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ADVANCES IN

Immunology VOLUME 78

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 78

San Diego San Francisco New York Boston London Sydney

Tokyo

∞ This book is printed on acid-free paper.

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

Academic Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com International Standard Book Number: 0-12-022478-X PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 EB 9 8 7 6 5 4 3

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1

CONTENTS

ix

CONTRIBUTORS Toll-like Receptors and Innate Immunity

SHIZUO AKIRA

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX.

Introduction Toll Receptors in Drosophila Development Toll Receptors in Innate Immunity of Drosophila Mammalian IL-1R-Signaling Pathway: Its Similarity with Drosophila Toll Signaling Discovery of Toll-like Receptors in Mammals LPS and Its Binding Molecules Intracellular Events Following LPS Stimulation Nuclear Factors Activated by LPS TLR4 and LPS Signaling Role of MyD88 in LPS Signaling MyD88-Dependent and -Independent Pathways in LPS Signaling LPS Internalization Species Differences in LPS Response Taxol and LPS LPS Tolerance TLR2 and LPS Signaling Recognition of Microbial Cell Wall Components by TLRs Toll-like Receptors and Host Resistance to Microbial Infection Conclusion References

1 2 2 4 7 9 12 18 21 22 23 24 25 26 27 29 30 34 35 36

Chemokines in Immunity

OSAMU YOSHIE, TOSHIO IMAI, AND HISAYUKI NOMIYAMA

I. Introduction II. Chemokine Superfamily

57 59 v

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CONTENTS

III. Migratory Properties of Lymphocytes and Dendritic Cells IV. Primary Lymphoid Organs and Chemokines V. Secondary Lymphoid Organs and Chemokines VI. Effector/Memory Cells and Chemokines VII. Dendritic Cells and Chemokines VIII. Concluding Remarks References

73 74 77 80 88 90 92

Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses

CHRISTOPH SCHANIEL, ANTONIUS G. ROLINK, AND FRITZ MELCHERS

I. Introduction II. Structures of Chemokines and Their Receptors III. Rules to Understand Receptor–Ligand Interaction and Migration in Vivo IV. The Generation of Cells Involved in the Humoral Defense against Foreign Invaders V. The Population of Secondary Lymphoid Organs by Lymphoid Cells VI. Compartmental Homing within Secondary Lymphoid Organs VII. Cellular Traffic Leading to a Humoral Immune Response: Finding the Right Partner VIII. Migration of Effector and Memory T and B Cells IX. Possible Clinical Relevance of the ABCD Chemokines X. Future Perspectives References

111 114 115 117 123 130 136 143 147 151 153

Factors and Forces Controlling V(D)J Recombination

DAVID G. T. HESSLEIN AND DAVID G. SCHATZ

I. Introduction II. Basic Features of V(D)J Recombination III. Forces Controlling Chromatin Structure and Accessibility IV. Cis-Acting Elements and the Assembly of Antigen Receptor Loci V. The Factors VI. The Two Substrate Problem VII. Models References

169 170 179 193 201 209 213 216

CONTENTS

vii

T Cell Effector Subsets: Extending the Th1/Th2 Paradigm

TATYANA CHTANOVA AND CHARLES R. MACKAY

I. II. III. IV. V.

Introduction T Cell Effector Subsets What Determines Effector T Cell Differentiation? Transcription Factors for T Cell Differentiation The Link between Chemokine Receptors and T Cell Effector Function VI. Cell Surface and Costimulatory Molecules That Distinguish T Cell Effector Functions VII. Microarrays for the Identification of T Cell Subset Expressed Genes VIII. Conclusions References

233 233 239 240 242 248 250 253 253

MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens

INGELISE BJERRING KASTRUP, MADS HALD ANDERSEN, TIM ELLIOT, AND JOHN S. HAURUM

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

Introduction Posttranslational Modifications of Proteins Posttranslational Modifications and Antigen Processing Posttranslational Modifications and MHC Binding Posttranslational Modifications and T Cell Recognition Posttranslationally Modified Peptide Antigens: Are They Immunologically Relevant? References

267 268 270 272 277 284 286

Gastrointestinal Eosinophils in Health and Disease

MARC E. ROTHENBERG, ANIL MISHRA, ERIC B. BRANDT, AND SIMON P. HOGAN

I. II. III. IV.

Introduction Gastrointestinal Eosinophils in Healthy States Gastrointestinal Eosinophils in Disease States Experimental Dissection of Eosinophilic Gastrointestinal Inflammation V. Function of Eosinophils VI. Summary and Concluding Remarks References

291 293 299

INDEX

329

302 314 317 320


CONTRIBUTORS

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

Shizuo Akira (1), Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, CREST of Japan Science and Technology Corporation, Osaka 565-0871, Japan Mads Hald Andersen (267), Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen OE, Denmark Eric B. Brandt (291), Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-30309 Tatyana Chtanova (233), Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia Tim Elliott (267), Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom John S. Haurum (267), Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen OE, Denmark David G. T. Hesslein (169), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011 Simon P. Hogan (291), Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039 Toshio Imai (57), Kan Research Institute, Kyoto 600-8815, Japan Ingelise Bjerring Kastrup (267), Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen OE, Denmark Charles R. Mackay (233), Garvan Institute of Medical Research, Darlinghurst, NSW 2110, Australia Fritz Melchers (111), Basel Institute for Immunology, CH-4005 Basel, Switzerland ix

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CONTRIBUTORS

Anil Mishra (291), Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039 Hisayuki Nomiyama (57), Department of Biochemistry, Kumamoto University Medical School, Kumamoto 860-0811, Japan Antonius G. Rolink (111), Basel Institute for Immunology, CH-4005 Basel, Switzerland Marc E. Rothenberg (291), Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039 Christoph Schaniel (111), Basel Institute for Immunology, CH-4005 Basel, Switzerland David G. Schatz (169), Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011 Osamu Yoshie (57), Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

ADVANCES IN IMMUNOLOGY, VOL. 78

Toll-like Receptors and Innate Immunity SHIZUO AKIRA Department of Host Defense, Research Institute for Microbial Diseases, Osaka University; CREST of Japan Science and Technology Corporation, Osaka, Japan 565-0871 E-mail: [email protected]

I. Introduction

Immunity in higher organisms can be broadly categorized into adaptive immunity and innate immunity. Adaptive immunity is mediated by clonally distributed T and B lymphocytes which provide immunological specificity and memory. In contrast, innate immunity is mediated by the action of other cells such as macrophages and neutrophils, and traditionally has been characterized as nonspecific. However, recent findings have shown that innate immunity has some capacity for specific recognition. For example, insects, which lack the high level of immune specificity and memory characteristics of vertebrates, can nevertheless combat the invasion of microbes rapidly and efficiently by producing specific antimicrobial peptides. The synthesis of these specific antimicrobial peptides is triggered by the differential activation of different Toll family receptors present on the cell surface. More recently, various studies have demonstrated that many aspects of innate immune systems are shared between insects and mammals, and indeed have shown that such systems play a crucial role in the immune response in higher organisms such as mammals (Hoffmann et al., 1999). Microbial invasion in mammals is first handled by the innate immune system, which is designed to control and eventually resolve the infection. The cells of this system are not only responsible for the first-line mechanism of bacterial clearance, but also play an instructive role in adaptive immunity through the action of soluble factors or costimulatory signals (Fearon and Locksley, 1996). Janeway (1992) has hypothesized that recognition of a pathogen is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors would recognize conserved molecular patterns (pathogen-associated molecular patterns [PAMPs]) shared by large groups of microorganisms. Recognition of these patterns would allow the innate immune system not only to detect the presence of an infectious microbe, but also to determine the type of the infecting pathogen. Recently, the family of Toll-like receptors has been found to be present in mammals and to function in a manner similar to the PRRs envisioned by Janeway (1992). I will discuss the role of a group of the Toll receptors, which are phylogenically conserved mediators of innate immunity essential for microbial recognition. 1 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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II. Toll Receptors in Drosophila Development

Toll was originally identified as a transmembrane receptor that is critical to the determination of dorsoventral polarity in Drosophila embryo (Anderson et al., 1985; Belvin and Anderson, 1996). Toll is characterized by the presence of a leucine-rich repeat (LRR) in the extracellular domain and homology within the cytoplasmic domain to the interleukin-1 receptor (IL-1R) cytoplasmic domain. Stimulation of Toll activates the Rel family transcription factor, Dorsal, by inducing the degradation of cactus, via a process that involves the adaptor protein Tube and the serine-threonine protein kinase Pelle. Toll is expressed over the entire surface of the embryo, but is activated only in the ventral region during early embryogenesis by interaction with Spatzel, a specially restricted, extracellular ligand. Spatzel is a member of the cysteine-knot family of growth factors and cytokine-like proteins (Mizuguchi et al., 1998). Production of Spatzel requires activation by an extracellular serine protease cascade that is confined to the ventral side of the embryo. Spatzel is cleaved by the protease, Easter, which in turn is activated by another protease known as Snake (Anderson, 1998). This activation of the Spatzel–Toll pathway in the ventral region results in the generation of a nuclear gradient of Dorsal along the dorsovental axis of the embryo. This gradient regulates the localized expression of a set of zygotic genes that in turn specify ventral and dorsal fate. Certain dominant mutant alleles of Toll encode proteins that behave as partially ligand-independent receptors, and embyos expressing these proteins become ventralized. Toll is required for proper motoneuron and muscle specification in the late embryonic stage.

III. Toll Receptors in Innate Immunity of Drosophila

The Toll pathway also controls the production of potent antifungal peptides in the adult fly. In microbial infection of Drosophila, a battery of antimicrobial peptides are rapidly synthesized in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. Antimicrobial peptides are categorized into two groups: one group consists of several antibacterial peptides including cecropin, diptericin, drosocin, attacin, and insect defensin, and the other group consists of the major antifungal peptide, drosomycin (Hultmark, 1993; Hoffmann et al., 1996). The genes encoding these antibacterial and antifungal peptides are differentially expressed following infection by distinct microorganisms. Drosophila that are infected by insect-pathogenic fungi produce only peptides with antifungal activities as a result of the selective activation of the Toll pathway. Toll-deficient flies are defective in the induction of Drosomycin, and are poorly resistant to fungal infection (Lemaitre et al., 1996). The Toll pathway is also involved in the control of some of the antibacterial peptide genes

TOLL-LIKE RECEPTORS AND INNATE IMMUNITY

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(e.g., cecropin and attacin). However, the role of Dorsal in regulating the antimicrobial response is unclear, in contrast to its well-established and essential role in the regulation of the dorsoventral target genes. Indeed, the drosomycin gene remains fully inducible following immune challenge in Dorsal-deficient mutants, indicating that the control of antimicrobial peptide genes is redundant and that other Rel proteins can substitute for Dorsal in Dorsal-deficient mutants. In addition to Dorsal, Drosophila expresses two other Rel family proteins, Dif (for Dorsal-related immunity factor) and Relish (Ip et al., 1993; Dushay et al., 1996). Recently, Dif has been shown to act downstream in the Toll signaling pathway in induction of drosomycin (Meng et al., 1999; Manfruelli et al., 1999; Han and Ip, 1999; Rutschmann et al., 2000). Relish mutants are completely defective for the induction of all antimicrobial peptides and are more susceptible to fungal infection than wild-type Drosophila. Taken together, these observations indicate that Dorsal is essential to the regulation of the dorsoventral target genes, whereas the two other Rel transcription factors Dif and Relish apparently act downstream in the Toll signaling pathway for the induction of drosomycin. Another Drosophila gene, 18-wheeler (18W), exhibits homology to Toll, having both the intracellular IL-1R-like domain and the extracellular LRRs. 18W is required for Drosophila morphogenesis and is thought to function as a cell adhesion or receptor molecule that faciliates cell movements. 18W has also been shown to be involved in the host defense in Drosophila, since embryos carrying a mutated 18W gene are compromised in their antibacterial response (Williams et al., 1997). In 18W mutants, induction of attacin is reduced 95% and cecropin is reduced 65%, while diptericin expression is only slightly affected. However, drosomycin expression seems completely unaffected in 18W mutants. Nuclear localization of Dif is also blocked, whereas that of dorsal is normal. These results would suggest that 18W activates Dif, which in turn mediates the induction of attacin. However, other studies show that attacin induction is not affected by the absence of Dif. Thus, induction of attacin by the 18W pathway may involve another Rel transcription factor, possibly Relish, that can compensate for the loss of Dif. The imd mutants exhibit a severely reduced survival rate when injected with E.coli, compared to Toll deficient or wild-type flies. The mutants are also completely defective in the synthesis of antibacterial peptides, including cecropin, attacin, and diptericin, following challenge by pathogens (Lemaitre et al., 1995a). On the other hand, these mutants are more or less normal with respect to induction of the antifungal peptide gene drosomycin, and accordingly, their resistance to fungal infection is similar to that of wild-type flies. Recently, Relish was found to be a key factor in the induction of both antibacterial and antifungal peptides (Hedengren et al., 1999). Diptericin induction shows an absolute requirement for the Relish gene, suggesting that Relish is likely to be part of the imd pathway. Thus, the genes encoding the various antimicrobial peptides seem

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to be controlled by different combinations of Rel-transcription factors that, in turn, are activated via distinct signaling cascades elicited by specific microbial populations. IV. Mammalian IL-1R-Signaling Pathway: Its Similarity with Drosophila Toll Signaling

