloprotease (Leca et al., 1995). This shedding may suppress further leukocyte migration into tissues by removing the ligand from the cell surface and providing a soluble competitive ligand inhibitor. Furthermore, soluble forms may interact with the ability of integrins to provide signals, which alters cell function. Indeed, in T cells, immobilized VCAM-1 provides a costimulatory signal, whereas binding of soluble VCAM-1 initiates an inhibitory signal (Kitani et al., 1996). In sum, integrins and their ligands play indispensable roles in the development and functioning of the immune system. They do so primarily by mediating the cellular traffic instrumental to the immune response. Furthermore, integrins play important roles in modulating signals generated by clonotypic receptors and as effectors in processes such as cytolytic killing and phagocytosis. Many of the mechanisms that govern the functioning of integrins throughout the animal kingdom have been adapted to the specialized needs of the immune system. Furthermore, these receptors are readily accessible targets for small-molecule and antibody inhibitors. Because many integrin functions are surprisingly nonredundant (Hynes and Wagner, 1996), modification of integrin function is a promising avenue for therapeutic control of certain immune responses.

ACKNOWLEDGMENTS Work in our laboratories was funded by grants from the National Institutes of Health. DMR was supported by funds from the California Breast Cancer Research Program of the University of California, Grant No. 3FB-0164.

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INDEX

A Agammaglobulinemia,X-linked, 159-160 Allergies, 293 aEP7, 346-348 a4p7, 220-223 inflammation blocldng, 242-243 lymph node development, 343 lymphocyte homing, 220-222, 233-234 aLp2, 349-350 Antigens capture, 262 CD1,257-258 CLA, 265 intestinal, 237-240 presenting cells, 264 processing, 264-265 AP-1, 23 Apoptosis dendritic cells, 263-264 GTPases role, 47-49 Asthma, 293-294 Ataxia-telangiectasia gene, 180-181 lymphoid defects characterization, 179 dissecting, 181-185 tumorigenesis characterization, 179 dissecting, 181- 185 ATM, 180-181

B Bacteria, 294 B cells -dendritic cell interaction

B cell differentiation, 289-291 B cell proliferation, 289 dialogues, 288-291 follicular DC, 286-287 general, 285 germinal center DC, 287-288 development, 339-340 memory, 237-240 receptors, ITIM inhibition, 155-156 p7, 225-227 Blood mononuclear cells, 277 Btk, 160

C E-Cadherin, 333-334 Calcium BCR-triggered, 156-157 Btk effects, 160 Ca", dendritic cell, 273-274 ITAM effects, 150 SHIP effects, 159 Cathepsin D, 272 Cbl, 12-14 CD43, 226-227 CD44, 226-227 CD1 antigens, 257-258 Cdc-42, 33-34 CD40/CD40L, 269-270 Cdc42sp, 36-37 Cell migration dendritic, 265-268 diapedesis, 338 C3 exotoxin, 33-34 Chemolo'nes, lymphocyte trafficking, 227-228 Chlamydia truchomutis, 214 Colitis, ulcerative, 213 381

382

INDEX

Crohn's disease, 213 C-type lectin receptor, 263 Cutaneous lymphocyte antigen, 265 Cytomegalovirus, 296-297 Cytoskeleton reorganization, 27-34

D Delayed-type hypersensitivity, 349-350 Dendritic cells adhesion molecules, 265 antigen capture, 262 antigen presentation, 264 apoptosis, 263-264 -B cell interaction B cell differentiation, 289-291 B cell proliferation, 289 dialogues, 288 fokcular DC, 286-287 general, 285 germinal center DC, 287-288 Ca", 273-274 clinical studies allergies, 293 asthma, 293-294 bacteria, 294 parasites, 294-295 psoriasis, 291 retroviruses, 298-300 rheumatoid arthritis, 291 transplantation, 291-293 tumors general, 300-302 human, 303-305 mouse, 302-303 viruses, 295-298 cytomegdovirus, 296-297 herpesvirus, 296 influenza, 297 measles, 297-298 costimulatory molecules, 268-269 C-type lectin receptors, 263 development, 262 lymphoid pathway, 277-278 stages, 259-260 theories, current, 278-279 enzymes, 272-273 FCEreceptors, 262 Fcy receptors, 262