Recently, remarkable advances have been made in the field of IL-1 signal transduction (Fig. 1). The IL-1R system is composed of a ligand-binding subunit, IL-1RI, and a signal transducing subunit, IL-1RAcP (Greenfeder et al., 1995; Wesche et al., 1997b). Upon binding of IL-1, IL-1RI forms a complex with IL-1RAcP. The adaptor protein MyD88 is next recruited to this complex, which in turn facilitates the association with the IL-1R-associated kinase (IRAK) via its death domain (Cao et al., 1996a; Huang et al., 1997; Wesche et al., 1997a; Burns et al., 1998). IRAK then becomes autophosphorylated, disassociates from the receptor complex, and interacts with TRAF6 (Cao et al., 1996b), consistent with studies showing that phosphorylation of IRAK lowers its affinity to the activated receptor complex and increases its affinity to TRAF6 (Wesche et al., 1997a). Recently, several novel IRAK-like molecules, termed IRAK-2 and IRAK-M, have also been shown to interact with MyD88, and to be involved in IL-1-induced NF-␬B activation (Muzio et al., 1997). However, neither IRAK-2 nor IRAK-M has been demonstrated to be strongly phosphorylated (Wesche et al., 1999). Furthermore, a kinase-defective form of IRAK was demonstrated to activate NF-␬B, albeit with a lower potency, indicating that the IRAK kinase activity may not be required for IL-1 signaling (Wesche et al., 1999; Knop and Martin, 1999). TRAF6 is a member of the TRAF (TNF-receptor associated factors) family of adaptor proteins (Cao et al., 1996b). The TRAFs were first described as proteins recruited to the TNF receptors (Arch et al., 1998). Currently, six TRAF proteins have been identified. TRAFs 1–5 are recruited to the TNF receptor complex, whereas TRAF6 participates in signaling initiated by the IL-1 receptor and CD40. Overexpression of TRAF2, TRAF5, and TRAF6 causes activation of NF-␬B and AP-1. The IL-1-mediated NF-␬B and c-Jun pathways bifurcate at TRAF6. TRAF6 associates with NF-␬B-inducing kinase (NIK), a MAP 3 kinaserelated protein (Malinin et al., 1997). NIK activates the I␬B kinase complex (including IKK␣ and IKK␤) that directly phosphorylates I␬B, which allows dissociation and degradation of this inhibitory component of NF-␬B (Regnier et al., 1997; DiDonato et al., 1997; Mercurio et al., 1997). TAK1, a member of the MAP 3 kinase family, was first implicated as a mediator of the signal transduction pathways triggered by members of the transforming growth factor-␤ (TGF-␤) superfamily (Yamaguchi et al., 1995). Recently, TAK1 was also shown to be involved in the IL-1 signaling pathway (Ninomiya-Tsuji


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FIG. 1. IL-1R signaling pathway. Upon binding of IL-1, IL-1RI forms a complex with IL-1RAcP. This complex recruits IRAK via an adaptor MyD88. IRAK then becomes autophosphorylated, disassociates from the receptor complex, and interacts with TRAF6. TRAF6 associates with NIK. NIK activates the I␬B kinase complex (including IKK␣ and IKK␤) that directly phosphorylates I␬B, which allows dissociation and degradation of this inhibitory component of NF-␬B. TRAF6 also activates MAP kinases including ERKs, JNK, and p38. TAK1/TAB1, EKSIT, and aPKC/p62 are shown to associate with TRAF6 and are suggested to be involved in IL-1R signaling. IL1R: IL-1 receptor, AcP: IL-1 receptor accessory protein, TLR: Toll-like receptor, IRAK: IL-1 receptor-associated kinase, TAK1: TGF-␤-activated kinase, TAB1: TAK1 binding protein 1, TRAF6: TNF receptorassociated factor 6, NIK: NF-␬B-inducing kinase, MKK: Mitogen-activated protein kinase kinase, JNK: c-Jun N-terminal kinase, IKK: I␬B kinase.

et al., 1999). Following IL-1 stimulation, a complex consisting of TAK1 and its regulator TAB1 is recruited to the TRAF6 complex, resulting in TAK1 activation. Activated TAK1 then stimulates the MAP kinase cascades, leading to JNK activation (Shirakabe et al., 1997), and a NIK-IKK cascade leading to NF-␬B activation. This suggests that TAK1 is located at or near the point of bifurcation of the IL-1-induced JNK and NF-␬B activation pathways. More recently, the adaptor protein ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) has been identified as a novel TRAF6 binding

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protein (Kopp et al., 1999). ECSIT interacts with the conserved TRAF domain of TRAF6, and also with the MAP3 kinase, MEKK1, which activates both AP-1 and NF-␬B. MEKK1 has been implicated in the activation of NF-␬B through the phosphorylation of the IKK complex (Lee et al., 1998; Nakano et al., 1998), although a recent finding from MEKK1 gene knockout experiments demonstrates that MEKK1 is not essential for IL-1- or LPS-induced NF-␬B activation (Xia et al., 2000). Kopp et al. (1999) demonstrated that expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant negative form of ECSIT blocks MEKK-1 processing and activation of NF-␬B. This suggests that ECSIT functions as a bridge to connect TRAF6 to downstream signaling kinases as in the case of TAK1. A homolog of ECSIT has been identified in Drosophila, and was found to bind TRAF6 and induce the transcription of host defense genes in insect cells. This indicates that ECSIT is an evolutionarily conserved signaling component in the Toll/IL-1 pathway. The factor p62 was identified as a protein interacting with the atypical protein kinase C (aPKC). It has previously been shown to interact with RIP, linking these kinases to NF-␬B activation by tumor necrosis factor␣ (TNF␣), and has also been implicated in the transmission of IL-1 signaling to NF-␬B. p62 specifically interacts with TRAF6, and thereby appears to act as an adaptor connecting the aPKCs to the IKK complex (Sanz et al., 2000). The roles of various molecules in the IL-1 signaling pathway have been assessed by gene targeting. In the case of MyD88, mice deficient in the gene were found to be defective in T cell proliferation, and in induction of hepatic acute phase proteins and serum cytokines in response to IL-1 (Adachi et al., 1998). IL-18 is an IL-1-related cytokine that shares a number of biological functions with IL-12, including the activation of natural killer cells, induction of interferon-␥ (IFN-␥ ), and T helper1 (Th1) cell differentiation (Okamura et al., 1995). In MyD88-deficient mice, IL-18-induced increases in IFN-␥ production and natural killer cell activity were abrogated, and the Th1 response was impaired. Furthermore, IL-18-induced activation of NF-␬B and JNK was blocked in MyD88−/− Th1-developing cells. These results demonstrate that MyD88 is essential for IL-1- and IL-18-mediated function. IRAK-deficient embryonic fibroblasts show reduced IL-1-induced NF-␬B and c-Jun NH2-terminal kinase activities (Kanakaraj et al., 1999). IRAK knockout (KO) mice are also severely compromised in IL-1-induced neutrophilia and show little increase in serum TNF or IL-6 levels following IL-1 stimulation. Furthermore, splenocytes derived from IRAK KO mice produced significantly reduced amounts of IFN-␥ in response to IL-18 (Thomas et al., 1999). Nonetheless, IRAK KO mice and cells retain significant residual IL-1 and IL-18 responses, indicating the existence of molecules whose functions overlap with IRAK. Two likely candidates for such molecules are IRAK-2 and IRAK-M, which have both sequence and functional similarities to IRAK. It has been


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demonstrated that IRAK is recruited to IL-1RAcP, whereas IRAK-2 preferentially binds IL-1RI (Muzio et al., 1997). In contrast to the ubiquitously expressed kinases IRAK and IRAK-2, IRAK-M expression is limited mostly to cells of the monocytic lineage, and is upregulated during differentiation, suggesting that IRAK-M may function in a more cell-specific manner (Wesche et al., 1999). TRAF6 KO mice have been generated and found to suffer from osteopetrosis (Lomaga et al., 1999; Naito et al., 1999). Activation of both NF-␬B and MAP kinases was impaired in embryonic fibroblasts from these mice, and IL-1 signaling in thymocytes was also defective. These results demonstrate that TRAF6 is an important transducer in IL-1 signaling. There are remarkable structural and functional similarities between the Drosophila Toll- and mammalian IL-1R-mediated signaling pathways (O’Neill and Greene, 1998). The intracellular portion of Toll shares sequence homology with the intracellular portion of the mammalian IL-1R (Gay and Keith, 1991; Medzhitov and Janeway, 1997). Mutagenesis and deletion analyses have shown that the amino acid residues conserved between the IL-1R and Toll cytoplasmic domains are essential for signal transduction by these receptors (Heguy et al., 1992; Schneider et al., 1991). Toll activates Dorsal through a process involving an adaptor molecule, Tube, and the degradation of Cactus. Tube activates Pelle, a Ser/Thr kinase that catalyzes the dissociation of Dorsal from Cactus and allows migration of Dorsal to the ventral nuclei. Dorsal and Cactus are Drosophila homologues of NF-␬B and I␬B, respectively, while the Drosophila Pelle is highly homologous to IRAK. Both Pelle and IRAK have N-terminal death domains (Cao et al., 1996a). The Drosophila Tube is a functional homologue of mammalian MyD88 although these two proteins are structually unrelated except for the presence of a death domain. Recently, a Drosophila TRAF-like protein has been identified that is probably a functional homologue of TRAF6, and a Drosophila homologue of I␬B kinase named DLAK has been identified (Liu et al., 1999; Kim et al., 2000). V. Discovery of Toll-like Receptors in Mammals

In 1997, Janeway’s group reported the cloning of the human homologue of Toll (human Toll/TLR4), the first member of the TLR family (Medzhitov et al., 1998). So far, six human Toll-like receptors (TLR1-6) have been cloned (Rock et al., 1998; Chaudhary et al., 1998; Takeuchi et al., 1999b) (Fig. 2). Over 10 members of TLR family can be found in a search of the human and mouse public databases (our unpublished data). This is an orphan receptor family characterized by an extracellular domain containing leucine-rich repeats and a cytoplasmic domain significantly similar to the intracellular portion of the IL-1R family. These genes are dispersed throughout the genome and show diverse patterns of expression. TLR1 and TLR6 map to human chromosome 4p14; TLR2

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FIG. 2. IL-1R/TLR family members. The cytoplasmic portions of IL-1R and TLR family members are highly homologous. In contrast, the extracellular portions are different; those of IL-1R family are composed of three Ig-like domains, whereas those of TLRs are composed of leucine-rich repeats. So far, six TLRs are published. However, over 10 TLRs have been found in a search of the human and mouse public databases (our unpublished data). After submitting the manuscript, additional 3 novel TLRs have been reported (Chuang and Ulevitch, 2000; Du et al., 2000; Hemmi et al., 2000).

and TLR3 to 4q31.3-q35; TLR4 to 9q32-q33; and TLR5 to 1q33.3-q42 (Rock et al., 1998; Chaudhary et al., 1998; Takeuchi et al., 1999b). Muzio et al. (2000) examined the expression of TLR family in human leukocytes and showed that TLR can be classified into three groups based on expression pattern: ubiquitous (TLR1), restricted (TLR2, TLR4, and TLR5 in myelomonocytic cells), and specific (TLR3 in dendritic cells). TLR3 expression in dendritic cells (DCs) is noteworthy. DCs develop upon culture of precursor monocytes in the presence of granulocyte-macrophage-colony stimulating factor (GM-CSF) plus IL-4 or IL-13 for 7 days. Upon additional exposure to inflammatory signals (such as TNF␣, IL-1␤, or lipopolysaccharide (LPS)) they undergo functional maturation. TLR3 was found to be expressed exclusively on DCs, but absent in the precursor monocytes. Moreover, the expression of TLR3 dramatically increased during monocyte differentiation in vitro. Finally, when DCs were subsequently treated with inflammatory signals to induce full maturation, TLR3 expression significantly decreased. Most Langerhans cells in the skin do not express TLR3,


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as assessed by in situ hybridization. On the other hand, TLR3-expressing DCs are clearly detectable in the T cell area of the lymph nodes. Expression of the various TLRs is modulated in response to a variety of stimuli. For example, the effect of LPS stimulation on TLR2 and TLR4 mRNA expression has been examined in mouse macrophages (Nomura et al., 2000; Medvedev et al., 2000). LPS was found to cause a strong increase in the steady-state levels of TLR2 mRNA which was detectable as early as 1 hr. In this experiment, maximal response was reached at 3 hr after stimulation, and was sustained throughout the 12-hr course of LPS treatment. In contrast to TLR2, TLR4 mRNA was found to be constitutively expressed in the absence of LPS, modestly down-regulated for 3–6 hr after LPS stimulation, and then returned to the basal level measured in untreated and unstimulated macrophages after 20 hr. TLR2 synthesis is strongly induced in adipocytes by stimulation with LPS, TNF␣, or zymosan, a yeast cell wall extract (Lin et al., 2000). It has also been shown that TLR4 expression and TLR4-associated signaling increase in injured myocardium (Frantz et al., 1999). Based on the homology in the cytoplasmic region between the TLRs and IL-1Rs, it was expected that both might use the same signaling molecules. Indeed, signaling initiated by both human Toll/TLR4 and IL-1R results in recruitment of the adaptor molecule MyD88 followed by the kinase IRAK (Muzio et al., 1997, 1998; Wesche et al., 1997a; Adachi et al., 1998; Medzhitov et al., 1998; Burns et al., 1998). In both cases, the subsequent steps leading to NF-␬B activation involve TRAF6 and NIK (Cao et al., 1996b; Malinin et al., 1997). Overexpression of dominant negative mutants originally shown to inhibit the IL-1 signaling pathway, including MyD88, IRAK, and TRAF6, were found to also inhibit TLR4-induced NF-␬B activation in human THP-1 cells (Medzhitov et al., 1998; Zhang et al., 1999). Furthermore, a constitutively active mutant of TLR4 was found to cause activation of AP-1, expression of the inflammatory cytokines IL-1, IL-6, and IL-8, and expression of the costimulatory molecule B7.1, which is required for the activation of naive T cells. Thus, these results indicate that TLR4 is a nonclonal receptor that functions in the innate immune system of vertebrates to elicit the adaptive immune response (Medzhitov and Janeway, 1997). VI. LPS and Its Binding Molecules

Lipopolysaccharide is an integral component of the outer membranes of Gram-negative bacteria and is a potent activator of macrophage functions (Ulevitch and Tobias, 1995). LPS is a complex glycolipid composed of a hydrophilic polysaccharide portion and a hydrophobic domain known as lipid A. The lipid A portion is responsible for the biological activity of LPS. LPS is positioned in the outer leaflet of the outer membrane of Gram-negative bacteria with the lipid A portion oriented inside. Stimulation of macrophages with LPS results in the production of various cytokines such as TNF␣, IL-1, IL-6,

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IL-10, macrophage inflammatory protein-1␣/␤ (MIP-1␣/␤), and inflammatory effector substances such as protanoids, leukotrienes, and nitric oxide (NO). Macrophages stimulated with LPS also show enhanced expression of cell surface antigens such as major histocompatibility complex (MHC) class II and B7-1/2 (Ulevitch and Tobias, 1995). Although small amounts of LPS can be favorable to the host, by triggering responses that augment the microbicidal activities of macrophages, overactivation of macrophages by large amounts of LPS results in a life-threatening condition called endotoxin shock. Endotoxin shock is characterized by hemodynamic instability, activation of the complement and clotting cascades, and multiorgan failure. Several LPS binding molecules that are involved in the LPS response have been cloned and characterized (Fenton and Golenbock, 1998), and are described below. A. CD14 The major cell surface receptor for LPS on macrophages is CD14, a 55-kD glycosylphosphatidyl inositol (GPI)-anchored glycoprotein that is intercalated in the outer lipid layer of the plasma membrane and thus contains no cytoplasmic region (Wright et al., 1990b). Binding of LPS to CD14 is greatly enhanced in the presence of the serum factor LPS-binding protein (LBP). In CD14-negative cells, such as endothelial cells, astrocytes, and fibroblasts, a soluble form of CD14 (sCD14) present in serum can functionally replace membrane-bound CD14 (Tapping and Tobias, 1997). sCD14 is present at a concentration of 1–3 ␮g/ml in the plasma of normal adults. Similar to the Toll receptor, CD14 is a member of a group of proteins containing LRRs. Mapping studies with CD14 have revealed that 7 out of its 10 LRRs can be deleted without affecting LPS binding (Juan et al., 1995a). Subsequent mutation studies indicated that LPS binding involves the amphipathic portions in the amino terminal region separate from the LRR region of CD14 (Juan et al., 1995b; Cunningham et al., 2000). CD14 has also been shown to mediate recognition and phagocytosis of apoptotic cells (Devitt et al., 1998). The important physiological role of CD14 has been demonstrated by the use of blocking antibodies and by gene targeting. In macrophages treated with anti-CD14 monoclonal antibody (mAb), low doses of LPS (10 ng/ml) of LPS are provided, suggesting that there could exist a CD14-independent mechanism(s) of LPS stimulation. This possibility is supported by the observation that high doses but not low doses of LPS can activate macrophages derived from CD14-deficient mice (Haziot et al., 1996; Perera et al., 1997). B. LBP LBP is a 60-kDa serum protein that modulates the response to LPS (Tobias et al., 1986; Schumann et al., 1990). LBP is an acute phase protein that is


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produced by the liver. The addition of LBP dramatically enhances the sensitivity of CD14+ cells to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14. The NH2-terminal half of LBP is responsible for its LPS binding activity while the COOH-terminal half is responsible for transferring LPS to CD14 (Abrahamson et al., 1997; Lamping et al., 1996). LBP thus acts as lipid transfer protein, catalytically transferring LPS monomers from aggregates to CD14, and exhibits sequence homology to two other lipid transfer proteins: phospholipid transfer protein and cholesterol ester transfer protein. Additional studies have shown that LBP copurifies with high-density lipoprotein (HDL) particles and also acts to transfer LPS into complexes of HDL where LPS is functionally neutralized (Wurfel et al., 1994, 1995; Ulevitch et al., 1979). Furthermore, it has been demonstrated that a high concentration of LBP decreases the in vivo activity of LPS and can protect against septic shock caused by Gram-negative bacteria (Lamping et al., 1998). Thus, LBP may serve both to enhance and to neutralize the biological activity of LPS. The ratio of LBP to LPS is reported to determine a number of essential processes: the monocytic response (Lamping et al., 1998), the binding of LBP to HDL (Massamiri et al., 1997), and the inactivation of LPS by HDL (Wurfel and Wright, 1997). The role of LBP in vivo was assessed by generating LBP KO mice (Wurfel et al., 1997). Ex vivo whole blood samples from the LBP−/− mice were markedly hyporesponsive to LPS, requiring nearly 1,000-fold greater amounts of LPS to obtain the same half-maximal response obtained with LBP+/− mice. The addition of recombinant LBP or plasma from LBP+/− mice restored the responses of LBP−/− cells. However, despite these striking findings obtained ex vivo, wildtype and LBP KO mice produced similar amounts of plasma TNF␣ following LPS challenge. This result suggests the possible existence of LBP-independent mechanisms for responding to LPS. C. CD11b/CD18 CD11b/CD18 is a member of the leukocyte integrin family of heterodimeric adhesion molecules, which consist of a common ␤-subunit and a unique ␣-subunit. Wright and Jong (1986) first demonstrated that the integrins were capable of binding unopsonized bacteria and LPS. However, peripheral blood mononuclear cells from CD18-deficient patients responded normally to LPS (Wright et al., 1990a). Transfection of CD11/CD18 integrins into Chinese hamster ovary (CHO) cells, which lack CD14, was found to confer LPS-induced signal transduction to these cells, demonstrating that CD11/CD18 can mediate the response to LPS independent of CD14 (Ingalls and Golenbock, 1995). Addition of LPB enhanced the sensitivity of CD11/CD18-tranfected CHO cells to LPS and Gram-negative bacteria, implying that LBP is also capable of transferring LPS to CD11/CD18 (Ingalls et al., 1998). The cytoplasmic domains of CD11/CD18 were found to be essential for such functions as phagocytosis. However, the cytoplasmic domains are dispensable for signaling, as demonstrated

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by the fact that a mutant CD11/CD18 lacking the cytoplasmic domains and incapable of internalization of Gram-negative bacteria still exhibits LPS-induced cellular activation (Ingalls et al., 1997). Thus, the function of CD11/CD18, like CD14, may be to transfer LPS to a second receptor that transduces the LPS signal. D. SCAVENGER RECEPTOR The macrophage scavenger receptor type A (SR-A) is a trimeric integral membrane glycoprotein that has the capacity to bind modified lipoproteins (Kodama et al., 1990). SR-A is expressed on a wide range of tissue macrophages. The fact that SR-A can be detected in atheromatous plaques, and its ability to mediate uptake of modified low-density lipoprotein (LDL) by arterial wall macrophages, has implicated it in the pathogenesis of atherosclerosis. Hampton et al. (1991) showed that macrophages can bind, internalize, and partially break down LPS using the macrophage scavenger receptor. SR-A can recognize a variety of polyanions in addition to LPS, including bacterial cell wall products such as lipoteichoic acid (LTA) (Dunne et al., 1994). SR-A also mediates phagocytosis of apoptotic thymocytes (Platt et al., 1996) and adhesion of macrophages to surfaces coated with serum proteins (Fraser et al., 1993), glucose-modified basement membrane proteins (El Khoury et al., 1994), or ␤-amyloid fibrils (El Khoury et al., 1996). SR-A KO mice have been shown to be more susceptible to intraperitoneal infection by the Gram-positive pathogen Staphylococcus aureus than control mice. These mutant mice were impaired in their ability to clear bacteria from the site of infection (Thomas et al., 2000). SR-A KO mice also exhibit increased susceptibility to infection by Listeria monocytogenes (Suzuki et al., 1997). Nevertheless, the role of these receptors in LPS-induced signal transduction appears to be minor, despite their demonstrated ability to bind LPS. Blocking these receptors with acetyl-LDLs had no effect on the LPS-induced release of TNF release in RAW cells. It is more likely that these receptors participate in LPS catabolism and detoxification. Indeed, not only are SR-A KO mice more susceptible to endotoxin shock, but they produce greater amounts of TNF␣ and IL-6 in response to LPS, consistent with the idea that SRA plays a protective role in host defense by scavenging LPS and by reducing the release of proinflammatory cytokines by activated macrophages (Haworth et al., 1997). VII. Intracellular Events Following LPS Stimulation

The formation of the LPS/LBP/CD14 ternary complex on the cell surface activates multiple signal transduction pathways as summarized below. However, given the rapid pace at which these pathways are currently being elucidated, our understanding of the roles of these molecules in LPS signaling will probably need to be modified in the near future.


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A. NONRECEPTOR PROTEIN KINASES Several lines of evidence suggest that three members of the Src-family of protein tyrosine kinases, Hck, Fgr, and Lyn, play critical roles in LPS-initiated signaling pathways (DeFranco et al., 1998). All three kinases are rapidly activated after LPS treatment. The p53/56lyn protein kinase has been shown to directly coassociate with CD14 (Stefanova et al., 1993). Moreover, expression of a constitutively active mutant of Hck in macrophages was shown to augment TNF␣ production, while antisense oligonucleotides against Hck mRNA inhibited LPS-induced responses (English et al., 1993). Hck is also shown to enhance the adherence of LPS-stimulated macrophages, at least in part, via Cbl phosphorylation and subsequent association of the p85 subunit of PI3-kinase with Cbl (Scholz et al., 2000). However, these kinases are not essential to signal transduction, since the normal LPS response was observed in macrophages derived from hck−/−fgr−/−lyn−/− triple KO mice (Meng and Lowell, 1997). Another intracellular tyrosine kinase, Syk, also becomes tyrosine phosphorylated upon LPS stimulation of macrophages (Crowley et al., 1996); however, cytokine production and signaling events in macrophages from Syk KO mice were found to be normal, including activation of MAP kinase family and tyrosine phosphorylation of the adapter protein Shc (Crowley et al., 1997). B. PHOSPHOLIPASE C Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphatidylinositol triphosphate, releasing diacylglycerol and inositol 1,4,5-triphosphate (IP3). Diacylglycerol (DAG) acts as the signal for activation of PKC, and also activates an acidic sphingomyelinase with the resultant production of ceramide. IP3 acts primarily as a messenger that induces the release of Ca2+ from intracellular storage sites. Phosphatidylcholine-specific phospholipase C (PC-PLC) also generates diacylglycerol, but does not generate IP3. Several reports suggest that the generation of DAG induced by LPS involves the PC-PLC, but not the PI-PLC pathway (Sands et al., 1994; Grove et al., 1990). Lack of IP3 generation may be consistent with either trivial changes or no increase in Ca2+ in response to LPS. C. PROTEIN KINASE C There are many reports describing activation of protein kinase C (PKC) in macrophages exposed to LPS. PKC is a family of at least 12 closely related isoenzymes that are categorized into three main groups based on their primary structure and activation requirements: conventional, novel, and atypical (Ron and Kazanietz, 1999). Macrophages and monocytes express the conventional ␣, ␤I, and ␤II isoenzymes, the novel ␦ and ⑀ isoenzymes, and the atypical ␨ isoenzyme (Fujihara et al., 1994; Liu et al., 1994; Zheng et al., 1995; Mischak et al., 1991). Several groups have proposed that particular PKC isozymes are

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involved in the LPS-induced production of cytokines and NO as well as in the activation of NF-␬B and MAP family kinases (St-Denis et al., 1998; Chen et al., 1998a; Kontny et al., 2000; Valledor et al., 2000; Lozano et al., 1994). D. PI3-KINASE PI3-kinase is a heterodimer composed of an 85,000 M regulatory subunit (p85) and a 110,000 M catalytic subunit (p110). The latter catalyzes the phosphorylation of inositol phospholipids on the D3 position of the inositol ring of phosphatidylinositol. PI3-kinase is activated by LPS and has been shown to associate with p53/56lyn (Herrera-Velit and Reiner, 1996). Signaling events triggered by PI3-kinase include the activation of the serine/threonine kinase, PKB, which in turn activates p70 S6-kinase through a mechanism controlled by the rapamycin target (mTOR) (Duronio et al., 1998). PI3-kinase also stimulates protein kinase C-␨ . However, the role of PI-3K in LPS signaling is controversial, and studies using the PI3-kinase inhibitors wortmannin and Ly294002 have arrived at different results. Some reports demonstrate a positive role for PI3-kinase in the response to LPS (Williams and Ridley, 2000; Venkataraman et al., 1999; Tengku-Muhammad et al., 1999; Weinstein et al., 2000; Manna and Aggarwall, 2000), whereas other reports show that PI-3 kinase plays a negative role in the activation of macrophages by LPS and induction of iNOS expression (Park et al., 1997; Chen et al., 1998b; Salh et al., 1998; Diaz-Guerra et al., 1999; Pahan et al., 1999). E. MITOGEN-ACTIVATED PROTEIN KINASE (MAP KINASE) FAMILY Mitogen-activated protein kinases (MAPK) are a family of kinases involved in intracellular signaling. Mammalian MAP kinases are largely divided into three groups based on their structure and function: (1) extracellular signal-regulated kinases (ERKs), (2) c-Jun N-terminal kinase or stress activated protein kinase (JNKs or SAPKs), and (3) the p38 group (Waskiewicz and Cooper, 1995; Cano and Mahadevan, 1995). These kinases are activated as part of a cascade consisting of at least three kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). MAPKK activates MAPK by phosphorylating it on both serine/threonine and tyrosine residues. MAPKK is in turn activated by the serine/threonine kinase MAPKKK. Many reports have shown that the MAP kinases ERK, JNK, and p38 are activated by LPS stimulation (DeFranco et al., 1998; Downey and Han, 1998). The activation of these MAPKs appears to be linked to the regulation of cytokine gene expression, as well as other important cellular functions. Recently, using JNK2-deficient fibroblasts, JNK2 has been shown to be essential for cytokine production in IL-1- and LPS-stimulated fibroblasts (Chu et al., 1999). Another report has demonstrated that the p38-dependent MAPKAP kinase 2 (MK2) is critical for LPS-induced TNF-␣ biosynthesis and that mice lacking MK2 survive LPS-induced endotoxin shock (Kotlyarov et al.,