FLT-3 ligmd, 278 generation blood mononuclear cells, 277 hematopoietic progenitor cells, 274-277 murine cell lines, 274 humoral responses in uitm, 289 in uiuo, 288-289 identification, 255 immune response, 255 macropinocytosis, 262 mannose receptor, 263 maturation IL-10 inhibition, 280-281 stimulators, 279-280 MHC class I loading, 264-265 migration control, 266-268 patterns, 265-266 morphology, 257 NK phenotype, 273 physiology, 255-257 signaling CD40/CD40L, 269-271 FadFasL, 272 OX4O/OX401, 271 RANK-UTRANCE/ODF, 271-272 RANKA'RANCE-Wosteoprotein, 271-272 TNFmNF-R, 269 -T cell interaction cytokine role, 282 in uiuo association, 281-282 T cell priming, 282-283 tolerance central, 283-284 peripheral, 284 Dinitrofluorobenzene, 282-283 DNFB, see Dinitrofluorobenzene

E ECM proteins, see Extracellular matrix proteins Encephalomyelitis, experimental autoimmune, 348-353 Endothelial cells HEV, levels, 213-214 -lymphocyte interaction p7 role, 225-227

383

INDEX

characterization, 211 IEL role, 225-227 LFA-1 role, 225 E-selection role, 225-227 L-selection role, 224-225 P-selection role, 225-227 sialomucin role, 226-227 ERKs, Extracellular-regulated kinases Exotoxin, C3, 33-34 Experimental autoimmune encephalomyelitis, 348-353 Extracellular matrix proteins, 27-28 Extracellular-regulated kinases IRR activation, 3 NFAT activation, 34-35 T cell activation, 16-17

F Fas, 47-49 Fas/FasL, 272 FCEreceptors, 262 Fcy receptors characterization, 154-158 dendritic cells, 262 Fetal liver kinase 2, 278-279 FLT-3, 278 Focal adhesion kinase, 66-68 Fyn, 117-119

G GAPS,see GTPase-activation proteins

Gastrointestinal tract inflammation a4P7 blocking, 234-237 lymphocyte recruitment role, 242-243 Grb2, 37-38 Grb2iSo.s Ra9 activation, 8-11 T cell activation, 11-12 GTPase-activation proteins function, 2 Ras activation, 14-15 GTPases Fas apoptosis, 47-49 HIV infection, 59-63 Rab family, 52-53 Ras family CD28 signaling, 49-51

cytokine receptor signaling, 25 function, 1-2 IRR activation, 3-5 Cbl role, 12-14 Grb2/Sos role, 8-11 PTK role, 5-8 SLP-76 role, 12-14 Vav role, 12-14 IRR signaling effectors, 15-18 negative factors, 23-25 transcription factors, 18-23 lymphocyte development, 64-66 regulation, 1-2 T cell anergy, 53-59 regulation, 68-69 Rho family CD28 signaling, 51-52 cell adhesion, 41-43 chemoattractant responses, 44-46 hematopoietic cells regulation, 38-39 Vav integration, 34-35 integrin adhesion, 41-43 IRR signaling activation, 39-41 characterization, 25-26 leukocytes cytoskeleton, 27-34 lymphocyte development, 66-68 p38 pathway, 68 SEKl pathway, 68 selections, 43-44 signal control, 34-35 Wiskott-Aldrich syndrome, 63-64