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1999). Although signaling from the TNF receptor is unchanged in these mice, production of TNF␣ is reduced approximately 90%. The amount and stability of TNF␣ mRNA and the secretion of the TNF␣ protein are unaffected in these mutants, indicating that MK2 must regulate TNF␣ biosynthesis at the posttranscriptional level. These studies show that MK2 is an essential component in the inflammatory response. F. PHOSPHOLIPASE A2 Phospholipase A2 (PLA2) consists of a family of enzymes that catalyze the hydrolysis of phosphatidlycholine and /or phosphatidylethanolamine. At least two forms of the enzyme, cytosolic PLA2 (cPLA2) and secretory PLA (sPLA) are activated following exposure to LPS (Forehand et al., 1993; Doerfler et al., 1994; Rodewald et al., 1994). cPLA2, a ubiquitous 85-kDa enzyme, is activated by MAP kinases, upon which it is translocated from the cytosol to membrane (Clark et al., 1991; Nemenoff et al., 1993). sPLA2 is a secreted enzyme that is detected in inflammatory fluids such as the synovial flulid of patients with rheumatoid arthritis (Seilhamer et al., 1989), and in plasma, where its level is increased in inflammatory diseases. Hydrolysis of phospholipids by PLA2 results in the release of arachidonic acid (AA) and lysophospholipids. Both of these products function as intracellular second messengers and /or serve as substrates for the generation of other bioactive lipid mediators such as the eicosanoids or plateletactivating factor (Balsinde et al., 1999; Murakami et al., 1997). G. PHOSPHOLIPASE D Phospholipase D (PLD) hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. PA is rapidly converted to DAG through the action of phosphatidate phosphohydrolase. Phospholipase D activity has been shown to increase after LPS treatment (Chu, 1992). Yamamoto et al. (1997) showed that PLD-dependent production of DAG from PC is involved in LPS-dependent activation of NF-␬B. Phosphatidylinositol 3-kinase (PI3-K) is shown to be located upstream of PLD. Procyk et al. (1999) have indicated that LPS activation of MAP kinases is mediated by PI3-K-mediated stimulation of PLD-dependent PC hydrolysis and subsequent activation of DAG-dependent PKC isoforms. PA is also the precursor of lysophosphatidic acid (LPA), generated by the action of PLA2. LPA is a potent phospholipid mediator with diverse biological activities and is known to bind to a G protein-coupled receptor. Thus, stimulation of PLD may result in the secondary generation of LPA, which stimulates cells as a paracrine. H. G PROTEIN The guanine nucleotide binding proteins (G proteins) consist of a family of heterotrimeric membrane proteins that link the activation of receptors to

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enzymes that generate secondary messengers or to ion channels. The Gi family of G proteins appears to be involved, at least in part, in the activation of macrophages by LPS. Gi family proteins have been implicated in the LPS-induced responses of B cells and macrophages (Jakway and DeFranco, 1986), LPS-stimulated thromboxane B2 (TXB2) synthesis in rat peritoneal macrophages (Coffee et al., 1990), prostaglandin E2 (PGE2) production in rat mesangial cells (Wang et al., 1988), and induction of IL-1␤ mRNA in U937 cells (Daniel-Issakani et al., 1989), and LPS-mediated iNOS induction (Schroeder et al., 1997). However, G protein may not always play a stimulatory role. Zhang and Morrison (1993) showed that pretreatment of mouse peritoneal macrophages with pertussis toxin markedly enhanced LPS-induced TNF␣ production but inhibited LPS-dependent NO production. Solomon et al. (1998) showed that CD14 coimmunoprecipitates with Gi/Go heterodimeric G proteins. Mastoparan, a peptide that specifically stimulates Gi and Go heterodimeric G proteins, was found to inhibit LPS-induced TNF␣ and IL-6 production in human monocytes. Furthermore, Mastoparan treatment of peripheral blood mononuclear cells and monocytes suppressed LPS-induced phosphorylation of p38 MAPK. Finally, Mastoparan was shown to protect rats from LPS-induced mortality. Thus, it seems likely that Gi/Go heterodimeric G proteins are involved in both positive and negative regulatory signaling by LPS. I. PROTEIN KINASE A Alteration of intracelllar cAMP levels is an important intracellular signaling mechanism involved in the regulation of gene expression. The effect of cAMP levels on iNOS production is varied, with some in vitro studies showing that an increase in cAMP levels causes iNOS induction (Koide et al., 1993; Imai et al., 1994; Nusing et al., 1996; Klein et al., 1998), while other studies have shown that increased cAMP levels cause a reduction in iNOS (Smith et al., 1997; Pahan et al., 1997). It has been shown that the increase in cAMP levels occurs after 6 hr of treatment by LPS (Okada et al., 1995), and that PKA is involved in the LPSinduced activation of junB and NF-␬B (Fujihara et al., 1993). Recently, Chen et al. (1999a) further showed that induction of PGE2 synthesis precedes the increase in cAMP, and that PGE2 acts as an autocrine factor causing activation of adenylate cyclase in the process of NF-␬B activation and iNOS induction by LPS. J. CERAMIDE The lipid ceramide has been studied as a secondary messenger in the TNF␣, IL-1␤, and IFN-␥ signaling pathways. Ceramide is generated from membrane sphingomyelin by the action of sphingomyelinase (SMase). One recent hypothesis, the molecular mimicry hypothesis, proposes that LPS may in fact act as a structural mimic of ceramide, directly interacting with ceramide-responsive


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enzymes at the plasma membrane (Wright and Kolesnick, 1995). This is supported by the observation that bacterial SMase and synthetic ceramide analogs could induce expression of an array of LPS-inducible mRNAs in macrophages from LPS-responsive (Lpsn) mice but not from LPS-hyporesponsive C3H/HeJ (Lpsd) mice (Barber et al., 1995). In the hyporesponsive macrophages, intracellular trafficking of both fluorescently labeled LPS and labeled ceramide analogs is reportedly altered, although LPS binding and internalization occur with normal kinetics (Kitchens and Munford, 1998). However, recent findings argue against the molecular mimicry hypothesis to explain LPS action. Ceramide analogs and LPS, while having some overlapping effects, have been shown to induce different patterns of gene expression (Barber et al., 1996). Furthermore, it has recently been demonstrated that LPS induces an increase in ceramide itself, rather than interacting directly with ceramide-responsive enzymes. LPS treatment of LPSd macrophages induced a rise in ceramide similar to that observed in LPS-responsive cells, demonstrating that LPS-induced ceramide generation is normal in these cells and that an increase in ceramide, by itself, is not responsible for the observed response to LPS (MacKichan and DeFranco, 1999). Ceramide does partially mimic LPS in its ability to activate the MAP kinases and AP-1, but it does not induce NF-␬B activation or cytokine production, suggesting that its role in LPS signaling is a limited one (Medvedev et al., 1999). K. RAPAMYCIN The p70 S6 kinase is responsible for phosphorylation of the S6 protein in response to various stimuli. Activation of the p70 S6 kinase involves TOR or FKBP12-rapamycin-associated protein (FRAP). Salh et al. (1998) demonstrate that in RAW cells, production of NO in response to LPS is independent of the PI3-kinase pathway, but requires the FRAP-dependent pathway. In this study, LPS was observed to induce p70 S6 kinase activity five-fold, but both kinase induction and NO production were blocked by rapamycin treatment. L. RHO FAMILY OF SMALL GTPASES Rho proteins regulate critical biological processes such as cell growth, transformation, metastasis, apoptosis, response to stress, and certain aspects of development. Rho proteins are known to activate NF-␬B in diverse cell types (Montaner et al., 1998; Perona et al., 1997). Recently, it has been demonstrated that Rho is involved in LPS-induced ICAM-1 expression in bovine aortic endothelial cells (Takeuchi et al., 2000c), adherence of monocytes to endothelial cells (Hmama et al., 1999), LPS-induced IL-8 expression in human endothelial cells (Hippenstiel et al., 2000), and LPS-induced endothelial cell contraction via inhibition of myosin light chain phosphatase (Essler et al., 2000).

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VIII. Nuclear Factors Activated by LPS

The combinatorial effects of transcription factors are very important in gene regulation. Many promoter cis-elements, and the factors which bind them, have been shown to function in a cooperative or synergistic manner. Cooperativity between transcription factors and higher order complex formation on the promoter appears to be necessary for proper gene regulation. In the case of genes mediating inflammatory and immune responses, NF-␬B, NF-IL6, and AP-1 are commonly found to bind to the same promoters and act in concert to regulate gene expression. This is in accord with the finding that LPS treatment leads to the activation of both NF-␬B, C/EBP, and AP-1 family of transcription factors. The transcription factors that are important nuclear targets in LPS signaling are described as follows. A. NF-␬B NF-␬B plays a critical role in immune and inflammatory responses (Lenardo and Baltimore, 1989; Baldwin, 1996). NF-␬B is activated by many types of extracellular stimuli, including TNF␣, IL-1, bacterial and viral infection, ultraviolet (UV) radiation, free radicals, and hypoxia. NF-␬B is a dimer composed of members of the Rel family of DNA-binding proteins, RelA/p65, p50, p52, c-Rel, RelB, and Bcl-3 (Ghosh et al., 1998). In most cell types, NF-␬B is sequestered in the cytoplasm as a result of its association with the inhibitory I␬B proteins. Activation of NF-␬B by various stimuli involves the phosphorylation of I␬B proteins by I␬B kinases, triggering their degradation and the release of NF-␬B, which translocates into the nucleus and activates various target genes. The promoters of most inflammatory genes (i.e., TNF␣, IL-1␤, IL-6, IL-8, iNOS, COX-2, etc.) contain the binding site(s) for NF-␬B. NF-␬B has been observed to translocate to the nucleus in LPS-stimulated macrophages, and has been implicated in the LPS-induced gene regulation of proinflammatory cytokines and inflammatory mediators (Xie et al., 1994; Liu et al., 1997; Dendorfer et al., 1994; Ni et al., 1998). The idea that NF-␬B contributes to LPS-induced gene regulation is supported by several observations. Infection of primary macrophages with an adenoviral vector expressing I␬B␣ or a dominant negative NF-␬B p65 was shown to inhibit LPS-induced TNF␣ secretion (Foxwell et al., 1998; Bondeson et al., 1999; Liu et al., 2000). The expression of multiple immune response genes by LPS is reduced in RelA−/− embryonic fibroblasts (Ouaaz et al., 1999). Expression of a dominant negative form of p65 in bovine endothelial cells inhibits LPS-induced upregulation of E-selectin, P-selectin, and I␬B␣ (Anrather et al., 1997). Finally, NF-␬B inhibitors such as pyrrolidine dithiocarbamate (PDTC), dimethylsulfoxide, and proteasome inhibitors have been shown to block LPS-mediated induction of cytokines and iNOS (Liu et al., 1997; Xie et al., 1994; Ziegler-Heitbrock et al., 1993; Schreck et al., 1992; Kelly et al., 1994; Haas et al., 1998; Griscavage


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et al., 1996). I␬B kinases are present as part of high molecular weight complex in the cytoplasm, and consist of two subunits, IKK␣ and IKK␤. Gene inactivation studies have revealed unique roles for each subunit: IKK␤ is critical to NF-␬B activation by proinflamatory cytokines (Li et al., 1999; Tanaka et al., 1999), whereas IKK␣ is essential to limb formation and skin development (Hu et al., 1999; Takeda et al., 1999). IKK␤ was also shown to be a major kinase involved in activation of NF-␬B by LPS. In transfection experiments, overexpression of wild type IKK␣, a dominant negative mutant IKK␣ (K44M), or wild type IKK␤ had no affect on LPS-induced NF-␬B-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK␤ inhibited LPS induction of NF-␬Bdependent transcription in a dose-dependent manner (O’Connell et al., 1998). In addition, LPS-induced IL-6 and IL-12 production was drastically reduced in IKK␤-deficient fibroblasts as compared with wild-type fibroblasts, showing that IKK␤ is essential for LPS-induced cytokine production (Chu et al., 1999). B. NF-IL6 NF-IL6 (also called C/EBP␤) is a member of the C/EBP family of proteins, which contain basic and leucine zipper domains in their carboxy-termini (Akira, 1997). NF-IL6 was first cloned as a nuclear protein that bound to the IL-1 responsive element of the IL-6 gene (Akira et al., 1990). NF-IL6 is activated posttranslationally, due mainly to phosphorylation by PKC, CAM kinase, and MAP kinases including ERK and p38 (Trautwein et al., 1993; Wegner et al., 1992; Nakajima et al., 1993; Engelman et al., 1998). NF-IL6 is expressed in macrophages, and has been implicated in the LPS-induced expression of proinflammatory cytokines and inflammatory mediator genes (Akira and Kishimoto, 1992, 1997). C. CREB (ATF) AND AP-1 FAMILY OF TRANSCRIPTION FACTORS The CREB/ATF family of transcription factors are leucine zipper proteins that bind to the cAMP response element (CRE). CREB, the most extensively studied CRE-binding protein, is phosphorylated at serine 133 by protein kinase A, calmodulin kinase, and RSK2, the latter of which is activated by MAP kinases. ATF-1, another member of this family of transcription factors, has significant sequence similarity to CREB, including a homologous phosphorylation domain. ATF-1 forms heterodimers solely with CREB. The AP-1 family of transcription factors consists of the cJun and Fos components and binds the 12-O-tetradecanoylphorbol-13-acetate response element (TRE). Jun forms heterodimers with other members of the AP-1 family, and Jun protein forms heterodimers with certain members of some other transcription factor families, such as ATF and C/EBP. Various studies have demonstrated that the CREB/ATF and AP-1 families of transcription factors, and the promoter elements they recognize, are important for the transcriptional activation of inflammatory mediators and

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proinflammatory cytokine genes in response to LPS (Mackman et al., 1991; Schanke et al, 1994; Shin et al., 1994; Gray et al., 1993; Yao et al., 1997; Proffitt et al., 1995; Pierce et al., 1996; Pan et al., 1998). The c-Jun and c-Fos genes are activated by the transcription complex Ternary Complex Factor/Elk-1, which is phosphorylated and activated by JNK, in the case of the c-Jun promoter, and JNK or ERK, in the case of the c-Fos promoter (Karin, 1995). Thus, activation of AP-1 is likely to be mediated by activation of JNK and ERK in response to LPS. The mechanism by which LPS activates CREB and ATF-1 is less clear, but recent experiments have suggested the involvement of mitogen and stress-activated protein kinase-1 (MSK-1), a kinase which is activated in vivo by a wide variety of stimuli including growth factors, phorbol esters, and cytokines. MSK-1 is activated by two different classes of MAP kinases, namely the ERKs and p38. This MAP kinase-MSK-1 pathway may be responsible, in part, for the LPS-stimulated induction of COX-2 and IL-1␤ via activation of CREB and ATF1 (Caivano and Cohen, 2000). D. OTHER TRANSCRIPTION FACTORS Sp1 has been implicated as a nuclear target of LPS signaling pathways. Bethea et al. (1997) showed that the Sp1 site is necessary for LPS-induced transcription of the TNFRII gene. Brightbill et al. (2000) analyzed the murine IL-10 promoter in the RAW264 macrophage line activated with LPS. They demonstrated the exclusive requirement for the Sp1 binding site, the ability of the Sp1 site to confer induction to a heterologous promoter, and the ability of the Sp1B domain to support inducible transcription when fused to a Gal4 DNA binding domain, suggesting that Sp1 may be a central mediator of LPS induction of IL-10. Peroxisome proliferator-activated receptor ␥ (PPAR-␥ ) is a member of the nuclear hormone receptor superfamily and is predominantly expressed in adipose tissue, adrenal gland, and spleen. PPAR-␥ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-␦ prostaglandin J2 (15␦-PGJ2) bind to PPAR-␥ and stimulate transcription of target genes. Several lines of evidence suggest that PPAR-␥ may exert anti-inflammatory effects by negatively regulating the expression of proinflammatory genes that are induced during macrophage differentiation and activation. It has been shown that LPS stimulation of macrophages increases the level of PPAR-␥ . Treatment of peritoneal macrophages with 15␦-PGJ2 or several synthetic PPAR-␥ ligands reduced the expression of iNOS and gelatinase B transcription in response to LPS ( Ricote et al., 1998). PPAR-␥ is thought to inhibit the inflammatory response genes by antagonizing the activities of AP-1, NF-␬B, and STAT1 transcription factors.


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IX. TLR4 and LPS Signaling

The fact that the Drosophila Toll family plays an essential role in the antimicrobial response suggested that Toll-like receptors (TLRs) in mammals may participate in innate immunity. Although LBP and CD14 were identified as factors that recognize LPS, the former is a soluble protein and CD14 lacks a transmembrane domain, suggesting that initiation of the LPS signal may involve a coreceptor ( Haziot et al., 1996; Perera et al., 1997). Furthermore, CD14 KO mice were found to have severely diminished responses to LPS, although cellular activation was seen with high doses of LPS (Haziot et al., 1996). C3H/HeJ is a mutant mouse strain that was reported ∼30 years ago to be hyporesponsive to LPS, and has been extensively characterized since (Sultzer, 1968; Sultzer et al., 1993). These mice also exhibit greatly increased susceptibility to bacterial infections. Analysis of backcross hybrids from crosses between LPS-responsive and LPS-hyporesponsive mice localized the defect to a single autosomal gene (LpS) on chromosome 4 (Watson and Riblet, 1974; Watson et al., 1978). The defect in another LPS hyporesponsive strain, C57Bl/10ScCr, and its progenitor, C57BL/10ScN, was also mapped to the same chromosomal location on mouse chromosome 4 (Coutinho et al., 1977; Coutinho and Meo, 1978). The Lps gene was recently cloned and shown to encode one of the Tolllike receptors, TLR4 (Poltorak et al., 1998, Qureshi et al., 1999). The mouse TLR4 gene contains one open reading frame of 2505 nucleotides, predicted to encode a protein of 835 amino acids. This protein consists of an extracellular domain formed by a tandem arrangement of a 22 leucine-rich repeat motif and an IL-1R-homologous intracellular domain. The C3H/HeJ mouse strain carries a missense point mutation within the cytoplasmic portion of the TLR4 gene, which changes a proline highly conserved within the TLR family to histidine. C57BL/ScCr and C57BL/ScN strains were found to be competely deficient in TLR4 mRNA expression due to a chromosomal deletion of the gene. Transgenic mice deficient in the TLR4 gene were generated (Hoshino et al., 1999) and macrophages and B cells from TLR4 KO mice were found to be hyporesponsive to LPS to a similar extent as C3H/Hej mice, confirming that TLR4 is required for LPS signaling. Previous experiments demonstrated that overexpression of TLR4 did not confer LPS responsiveness on human embryonic kidney (HEK) 293 cells, although it did cause constitutive, low-level activation of NF-␬B (Kirschning et al., 1998). This suggested that an additional molecule, absent in HEK293 cells, is required for TLR4-mediated LPS signaling. Subsequently, Shimazu et al. (1999) identified a novel molecule, MD-2, that is required for TLR4-mediated LPS signaling, and this molecule was found to be absent from HEK293 and IL-3-dependent Ba/F3 cell lines. The role of MD-2 in TLR4-mediated signaling was demonstrated by the observation that transfection of 293 or Ba/F3 with either TLR4

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alone or MD-2 alone did not confer responsiveness to LPS, whereas cotransfection with both TLR4 and MD-2 did. Furthermore, these authors showed that MD-2 is physically associated with TLR4 on the cell surface. Yang et al. (2000) also demonstrated that coexpression of MD-2 is absolutely required for LPS activation of MAP kinase proteins and Elk-1. Furthermore, while expression of TLR4 alone can mediate the activation of NF-␬B and IL-8 production in HEK293 cells, these responses are enhanced further when MD-2 is coexpressed. In addition to TLR4, two other genes have been demonstrated to be responsible for LPS hyporesponsiveness in C3H/HeJ mice. One is the gene encoding the GTPase Ran which contains a point mutation at position 870 (Kang et al., 1996). The Ran gene also maps to mouse chromosome 4. Retroviral transfer of wild-type Ran cDNA into C3H/HeJ mice render them sensitive to LPS, and adenovirus transfer into normal mice makes them hyporesponsive (Wong et al., 1999). Ran may therefore be involved in some manner in the LPS response. The other gene encodes the secretory leukocyte protease inhibitor. Differential display analysis of matched macrophage cell lines from C3H/HeJ and C3H/HeN mice resulted in the identification and isolation of the SLPI cDNA, which was overexpressed in the C3H/HeJ macrophage cell line (Jin et al., 1997). Human SLPI is an 11.7-kDa cystein-rich protein found in saliva, seminal plasma, and cervical, nasal, and bronchial mucus. SLPI is a potent inhibitor of leukocyte serine proteases, notably elastase and cathepsin G from neutrophils, and chymase and tryptase from mast cells, as well as trypsin and chymotrypsin from pancreatic acinar cells. Transfection of macrophages with SLPI suppressed LPS-induced activation of NF-␬B and production of NO and TNF␣. The murine SLPI gene does not map to chromosome 4. The relation of Ran and SLPI to TLR4 awaits further studies. X. Role of MyD88 in LPS Signaling

MyD88 was originally isolated as a myeloid differentiation primary response gene, which is rapidly induced upon IL-6-stimulated differentiation to macrophages in M1 myeloleukemic cells (Lord et al., 1990). This immediate-early activation profile suggested that MyD88 functions in the regulated progression of myeloid differentiation. Subsequently, MyD88 was found to be structurally related to the IL-1 receptor family, particularly Toll (Hultmark, 1994). Unlike the IL-1 receptor family, MyD88 does not have a transmembrane portion, but instead, contains a death domain in its N-terminus (Hardiman et al., 1996; Bonnert et al., 1997). The C-terminal domain of the protein is highly homologous to the cytoplasmic segments of the IL-1 receptor family. Recently, MyD88 was isolated as an adapter molecule responsible for recruiting IRAK to the IL-1 receptor complex and activating NF-␬B after IL-1 stimulation (Muzio et al., 1997; Wesche et al., 1997a). MyD88 is therefore a functional homologue of the Drosophila factor Tube, although these two molecules are not related by amino acid sequence.


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MyD88 KO mice have been generated (Adachi et al., 1998) and found to be completely defective in T cell proliferation and induction of hepatic acute phase proteins and serum cytokines in response to IL-1. In addition, the response to IL-18 was found to be defective. IL-18 is an IL-1-related cytokine which shares many biological functions with another interleukin, IL-12. These functions include the activation of natural killer cells, induction of IFN-␥ , and Th1 cell differentiation. In MyD88-deficient cells, IL-18-induced increases in IFN-␥ production and natural killer cell activity were abrogated. In vivo, the Th1 response was also impaired. Furthermore, in developing Th1 cells from MyD88 KO mice, IL-18-induced activation of NF-␬B and JNK was blocked. These results demonstrate that MyD88 is a critical component in the signaling cascades mediated by IL-1R and IL-18R. The similarities between the signaling pathways mediated by the TLRs and IL-1Rs suggested that MyD88 may also be involved in TLR4-mediated LPS signaling. Examination of MyD88 KO mice showed that LPS-mediated functions were almost completely abolished (Kawai et al., 1999). MyD88 KO mice injected with LPS did not produce increased serum levels of cytokines such as IL-6, TNF␣, or IL-1␤, nor did they develop endotoxin shock. Similarly, macrophages isolated from MyD88 KO mice and stimulated with LPS did not produce any detectable levels of IL-6 or TNF␣, and produced only minor amounts of NO2− in response to LPS plus IFN-␥ . Furthermore, B cells isolated from these mice were hyporesponsive to LPS. These results demonstrate that MyD88 is critical to LPS-mediated, as well as IL-1 and IL-18-mediated cellular functions. The role of IRAK was investigated using IRAK KO mice (Swantek et al., 2000). IRAK-deficient macrophages displayed impaired TNF␣ production in response to LPS, accompanied by defective LPS-induced activation of NF-␬B and the MAP kinases. IRAK KO mice are hyporesponsive to LPS. However, the mutant mice and cells exhibited a less pronounced resistance to LPS than MyD88 KO mice and cells, indicating that lack of IRAK may be compensated by other IRAK-related molecules such as IRAK-2 or IRAK-M. A critical role for TRAF6 in LPS signaling was also demonstrated by gene targeting (Lomaga et al., 1999). TRAF6-deficient B cells exhibited little proliferation in response to LPS stimulation, and TRAF6-deficient bone marrow macrophages were impaired in their ability to induce iNOS. Furthermore, LPS-induced NF-␬B activation in TRAF6-deficient Abelson pre-B cells was reduced significantly. XI. MyD88-Dependent and -Independent Pathways in LPS Signaling

Not only do MyD88-deficient mice fail to respond to LPS, IL-1, and IL-18, they also fail to respond to other microbial cell wall components such as peptidoglycan and lipopeptides. However, the signaling pathways triggered by these latter cell wall components appear to be different from those stimulated by LPS.

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FIG. 3. MyD88-dependent and -independent pathways in dendritic cell maturation. At least two signaling pathways originate from the cytoplasmic portion of TLR4. One is the MyD88-dependent pathway, which subsequently activates IRAK, TRAF6, and NF-␬B. This pathway is essential for cytokine production. The other is the MyD88-independent pathway, which cannot activate IRAK or TRAF6. Although the signaling molecules involved are unknown, this pathway can also induce NF-␬B activation. This NF-␬B activation cannot lead to cytokine induction but to induction of costimulatory molecules such as CD40, CD80, and CD86 in dendritic cells.