H Hematopoietic cells Ras signals, 34-35 Rho signals regulation, 38-39 Vav integration, 34-35 Hematopoietic progenitor cells, 274-277 Hematopoietic stem cells, 338-339 Herpes simplex virus, 295-296 Human immunodeficiency virus dendritic cells, 298-300 G protein role, 59-63 Hypersensitivity, delayed-type, 349-350

384

INDEX

I ICAMs, see Intercellular adhesion molecules Immuglobulins IgA, 290 IgM, 289-290 Immune receptor tyrosine-based inhibitory motifs characterization, 149-150 families, 150, 152, 154 inhibitory signal, FcyRII-mediated, 154- 158 KIR receptor inhibition, 161-163 mouse models, 161-165 Immune recognition receptors Ras activation, 3-5 Cbl role, 12-14 Grb2/Sos role, 8-11 PTK role, 5-8 Shc role, 8-11 SLP-76 role, 12-14 Vav role, 12-14 signaling Ras effectors, 15-18 negative factors, 23-25 transcription factors, 18-23 Rho characterization, 25-26 effectors, 39-41 signal transduction, 2-3 Immune system integrin ligands function, 330-331,337-343 localization, 333-334 signaling, 335-337 structure, 331-333 Inflammation a4p7 blocking, 242-243 integrin-mediated, 350-353 lymphocyte-mediated clinical study, 350-351 recruitment role, 234-237 Inff uenza virus, 297 Inositol 5-polphosphate phosphatase SH2-containing FcyRII inhibition, 156 mechanism, 158-161 mouse models, 163-165 phosphoinositol hydrolyzation, 150 properties, 24-25

Integrin adhesion receptors ~~4p7,220-223 autoimmune blocking, 242-243 gastrointestinal tract inflammation, 242-243 inflammation blocking, 242-243 lymph node development, 343 lymphocyte homing, 220-222, 233-234 lymphoid malignancies, 241 vaccine development, 241-242 p7,225-227 LFA-1 cascading, 232-233 characterization, 225 signaling, 335-337 Integrins B cell development, 339-340 characterization, 325-326 aEP7, 346-348 hematopoietic organ seeding, 338-339 inflammatory disease, 350-351 aLp2, 349-350 ligand binding sites, 326-330 function, 330-331 inflammatory response, 351-353 locahzation, 333-334 mucosal immunity, 334-335 structure, 331-333 lymphocyte recirculation, 344-348 signaling, 335-337 trafficking control, 337-338 IntercelIular adhesion molecules dendritic cells, 265 inflammatory response, 351-353 integrin ligand domains, 333-334 Rho function, 33-34 Interleukin-2, 4-5, 289-290 Interleukin-10, DC, 280-281 Intestinal antigens, 237-240 Intraepithelial lymphocytes, 346-347 y-Irradiation, 181 IRRs, see Immune recognition receptors ITIMs, see Immune receptor tyrosine-based inhibitory motifs

J JNK cascade, 36-37

INDEX

-c-Jun, 21 IRR signaling, 39-41

Killer cell inhibitory receptors, 161-163

L LAT, see Linker activation of T cells Lck HIV, 61-62 pre-TCR sensor, 117-119 Lectin receptor, C-type, 263 Leishmania spp., 294-295 Leukocytes, see also specijic leukocyte cytoskeletons, 27-34 Rab function, 52-53 trafficking, 337-338 LFA-1 cascading, 232-233 characterization, 225 dependent adhesion, 33-34 signaling, 335-337 Ligands, integrin function, 330-331 inflammatory response, 351-353 localization, 333-334 mucosal immunity, 334-335 structure, 331-333 Linker activation of T cells, 12 Liver, fetal, 338-339 LPA, see Lysophosphatidic acid Lymphocytes, see also specijic lymphocyte blood-borne, recruitment chemotactic factors, 227-229 integrin-triggering, 227-229 required steps, 211-212 development Ras role, 64-66 Rho role, 66-68 -endothelium interaction P7 role, 225-227 characterization, 211 IEL role, 225-227 LFA-1 role, 225 E-selection, 225-227 L-selection role, 224-225 P-selection, 225-227 sidornucin role, 226-227