Mycoplasmal lipopeptide activation of NF-␬B and MAP kinases, which is mediated by TLR2, are completely abolished in TLR2-deficient or MyD88-deficient macrophages (Takeuchi et al., 2000b). However, LPS activation of MAP kinases and NF-␬B remain intact in MyD88-deficient macrophages, although the activation of these molecules was delayed when compared with wild-type mice (Kawai et al., 1999). This indicates that the LPS response may be mediated by both MyD88-dependent and -independent pathways, each of which leads to the activation of MAP kinases and NF-␬B. However, the MyD88-dependent pathway is essential for the inflammatory response mediated by LPS. Although the nature of the MyD88-independent pathway remains largely unknown, it has recently been found that MyD88-deficient DCs can undergo functional maturation in response to LPS, indicating that a MyD88-independent pathway plays a functional role in this response (Kaisho & Akira, 2001; Kaisho et al., in press) (Fig. 3). XII. LPS Internalization

Wright’s group has shown that fluorescent LPS presented to peripheral mononucleocytes (PMNs) in the form of LPS–sCD14 complexes results in the rapid transport of LPS to a perinuclear site (Detmers et al., 1996). Procedures shown to block this transport (lowering temperature or addition of cytochalasin D or wortmannin) were also found to block the cellular response to LPS, suggesting that LPS internalization may be required for signal transduction. These authors


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also showed that LPS transport is absent in peritoneal macrophages recovered from LPS-hyporesponsive (Lpsd) mice (Thieblemont and Wright, 1997), and that structural analogues of LPS that are biologically inactive are not transported to the perinuclear site (Thieblemont et al., 1998). These results demonstrate a strong correlation between LPS internalization and cell signaling. However, other published reports indicate that LPS internalization and LPS-dependent cell activation are dissociated (Kitchens et al., 1992; Gegner et al., 1995; Poussin et al., 1998; Underhill et al., 1999). LPS internalization is involved not only in cell activation, it is critical to cell detoxification. LPS is known to be deacylated by the specialized enzyme, acyloxyacyl hydrolase (AOAH), whose expression is restricted to a low density intracellular compartment in PMNs (Munford, 1991). It has been shown that LPS deacylation by macrophages from C3H/HeN and C3H/HeJ mice was qualitatively and quantitatively similar, showing that TLR4 engagement is not required for this detoxification (Munford and Hall, 1985). XIII. Species Differences in LPS Response

Some mammalian species show an ability to discriminate between different LPS structures. The naturally occurring lipid A types Rhodobacter sphaeroides lipid A (RSLA) and lipid IVA have been found to be potent LPS antagonists in LPS-responsive human cells (Golenbock et al., 1991; Takayama et al., 1989; Kovach et al., 1990). In contrast, these compounds have very different effects in hamster and mouse cells: in hamsters, both compounds are LPS mimetics (Delude et al., 1995), while in mice, lipid IVA is an LPS mimetic and RSLA is an LPS antagonist (Golenbock et al., 1991; Takayama et al., 1989; Kirkland et al., 1991; Lynn and Golenbock, 1992). It has also been shown that Salmonellatype lipid A is inactive in human cells but active in mouse cells as assessed by TNF␣ induction and NF-␬B activation. The species-specific effects of the lipid A-like compounds are determined not by the species of CD14. In addition, the inhibitors are not simply competing with LPS for binding to CD14, but they are antagonizing LPS at a site distinct from CD14 (Kitchens et al., 1992; Kitchens and Munford, 1995). Indeed, recent papers indicate that TLR4 is directly involved in recognition of LPS (Poltorak et al., 2000; Lien et al., 2000). Mice respond to LPS and its congener tetra-acyl LPS, which lacks secondary acyl chains, while humans fail to respond to this variety. The lipid A analogues lipid IVA and RSLA are both potent antagonists in LPS-responsive human cells, whereas both are LPS mimetics in hamster macrophages. In cells expressing TLR4 from heterologous species, the specificity of the response to different lipid A species and analogues correlates with the species from which the TLR4 is derived, and not to the species of the host cells. However, conclusive evidence showing that LPS is directly recognized by TLR4 will require further experimentation.

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XIV. Taxol and LPS

Taxol, a diterpene purified from the bark of the Western yew (Taxus brevifolia), is an antitumor agent that blocks mitosis by binding and stabilizing microtubules. Ding et al. (1990) found that Taxol induces the secretion of TNF and downregulation of TNF receptors in murine macrophages. Although the structure of Taxol is quite different from that of LPS, it possesses many LPS-like activities, such as tyrosine phosphorylation of MAP kinases, induction of LPSinducible gene expression, and activation of NF-␬B (Manthey et al., 1992; Ding et al., 1993; Hwang and Ding, 1995). Interestingly, Taxol mimics the actions of LPS on murine macrophages but not on human LPS-responsive cells including macrophages (Manie et al., 1993). Taxol-induced signaling events in murine macrophages are blocked by LPS antagonists, suggesting that LPS and Taxol share a receptor or signaling molecule (Manthey et al., 1993). Given that LPS antagonists suppress LPS-induced signaling without preventing LPS binding to CD14, CD14 might not be the common target recognized by LPS and Taxol. Furthermore, the LPS-mimetic activity of Taxol is not observed in macrophages obtained from the LPS-hyporesponsive C3H/HeJ mice (Ding et al., 1990). Therefore, TLR4 appeared to be involved in both Taxol and LPS signaling. To determine whether TLR4 mediates a Taxol-induced signal, Kawasaki et al. (2000) constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, or both mouse TLR4 and mouse MD2, and then examined Taxol-induced NF-␬B activation in these transformants. NF-␬B activation by Taxol was detected in Ba/F3 cells coexpressing mouse TLR4 and MD2 but not in cells expressing TLR4 alone. In contrast, coexpression of human TLR4 and human MD2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4/MD2 complex is responsible for conferring species specificity with respect to Taxol responsiveness. Taxolinduced NF-␬B activation via TLR4/MD2 was also blocked by an LPS antagonist. These results demonstrate that coexpression of mouse TLR4 and mouse MD2 is required for Taxol responsiveness and that the TLR4/MD2 complex is the molecule that is shared between Taxol and LPS signal transduction in mice. By use of a photoactivable Taxol analgoue, Bhat et al. (1999) have further shown that CD18 binds Taxol in mouse macrophage membranes. Macrophages from Mac-1(CD11b/CD18, CR3) KO mice were found to be defective in Taxolinduced IL-12 p40 mRNA induction, while treatment of normal macrophages with anti-Mac-1 Ab blocked Taxol-induced IL-12 p70 secretion. These results suggest that CD18 is critical for the action of Taxol as a full LPS mimetic. Byrd et al. (1999), using biotin-labeled Taxol and avidin-agarose affinity chromatography, have also identified heat shock protein 90 (Hsp 90) as a Taxol-binding protein. Geldanamycin, a specific inhibitor of the Hsp 90 family, blocked the nuclear


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translocation of NF-␬B and expression of TNF-␣ in macrophages treated with Taxol or with LPS. The role of CD18 and Hsp 90 in the TLR4-mediated signaling pathways remains unknown. XV. LPS Tolerance

Exposure to LPS is known to cause reduced sensitivity to subsequent LPS challenge. This phenomenon is termed LPS tolerance (also called as LPS hyporesponsiveness or refractoriness). LPS tolerance in vivo is manifested by decreased febrile response and an escape from lethality and in vitro by reduced production of inflammatory cytokines in response to secondary stimulation with LPS. However, not all responses are attenuated by LPS tolerance, and in some cases LPS-induced gene expression even increases. Such genes include those encoding the p50 subunit of NF-␬B, IL-1 receptor antagonist, TNF receptor type II, and IL-10. The molecular mechanisms for LPS tolerance have been investigated for a number of years in two aspects: the involvement of cytokines and mediators that suppress inflammatory responses, and altered intracellular signal transduction events (Ziegler-Heitbrock, 1995). IL-10 and TGF-␤ are the potent inhibitors of IL-12 synthesis by macrophages. Randow et al. (1995) have identified IL-10 and TGF␤ as endogenous mediators responsible for the inhibition of LPS-induced TNF production during LPS tolerance. However, Wittmann et al. (1999) found that IL-12 production induced by LPS was not enhanced by neutralization of endogenously produced IL-10. Consistent with the latter result, IL-10 KO mice were found to become LPS tolerant with respect to the induction of TNF␣ similar to wild-type mice (Berg et al., 1995). Karp et al. (1998) also showed that treatment of human monocytes with neutralizing antibodies to IL-10 and TGF-␤ failed to prevent LPS tolerance with respect to the induction of IL-12, although they did prevent LPS tolerance with respect to TNF␣. IL-4 is another antagonist of proinflammatory monokines, and has been shown to downregulate IL-12. As observed with antibodies against IL-10, neutralizing antibodies against IL-4 failed to abrogate LPS tolerance in monocyte cell cultures, as measured by IL-12 induction. Therefore, induction of LPS tolerance does not seem to be mediated by anti-inflammatory ctyokines such as IL-10, TGF-␤, and IL-4. Recently, Baer et al. (1998) have detected an autocrine factor that attenuates TNF␣ transcription in macrophages and that is distinct from IL-4, IL-10, and TGF-␤. Inhibition of TNF␣ by this factor is associated with enhanced expression of NF-␬B p50 in the nucleus. The biochemical characterization and cloning of this novel factor await further study. PGE2 is also known to inhibit macrophage activation and inflammatory cytokine expression in macrophages (Metzger et al., 1981; Taffet and Russell, 1981; Kunkel et al., 1988; Hancock et al., 1988). However, it is unlikely that PGE2

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plays a major role in LPS tolerance, because indomethacin, a nonselective COX-1/COX-2 inhibitor did not reverse LPS tolerance (Bogdan et al., 1993; Wittmann et al., 1999) Several reports suggest that LPS tolerance is due to downregulation of postreceptor signaling mechanisms. Although expression of an LPS receptor such as CD14 was not altered in LPS-tolerant monocytes and macrophages (Mathison et al., 1993, Ziegler-Heitbrock, 1995), LPS signaling pathways have been shown to be affected in several respects. For example, LPS tolerance is associated with reduced activation of PKC in macrophages (West et al., 1997), selective reduction in macrophage membrane G proteins (Makhlouf et al., 1996), and induction of a protein phosphatase (Kravchenko et al., 1996). Defects in NF-␬B activation may play a key role in LPS tolerance. Pretreatment of macrophages with LPS was found to increase levels of I␬B␣, an inhibitor of NF-␬B, and was proposed to be responsible for downregulation of IL-1␤ (LaRue and McCall, 1994). Furthermore, it has been demonstrated that in LPS-tolerant cells, NF-␬B is composed predominantly of p50/p50 homodimers (Ziegler-Heitbrock et al., 1994; Goldring et al., 1998; Kastenbauer and Ziegler-Heitbrock, 1999), in contrast to normal cells where p50/p65 heterodimers predominate. The p50/p65 heterodimers found in normal cells are transcriptionally active, whereas the p50/p50 homodimers are inactive and prevent promoter binding by p50/p65 dimers. Recently, Nomura et al. (2000) investigated the molecular mechanism of LPS tolerance in mouse peritoneal macrophages, and showed that LPS tolerance in the macrophages is primarily due to the downregulation of surface TLR4 expression. They also found that cytoplasmic signaling pathways are downregulated during LPS tolerance. Sato et al. (2000) showed that macrophages pretreated with the mycoplasmal lipopeptide MALP-2 exhibit reduced production of TNF␣ in response to LPS. However, in contrast to LPS tolerance induced by LPS, LPS tolerance induced by MALP-2 was not associated with decreased expression of surface TLR4, indicating that tolerance is due to modulation of the downstream cytoplasmic signaling pathways. Medvedev et al. (2000) also showed that LPS and IL-1 induce cross-tolerance, whereas LPS and TNF␣ do not. This suggests that LPS tolerance results from inhibition of the function of signaling intermediates common to LPS and IL-1 signaling. In this respect, it is noteworthy that endogenous IRAK levels are consistently expressed at reduced levels in the LPS-tolerant human macrophage cell line, THP-1, and IRAK no longer associates with MyD88 in tolerant THP-1 cells in response to LPS (Li et al., 2000). Thus, LPS tolerance seems to be the consequence of changes that take place at various points in the LPS signaling pathways, and the precise mechanism presumably varies with the cell type and experimental model. The detailed mechanisms by which LPS tolerance is achieved await further studies.


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XVI. TLR2 and LPS Signaling

TLR2 has also been shown to be involved in LPS-mediated signaling (Yang et al., 1998; Kirschning et al., 1998). Human TLR-2 binds LPS in the presence of LBP and CD14 and induces NF-␬B activation. Overexpression of human TLR2 conferred LPS responsiveness on the human embryonic kidney cell line 293, whereas overexpression of human TLR4 failed to confer LPS responsiveness, although it did lead to a constitutive activation of NF-␬B. In addition, evidence was obtained suggesting that human TLR2 interacts with CD14 to form an LPS receptor complex. LPS treatment leads to the oligomerization of this receptor and to the subsequent recruitment of IRAK (Yang et al., 1999). This activation of NF-␬B by LPS was blocked by expression of a dominant-negative form of TLR2. However, results from TLR4 mutant mice demonstrated that the TLR4 receptor is required for the LPS response. This discrepancy might be attributed to species-specific differences in the role of the TLRs, i.e., LPS signaling is mediated by TLR4 in mice and by TLR2 in human. However, transfection of cell lines with human TLR4, while not sufficient to confer responsiveness to LPS, did confer dose-dependent responsiveness when cotransfected with MD2, a factor shown to be physically associated with TLR4 on the cell surface (Shimazu et al., 1999). Finally, the TLR2 gene was found to be defective in Chinese hamster CHO cells, yet these cell lines exhibit normal LPS responsiveness (Heine et al., 1999). Thus, the initial proposal that TLR2 is the LPS receptor was not supported by these subsequent studies. In order to determine the role of TLR2 in LPS signaling, we generated TLR2 KO mice by gene targeting (Takeuchi et al., 1999a). We found that TLR2deficient macrophages produced IL-6 and TNF␣ in response to LPS or lipid A to the same extent as wild-type macrophages. This is in contrast to TLR4-deficient macrophages, which did not produce any detectable levels of IL-6 or TNF␣. TLR2-deficient B cells showed normal proliferation and increased class II expression in response to LPS, and TLR2 KO mice were not resistant to LPSinduced shock. The in vivo results demonstrate that TLR2 is not involved in the LPS response in mice, and should be considered more reliable than the earlier overexpression studies. Similarly, Underhill et al. (1999) demonstrated that expression of a dominant negative form of TLR2 in mice abrogates the inflammatory responses to yeast and Gram-positive bacteria, but not to Gramnegative bacteria. In addition, Faure et al. (2000) showed that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express high levels of TLR4 but extremely low levels of TLR2 and respond vigorously to LPS but not to the Mycobacterium tuberculosis 19-kD lipoprotein. The latter lipoprotein is known to specifically activate cells via TLR2. Transient transfection of HMEC cells with a mutant TLR4 expression vector or treatment with an anti-TLR4 mAb inhibited activation of NF-␬B by LPS, whereas

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treatment with a mAb against TLR2 was ineffective. Furthermore, it has been shown that TLR4 mutations are associated with LPS hyporesponsiveness in humans (Arbour et al., 2000). Recently, Hirschfeld et al. (2000) suggested that the overexpression of either human or murine TLR2 causes cell lines to become extremely sensitive to minor contaminations in many commercial LPS preparations, and that LPS-mediated TLR2 signaling described in previous papers might be due to contamination of microbial components which activate cells via TLR2. Although it remains possible that TLR2 may play some role in LPS signaling in other cell types, we may reasonably conclude from the above results that TLR4 plays a crucial role in LPS signaling.

XVII. Recognition of Microbial Cell Wall Components by TLRs

A. GRAM-POSITIVE BACTERIAL CELL WALL COMPONENTS Gram-positive bacteria do not contain LPS, yet they trigger a toxic shock syndrome similar to that induced by LPS. This response is caused by cell wall components of Gram-positive bacteria, such as peptidoglycan (PGN) and LTA (Fig. 4). PGN is an alternating ␤(1, 4) linked N-acetylmuramyl and N-acetylglucosaminyl glycan whose residues are crosslinked by a short peptide. LTA is a macroamphiphile, equivalent to LPS in Gram-negative bacteria, containing a substituted polyglycerophosphate backbone attached to a glycolipid, and is anchored in the membrane by the glycolipid. Recently it has been demonstrated that TLR2 may act as a receptor for PGN and LTA from Gram-positive bacteria. Whole Grampositive bacteria, soluble PGN, and LTA induced the activation of NF-␬B in 293 cell expressing TLR2 but not in cells expressing TLR1 or TLR4 (Schwander et al., 1999). Similarly, CHO fibroblast cells expressing TLR2 were activated

FIG. 4. Recognition of microbial components by TLRs. PGN, peptidoglycan; LAM, lipoarabinomannan; LTA, lipoteichoic acid; LPS, lipopolysaccharide; HSP, heat shock protein.


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by heat-killed Staphylococcus aureus and Streptococcus pneumonia and peptidoglycan from S. aureus, whereas CHO expressing TLR4 did not respond to these stimuli (Yoshimura et al., 1999). TLR-mediated activation by these Grampositive cell wall components was further assessed using TLR2 and TLR4 KO mice (Takeuchi et al., 1999a). These results demonstrated that the response to Gram-positive bacterial peptidoglycan is mediated by TLR2, but not TLR4. In contrast to the results from in vitro overexpression studies, the LTA response was found to be mediated by TLR4, but not TLR2. This finding shows that both TLR2 and TLR4 are responsible for the recognition of Gram-positive bacteria. B. MYCOBACTERIAL CELL WALL COMPONENTS Like Gram-positive bacteria, mycobacterial cell walls do not contain LPS, yet are potent stimulators of the mammalian immune system. Pathogenic mycobacteria, such as Mycobacterium tuberculosis, are part of a family of slow-growing mycobacteria that contain mannose-capped lipoarabinomannan (ManLAM), a glycolipid, in their cell walls. In contrast, rapidly growing mycobacteria are nonpathogenic in immunocompetent hosts and their cell walls contain a different glycolipid, arabinofuranosyl-capped lipoarabinomannan (AraLAM) (Chatterjee et al., 1992). AraLAM, but not ManLAM, is a potent macrophage activator (Roach et al., 1993), and this difference in macrophage response is thought to determine virulence. AraLAM has been shown to activate cells via TLR2, but not TLR4. TLR2-dependent activation of the cells by AraLAM required the presence of CD14 and was markedly augmented in the presence of LBP (Means et al., 1999a). Underhill et al. (1999) also demonstrated that TLR2 is the principal mediator of macrophage activation in response to mycobacteria. Expression of a dominant negative from of TLR2 blocked TNF␣ production induced by whole heat-killed M. tuberculosis. The mycobacterial cell wall can be separated into three major fractions: LAM, mycolylarabinogalactan-peptidoglycan complex (mAGP), and total lipids. Underhill et al. (1999) showed that the cellular responses to these components are mediated by TLR2. On the other hand, Means et al. (1999b) showed that cell activation by M. tuberculosis is mediated by both TLR2 and TLR4. However, unlike the case of Gram-positive bacteria, activation by the active components of M. tuberculosis is not augmented by the presence of CD14. As decribed above, LAM isolated from M. tuberculosis (ManLAM) does not trigger this TLR-dependent cellular activation. Further investigation by Means et al. found that cellular activation can be triggered by both soluble and cell wall-associated factors from mycobacteria via distinct TLR proteins: activation via TLR2 was found to be due to a soluble heat-stable, protease-resistant factor, probably a polysaccharide or a glycolipid, whereas activation via TLR4 was found to be due to a heat-sensitive cell-associated factor.

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C. LIPOPROTEINS Lipoproteins have been found extensively in both Gram-positive and Gram-negative bacteria, including Treponema pallidum, Mycoplasma species, and Borrelia burgdorferi, and are potent simulators of macrophages (Hoffmann et al., 1988; Radolf et al., 1991; Hauschildt et al., 1990; Muhlradt et al., 1997). The portion of lipoprotein responsible for its activity is located in the NH2-terminal triacylated lipopeptide region (Akins et al., 1993; Radolf et al., 1991). Removal of this lipid element renders the compound biologically inactive, whereas synthetically produced lipopeptides were found to activate B cells and macrophages. Stimulation of macrophages with lipoproteins or synthetic lipopeptides results in the activation of the MAP kinase family and NF-␬B (Norgard et al., 1996; Rawadi et al., 1998, 1999; Sacht et al., 1998). By fractionation of the soluble cell wall-associated proteins of M. tuberculosis, Brightbill et al. (1999) purified the component responsible for the induction of IL-12 production by macrophages, and showed it to be a 19-kD lipoprotein. Stable expression of TLR2 in HEK 293 cells conferred responsiveness to this 19-kD lipoprotein, whereas transfection of TLR2 dominant negative mutant into the RAW 264.7 macrophage cell line inhibited IL-12 p40 promoter activation by this lipoprotein. These data indicate that TLR2 can mediate gene activation by M. tuberculosis lipoprotein. Brightbill et al. (1999) also showed that several other lipoproteins stimulated TLR2-dependent transcription of iNOS and production of NO. Similarly, Hirschfeld et al. (1999) showed that transfection of TLR2 into cell lines conferred responsiveness to B. burgdorferi lipoproteins, lipopeptides, and sonicated B. furgdorferi, as measured by NF-␬B activation and cytokine production. Takeuchi et al. (2000a) demonstrated that the mycoplasmal lipopeptide-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. Aliprantis et al. (1999) further showed that induction of apoptosis by lipoproteins is also mediated through TLR2. D. ZYMOSAN Zymosan is a component of the yeast cell wall that, when phagocytosed by macrophages, induces secretion of TNF␣. Internalization of zymosan is mediated, in part, by the mannose receptor. TLR2 is recruited specifically to macrophage phagosomes containing zymosan, and cells expressing a dominant negative form of TLR2 failed to either produce TNF␣ or activate NF-␬B, indicating that TLR2 principally mediates the signals triggered by yeast zymosan (Underhill et al., 1999). E. HEAT SHOCK PROTEINS Heat shock proteins are highly conserved molecules that play an important role in protein folding and assembly, as well as in the translocation of proteins between different cellular compartments. Hsp synthesis is dramatically increased in microbes and in eukaryotic cells under conditions of stress. Microbial Hsp60


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is a major inducer of the immune system in infection (Kaufmann et al., 1991), and recent studies have suggested that mammalian Hsp60 also has potent immunostimulatory properties. Recombinant human Hsp60 was found to elicit a proinflammatory response when incubated with mouse or human macrophages, endothelial or smooth muscle cells (Chen et al., 1999b; Kol et al., 1999). This response included increased expression of adhesion molecules and the release of inflammatory mediators such as IL-6 and TNF␣. In addition, human Hsp60 caused increased gene expression of IL-12 and IL-15, both of which are essential in driving the Th1 response. Human Hsp60 has been shown to activate human macrophages in a process involving CD14 and p38 MAP kinase, similar to LPS (Kol et al., 2000). Mammalian Hsp60 can be expressed on the cell surface in response to stress or released from the cell interior as a result of necrosis, and could thereby play a role in initiating or sustaining inflammatory responses. This would suggest that damaged mammalian cells and microbial pathogens both activate innate immunity via the same recognition and signal transduction system. Indeed, macrophages of C3H/HeJ mice, which carry a mutant TLR4, have been found to be nonresponsive to human Hsp60. In this system, both Hsp60induced induction of TNF␣ and NO formation were found to be dependent on a functional TLR4 (Ohashi et al., 2000). This finding suggests that TLRs may not only function in the innate immune defense against microbial pathogens but also may mediate some physiological functions triggered by endogenous ligands. F. CpG Bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides can stimulate murine and human lymphocytes, whereas eukaryotic DNA and methylated oligonucleotides are nonstimulatory (Krieg, 1996; Lipford et al., 1998; Tokunaga et al., 1999). Unmethylated CpG motifs are much more common in bacterial DNA than in vertebrate DNA, and when present, they are more likely to be methylated. Some of the responses elicited by DNA containing unmethylated CpG motifs include B cell proliferation (Krieg et al., 1995; Sun et al., 1997), the release of TNF␣, IL-1, IL-6, and IL-12 from monocytes and macrophages (Halpern et al., 1996; Klinman et al., 1996; Stacey et al., 1996), the activation of NF-␬B and the MAP kinase family (Stacey et al., 1996; Yi and Krieg, 1998a,b; Hacker et al., 1998), and the maturation of DCs (Sparwasser et al., 1998; Jakob et al., 1998). The release of IL-12 by macrophages leads to the rapid production of IFN-␥ by NK cells as well as NK cell activation (Yamamoto et al., 1992a,b; Tokunaga et al., 1992). IFN-␥ , in turn, causes macrophage activation and consequent production of TNF␣ and IL-12. Thus, these CpG motifs stimulate a Th1-like inflammatory response characterized by the release of IL-12 and IFN-␥ (Roman et al., 1997; Chu et al., 1997; Carson and Raz, 1997; Jakob et al., 1998), and this has raised the possibility that CpG DNA might be useful as immunotherapy for Th2-dominant diseases such as allergies (Kline et al., 1998; Broide et al., 1998). However, the molecular mechanism by which CpG-DNA

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exerts these effects is not well defined. The fact that inhibitors of endosomal maturation and acidification can block CpG-DNA-induced signaling indicates that cellular uptake of CpG-DNA into the endosomes and subsequent endosomal maturation are required for signaling to be initiated (Hacker et al., 1998; Macfarlane and Manzel, 1998; Yi and Krieg, 1998a; Yi et al., 1998). Recently, it has been shown that MyD88-deficient macrophages do not respond to unmethylated CpG, indicating the involvement of TLR in the recognition of CpG (Hacker et al., 2000). Recently, TLR9 has been shown to be involved in CpG DNA recognition (Hemmi et al., 2000). Thus, the vertebrate immune system has developed to recognize elements unique to bacterial DNA, such as PAMP, and to use these to trigger innate immune defenses against infection by microorganisms. XVIII. Toll-like Receptors and Host Resistance to Microbial Infection

Although C3H/HeJ mice show little susceptibility to the lethal effect of LPS, they are highly susceptible to infection by Salmonella typhimurium. The 50% lethal dose (LD50) of S. typhimurium in C3H/HeJ mice is Naive T Activated T Th2 Th2, CLA+ Th0/Th1 B, CLA+, ␣4␤7+ B, naive T, Tcm Th2 ␣4␤7+, IEL CLA+

The chromosomal locus of each chemokine receptor is based on the latest information in Human Genome Browser and Ensembl project (see http://cytokine.medic.kumamoto u.ac.jp/).