385

growth signals, 34-35 homing 014017 preactivation, 233-234 014p7 characterization, 220-223 segregation, 237-241 adhesion cascade naive effectors, 232-233 nonintestinal tissue, 234 MAdCAM-1, 213-220 memory effectors, 231-232 naive effectors, 231-232 T cells in uiuo, 229-230 inflammatory disease, 350-351 mixed reactions, 268 trafficking alterations, 234-237 characterization, 209 muscularis patterns, 237 serosa patterns, 237 studies, 210-211 theory, 210 Lyn/FceRI ratio, 121 Lysophosphatidic acid, 33

Macropinocytosis, 262 MAdCAm-1,see Mucosal vascular addressin Major histocompatibility complex deiidritic cell loading, 264-265 maturation, 279-280 Mannose receptor, 263 MAPKAP kinase-2, 39-41 Measles virus, 297-298 MEK-1, 118 MEKK-1, 21-22, 39-41 Memory cells, 237-241 Metal ion-dependent adhesion site, 329 MIDAS, see Metal ion-dependent adhesion site MIPBP, 228 MKK7,39-41 Mucosal vascular addressin autoimmune blocking, 242-243 aEP7 integrins, 346-348 inflammation blocking, 242-243 integrin ligands, 334-335

INDEX

lymph node development, 343 lymphocyte-endotheliuminteraction, 213-220,233-234 Mutations adaptive responses, 138-140 redundancy, 138-140

N NADPH oxidase chemoattractant responses response, 46-47 Rho signal regulation, 38-39 Natural killer cells CD4 role, 108 DC phenotype, 273 as lymphoid DCs, 257 Rho signal regulation, 38-39 NFAT, Nuclear factor of activated T cells NKR-P1, 273 Nuclear factor of activated T cells characterization, 5 growth signals, 34-35 Ras signaling, 19-20, 23

0 OX4O/OX4OL, 271

P P38 -JNK, 39-41 -SEKI, 68 Parasites, 294-295 Phagocytosis, 34 Phospholipase D, 47 Phosphotyrosine phophatases, SH2containing, 24 Precursor cells, 337-338 Pre-T cell receptors delic exclusion, 127-128 assembly, 116-117 CD3 components, 114-115 cell survival, 131-138 function, 131 aPyS lineage role, 124-127

sensor characterization, 119, 121-122 downstream effectors, 117-119 TCR-@locus role, 127-128 Programmed cell death, see Apoptosis

R Rab, 52-53 Rac-1, 23 Raf-1 kinase characterization, 16 function, 22 RAG-1, 118 RAG-2, 182 RANK-L/TRANCE/ODF, 271-272 Rapl, 5 Ras CD28 signaling, 49-51 cytokine receptor signaling, 25 function, 1-2 IRR activation, 3-5 Cbl role, 12-14 GrbZ/Sos role, 8-11 PTK role, 5-8 Shc role, 8-11 SLP-76 role, 12-14 IRR signaling effectors, 15-18 negative factors, 23-25 transcription factors, 18-23 lymphocyte development, 64-66 regulation, 1-2 T cell anergy, 53-59 Rel-B, 280 Retroviruses, 298-300 Rheumatoid arthritis, 291 Rho CD28 signaling, 51-52 cell adhesion, 41-43 chemoattractant responses, 44-46 cytoskeleton reorganization, 27-34 hematopoietic cells regulation, 38-39 Vuu integration, 34-35

INDEX

integrin adhesion, 41-43 IRR signaling activation, 39-41 characterization, 25-26 p38 pathway, 68 SEKl pathway, 68 selections, 43-44 RHO family, 34-35

Scdl, 36-37 SDF-1, 227-228 SEKl, 68 E-Selection, 225-227 L-Selection, 224-225, 232-233 P-Selection, 225-227 Serum response element, 22 Serum response factor, 22 SHIP, see Inositol 5-polyphosphate phosphatase Sialomucins, 226-227 Signal transduction cytokine receptors, 25 dendritic cells CD40/CD40L, 269-271 FadFasL, 272 OX4O/OX401,271 RANK-L/TRANCE/ODF, 271-272

RANK/TRANCE-Wosteoprotein, 271-272 TNF/TNF-R, 269 FqRII, 154-158 integrin, 335-337 IRR characterization, 2-3 Ras effectors, 15-18 Rho effectors, 39-41 transcription factors, 18-23 SLCIGCkine, 228 SLP-76, 12-14 SRE, see Serum response element SRF, see Serum response factor Stem cells, hematopoietic, 338-339 Syk, 118, 122

T T cell receptors, see a h Pre-T cell receptors CD3 contribution, 116-117

CD3P chain components, 113-114 CD3.9 chain components, 112-113 CD3y chain components, 114 cell suMval, 131-138 FceRIy components, 115-116 ap chain sensors, 128-131 p chain allelic exclusion, 127-128 components, 109, 111 y6 chain, 124-127 mutations, 138-140 pTa chain components, 111-112 maturation independent of, 122-124 T cells activation linker, 11-12 anergy Rapl-induced, 5 Ras induced, 53-59 CD28 signaling Ras role, 49-51 Rho role, 51-52 -dendritic cell interaction cytokine role, 282 T cell priming, 282-283 in uiuo association, 281-282 development CD3 function, 103, 105-106 integrin function, 340-343 mouse model, 106-109 ERK activation, 16-17 memory, recirculation, 344345 naive, recirculation, 344 Raf-1 activation, 16-17 shape regulation, 43-44 Thymocytes development, 341342 a-Tubulin, 31-32 Tumorigenesis, AT characterization, 179 dissecting, 181-185 Tumor necrosis factors dendritic cells generation, 269-277 maturation, 279-280 signahng, 269-272

387

388

INDEX

U Ulcerative colitis, 213

W Wiskott-Aldrich syndrome, 63-64

V Vascular cell adhesion molecules inflammatory response, 351-353 integrin ligand domains, 331-332, 334 lyniphocyte homing, 220-221 Vav G protein regulation, 68-69 Ras activation, 12-14 Rho signals, 34-35 V(D)J recombination, 182-185 Viruses, 295-298

X X-linked agammaglobulinemia, 159-160

ZAP-70 -CD3 complex, 57 characterization, 118 recruitment, 121-122

CONTENTS OF RECENT VOLUMES

Volume 68

Immunological Treatment of Autoimmune Diseases J. R. KALDEN,F. C. BREEDVELD, AND G. R. BURMESTER H. BURKHARDT,

Posttranscriptional Regulation of mRNAs Important in T Cell Function JAMES S. MALTER

INDEX

Molecular and Cellular Mechanisms of T Lymphocyte Apoptosis JOSEF M. PENNINGER AND GUIDO KROEMER

Volume 69 Molecular and Cellular Events in Early Thymocyte Development A N D HANS HANS-REIMER RODEWALD JORG FEHLINC

Prenylation of Ras GTPase Superfamily Proteins and Their Function in Immunobiology ROBERTB. LOBELL

Regulation of Immunoglobulin Light Chain Isotype Expression FREDERICK W. ALT AND JAMES R. GORMAN

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules FRANK MOMBURG AND GONTHER J. HAMMERLING

Role of Immunoreceptor Tyrosine-Based Activation Motif in Signal Transduction from Antigen and Fc Receptors NOAHISAKOV

Adoptive Tumor Immunity Mediated by Lymphocytes Bearing Modified AntigenSpecific Receptors THOMAS BROCKER AND KLAUS KARJAIAINEN Membrane Molecules as Differentiation Antigens of Murine Macrophages ANDREWJ. M c K A N~D SIAMON ~ ~ ~ GORDON Major Histocompatibility Complex-Directed Susceptibility to Rheumatoid Arthritis GEHALD T. NEPOM

Atypical Serine Proteases of the Complement System G~RAR J. D ARLAUD,JOHN E. VOLANAKIS, NICOLEM. THIELENS, STHANAM V. L. NARAYANA, V~RONIQUE Ross], AND YUANYUANXU ~

Accessibility Control of V(D)J Recombination: Lessons from Gene Targeting M. HEMPEL, ISABELLE LEDUC, WILLIAM NOELLEMATHIEU, RAJKAMALTRIPATHI, AND PIERRE FERRIER 389

390

CONTENTS OF RECENT VOLUMES

Phylogenetic Emergence and Molecular Interactions between the Immune System Evolution of the Immunoglobulin Family and Gene Therapy Vectors: Bidirectional JOHNJ. MARCHALONIS, SAMUEL F. Regulation of Response and Expression SCHLUTER, RALPH M. BERNSTEIN, JONATHAN S. BROMBERG, LISADEBRUYNE, SHANXIANG SHEN,AND ALLENB. AND LIHUIQIN EDMUNDSON Major Histocompatibility Complex Genes Influence Individual Odors and Mating Preferences DUSTINPENNAND WAYNE Pons Olfactory Receptor Gene Regulation ANDREWCHESS

INDEX

Current Insights into the “Antiphospholipid Syndrome: Clinical, Immunological, and Molecular Aspects DAVIDA. KANDIAH,ANDREJSALI, YONGHUASHENG,EDWARD J. VICTORIA, DAVIDM. MARQUIS, STEPHEN M. COUTTS, AND STEVEN A. KRILIS

INDEX

Volume 71 Volume 70 Biology of the Interleukin-2 Receptor BRADH. NELSONAND DENNIS M. WILLERFORD Interleukin-12: A Cytokine at the Interface of Inflammation and Immunity GIORGIO TRINCHIERI Recent Progress on the Regulation of Apoptosis by Bcl-2 Family Members ANDYJ. MINN, RACHELE. SWAIN, AVERIL MA. AND CFLUGB. THOMPSON Interleukin-18: A Novel Cytokine That Augments Both Innate and Acquired Immunity HARUKI OKAMURA, HIROKO TSUTSUI, SHIN-ICHIRO KASHIWAMURA, TOMOHIRO YOSHIMOTO, AND KENJI NAKANISHI

a&S Lineage Commitment in the Thymus of Normal and Genetically Manipulated Mice HANSJORGFEHLING, SUSAN GILFILLAN, AND RHODRI CEREDIG Immunoregulatory Functions of yS T Cells WILLIBORN,CAROL CADY, JESSICA JONESCARSON, AKIKOMUKASA, MICHAEL LAHN, AND REBECCAO’BRIEN STATs as Mediators of Cytokine-Induced Responses TIMOTHY HOEYAND MICHAEL J. GRUSBY CD95(APO-UFas)-Mediated Apoptosis: Live and Let Die PETERH. KRAMMER A CXC Chemokine SDF-UPBSF: A Ligand for a HIV Coreceptor, CXCR4 TAKASHI NAGASAWA, KAZUNOBU TACHIBANA, AND KENJIKAWABATA

T Lymphocyte Tolerance: From Thymic CD4’ T-cell Induction and Effector Deletion to Peripheral Control Mechanisms Functions: A Comparison of Immunity BRIGITTA STOCKINGER against Soluble Antigens and Viral Infections ANNETTEOXENIUS, ROLFM. Confrontation between Intracellular Bacteria ZINKERNAGEL. AND HANSHENGARTNER and the Immune System ULRICHE. SCHAIBLE, HELENL. COLLINS, Current Views in Intracellular Transport: AND STEFAN H. E. KAUFMANN Insights from Studies in Immunology VICTORW. Hsu AND PETERJ. PETERS INDEX


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