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FIG. 2. Phylogenic relationship of the human chemokine receptors. The evolutionary distance was estimated by using the prrp and phylp programs (Gotoh, 1995).

tyrosine sulfation motif, a DRY (Asp-Arg-Tyr) motif (DRYLAIVHA) in the 2nd intracellular loop, two conserved cysteine residues, one in the amino-terminal domain and the other in the 3rd extracellular loop, forming a disulfide bond stabilizing the ligand binding pocket, and the C-terminal cytoplasmic tail rich in serine and threonine residues (Murphy, 1994; Baggiolini et al., 1997; Rollins, 1997). Like the ligand genes, the receptor genes were mostly mapped at specific loci on chromosomes 2 and 3 (Murphy, 1994; Baggiolini et al., 1997; Rollins, 1997). However, some of the recently identified chemokine receptors were also mapped to different chromosomal loci (see below). The classical “inflammatory chemokines” and their receptors tend to have highly redundant and promiscuous ligand–receptor relationships (Murphy, 1994; Baggiolini et al., 1997; Rollins, 1997). Thus, multiple chemokines can bind to a single receptor, whereas a single chemokine can bind to multiple receptors (Table II). In fact, such complex ligand–receptor relationships are one of the most unique features of the classical “inflammatory chemokines” and may indicate the involvement of multiple chemokines and chemokine receptors in induction of robust leukocyte infiltration in acute and chronic inflammatory responses (Mantovani, 1999). Notably, however, the emerging “immune chemokines” tend to have more specific ligand–receptor relationships (Table III) (Yoshie et al., 1997; Zlotnik et al., 1999; Zlotnik and Yoshie, 2000).

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CHEMOKINES IN IMMUNITY

TABLE III SUMMARY OF IMMUNE CHEMOKINES Chemokine CC Chemokine TARC MDC/STCP-1 SLC/6Ckine

Main Tissue Expression Thymus, Lung Thymus, LN LN, Spleen, PP

Main Producer Cell

Receptor

ELC/MIP-3␤

Thymus, LN, PP

TECK ILC/CTACK MEC/CCL28

Thymus, SI Skin Colon, SG, MG

LARC/MIP-3␣ PARC/DC-CK1

Lung, SI, Skin Lung, LN

DC, EK, Bronchial EP DC, M, Thymic EP HEV, Stromal cell, Lympatic EN DC, M, Thymic EP, Stromal cell Intestinal EP EK Salivary, Mammary and Colon EP EP, EK, M FDC, M

CCR4 CCR4 CCR7

CXC Chemokine IP-10 Mig I-TAC BLC/BCA-1 SDF-1/PBSF CXCL16

LN, Spleen, Thymus, Liver Spleen, Liver Pancreas, Spleen, Thymus Liver, Spleen, LN BM, Spleen, Thymus, SI LN, Spleen, SI, Lung

IFN-␥ -treated cell IFN-␥ -treated cell IFN-␥ -treated cell FDC, HEV Stromal cell DC

CXCR3 CXCR3 CXCR3 CXCR5 CXCR4 CXCR6

CX3C Chemokine Fractalkine

Brain, Heart, Lung, SI

Neuron, DC, EP, Activated EN

CX3CR1

C Chemokine Lymphotactin

Spleen, Thymus

Activated T

XCR1

CCR7 CCR9 CCR10 CCR10, CCR3 CCR6 Unknown

Abbreviations: LN, lymph node; PP, Peyer’s patch; SI, small intestine; SG, salivary gland; MG, mammary gland; BM, bone marrow; DC, dendritic cell; EP, epitehlial cell; EN, endothelial cell; EK, epidermal keratinocyte; M, macrophage; FDC, follicular dendritic cell.

Cellular responses to chemokines are generally sensitive to pertussis toxin (Baggiolini et al., 1994; Murphy, 1994; Rollins, 1997). This makes it likely that chemokine receptors are mainly coupled with a G␣i type G protein, which inhibits adenylyl cyclase. Thus, triggering of chemokine receptors leads to inhibition of cyclic adenosine monophosphate (cAMP) production and also activates multiple intracellular signaling pathways involving phospholipases C (PLC), phosphatidylinositol 3-kinase (PI3K), tyrosine kinases, small GTP-binding proteins, and mitogen-activated protein kinases (MAPKs)(Bokoch, 1995). Recently, targeted disruption of PI3K␥ or PLC-␤2/-␤3 in mice revealed that PI3K␥ , the unique G protein-coupled isoform of PI3K, plays a central role in chemoattractant-induced chemotaxis and respiratory burst of leukocytes, whereas PLC-␤2 and -␤3 are involved in chemoattractant-induced calcium mobilization and

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activation of classical protein kinase C, but not in chemotaxis (Sasaki et al., 2000; Li et al., 2000; Hirsch et al., 2000). Leukocyte responses to chemokines are also characteristically transient, and the receptors become rapidly desensitized. Phosphorylation of serine and threonine residues in the C-terminal cytoplasmic tails of chemokine receptors by a G protein-coupled receptor kinase has been shown to lead to arrestin-mediated clathrin binding and rapid internalization (Franci et al., 1996; Prado et al., 1996; Haribabu et al., 1997; Orsini et al., 1999). Signaling is also likely to depend on the cellular backgrounds. For example, germinal center B cells, even though expressing CXCR4 at high levels were shown to be refractory to stromal cell-derived factor 1 (SDF-1)/CXCL12triggered migration because of a high-level expression of regulator of G protein signaling 1 (RGS1)(Moratz et al., 2000). SDF-1/CXCL12 at high concentrations was also shown to induce repulsion instead of attraction in subpopulations of T cells via intracellular signaling biochemically different from that of attraction (Poznansky et al., 2000). C. EVOLUTION OF THE CHEMOKINE SUPERFAMILY Figures 3 and 4 show the chromosomal localization of the known chemokines and chemokine receptors. First of all, there are striking accumulations of chemokine genes mapped at the traditional CXC and CC chemokine clusters on chromosomes 4q and 17q respectively. Obviously, multiple gene duplication events have been taking place at these chromosomal sites, generating large numbers of CXC and CC chemokines (Fukuda et al., 1999; Nomiyama et al., 1999). These chemokines are mostly directed to neutrophils, monocytes, and eosinophils, and constitute the classical “inflammatory chemokines” (Baggiolini et al., 1994; Murphy, 1994; Rollins, 1997). Thus, there must have been strong evolutionary pressures to increase the number of chemokines capable of recruiting neutrophils (CXC chemokines), monocytes (CC chemokines), and eosinophils (CC chemokines). Extensive gene duplication events have also been taking place for the chemokine receptor genes on chromosomes 2 and 3 ( Baggiolini et al., 1994; Murphy, 1994; Rollins, 1997). It is thus probable that the characteristic redundant and promiscuous ligand–receptor relationships of the inflammatory chemokines have resulted from co-evolution of ligands and their receptors through multiple gene duplication and diversification as depicted schematically in Fig. 5. Furthermore, the extensive gene duplication events of the inflammatory chemokines have obviously took place even after the diversification of human and mouse species. This can be seen, for example, from the phylogenic relationship among the human and mouse MCP subgroup chemokines. As shown in Fig. 6, the human MCP proteins are more similar to one another than to mouse MCP proteins. Thus, it is often difficult and even nonsense for some inflammatory chemokines to assign genetic (not functional) species counterparts.

FIG. 3. The chromosomal localization of human chemokines.

70 FIG. 3. (Continued )

71 FIG. 4. The chromosomal localization of human chemokine receptors.

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FIG. 5. Coevolution of chemokine ligands and receptors by repeated gene duplication events. This figure is intended to explain how rapid and unequal increases in chemokine ligands and receptors in evolution could lead to redundant and promiscuous ligand–receptor relationships. L, ligand; R, receptor.

In contrast, the immune chemokines directed at lymphocytes and dendritic cells are mostly mapped at chromosomal sites different from the traditional major chemokine clusters (Yoshie et al., 1997; Zlotnik et al., 1999; Zlotnik and Yoshie, 2000). It should be noted that there are in fact two separate clusters on chromosome 4 with the one containing the immune chemokines (Lee and Farber, 1996). Furthermore, the immune chemokines have much more specific ligand-receptor relationships than the inflammatory chemokines (Table III). They are also well conserved between humans and mice, i.e., they have obvious species orthrogues. The phylogenic tree reveals that the immune chemokines were obviously diverged from the inflammatory chemokines before the extensive diversification of the latter (Fig. 1). Interestingly, there are also mini-clusters

FIG. 6. The phylogenic relationship of human and mouse MCP subgroup chemokines. The evolutionary distance was estimated by using the prrp and phylp programs (Gotoh, 1995).


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even among the chemokines located outside the major clusters (Fig. 3). The chemokines at each minicluster tend to be most closely related to each other in the phylogenic tree (Fig. 1) and act on the same receptor (Table III). Thus, the chemokines at these miniclusters are also likely to have been generated from a common ancestor and have a close functional relationship, as evident from their receptor sharing (Fig. 5). Taken together, the evolutionary distance reflected by the chromosomal and phylogenic distances among the chemokines correlates well with their different biological roles. In the following sections, we will now examine the pathophysiological roles of chemokines in various aspects of the immune system. III. Migratory Properties of Lymphocytes and Dendritic Cells

Lymphocyte precursors differentiate into B cells primarily in the fetal liver and later in the bone marrow. Some precursors are transferred to the thymus, where they differentiate into T cells. Thus, the bone marrow and thymus are the primary lymphoid organs where the major lymphopoiesis takes place. From there, mature lymphocytes enter the bloodstream and populate the secondary lymphoid organs, which include spleen, lymph nodes and lymphoid tissues associated with mucosal surfaces of the gut, and respiratory and genital tracts (the mucosa-associated lymphoid tissues). On the other hand, immature dendritic cells located in the peripheral tissues start maturation upon antigen-uptake and migrate via afferent lymphatic vessels into the secondary lymphoid organs where they become fully differentiated antigen-presenting cells capable of activating naive lymphocytes. Thus, the secondary lymphoid organs provide the strategic sites where naive lymphocytes encounter mature antigen-presenting dendritic cells. Naive lymphocytes, however, stay in the secondary lymphoid organs only shortly if not activated by antigen-presenting cells and migrate into lymphatic vessels and back to the bloodstream within hours to start another cycle of circulation, a process called lymphocyte recirculation (Springer, 1994; Butcher and Picker, 1996). When properly activated by antigen-presenting cells, on the other hand, naive lymphocytes start to proliferate and eventually differentiate into effector/memory lymphocytes, which are then seeded via the blood stream throughout the body. In contrast to naive lymphocytes, effector/memory lymphocytes migrate into peripheral tissues where they may re-encounter with their target antigens. Such migratory properties of small lymphocytes, which increases their chances to encounter with their target antigens, are essential for efficient immunological surveillance and effector functions. The migration of lymphocytes, which is closely related to their functional properties, is regulated in part by selective expression of certain cell adhesion molecules (Springer, 1994; Butcher and Picker, 1996). For example, naive T cells, which are characterized as CD45ROlowRAhigh and L-selectinhigh,

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preferentially migrate from blood into secondary lymphoid tissues such as lymph nodes and Peyer’s patches via high endothelial venules (HEVs). Naive T cells that home into lymph nodes express mainly L-selectin to interact with CD34 on HEVs in lymph nodes, while those homing to Peyer’s patches express both L-selectin and ␣4␤7 integrin to interact with MAdCAM-1 on HEVs in Peyer’s patches. Memory/effector T cells, which are characterized as CD45ROhighLselection+/−, tend to migrate into tissues such as skin and gut mucosa where they are more likely to reencounter their target antigens. T cells to be localized in the skin express cutaneous lymphocyte-associated antigen (CLA), while those directed to the gut lamina propria display high levels of ␣4␤7 integrin. Thus, differential expression of a variety of adhesion molecules, together with differential expression of various chemokine receptors as described below, plays major roles in regulation of nonrandom migration of various subsets of lymphocytes into specific tissue microenvironments (Springer, 1994; Butcher and Picker, 1996).

IV. Primary Lymphoid Organs and Chemokines

A. BONE MARROW In the parenchyma of bone marrow, various hematopoietic cells undergo proliferation and eventually differentiate into mature hematopoietic cells. Mature cells then emigrate through the sinus endothelium into the blood. Stromal cellderived factor 1 (SDF-1)/CXCL12 was originally identified from a murine bone marrow stromal cell line using a method coined “signal sequence trap” which aimed at selective cloning of secretory proteins and type I membrane proteins (Tashiro et al., 1993). The same molecule was also identified as pre-B cell stimulatory factor (PBSF) through its enhancing effects on IL-7-dependent growth of pre-B cells (Nagasawa et al., 1994). Later, SDF-1/CXCL12 was shown to be a highly efficient chemoattractant for various types of cells such as lymphocytes, monocytes, and CD34+ hematopoietic precursor cells (Bleul et al., 1996b; Aiuti et al., 1997). Thus, SDF-1/CXCL12 may be involved in homing of CD34+ hematopoietic precursor cells into the bone marrow. A seven-transmembrane G-protein-coupled receptor originally identified as fusin, the entry coreceptor for X4 type HIV-1 (Feng et al., 1996), was subsequently shown as the receptor for SDF-1/CXCL12 and is now termed CXCR4 (Bleul et al., 1996b; Oberlin et al., 1996). CXCR4 was shown to be expressed on T cells, B cells, monocytes, dendritic cells, megakaryocytes, and CD34+ hematopoietic precursor cells (Bleul et al., 1997; Hori et al., 1998; Aiuti et al., 1999). Among the CD34+ progenitor cells, the B cell lineage progenitors and precursors express CXCR4 at high density (Aiuti et al., 1999). SDF-1/CXCL12 was also shown to induce transendothelial migration of megakaryocytes and enhanced generation of platelets during this process (Hamada et al., 1998). SDF-1/CXCL12 knockout mice were generated


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and found to be defective in fetal B lymphopoiesis in the liver and bone marrow as well as in fetal myelopoiesis in the bone marrow in addition to having a defect in the cardiac septal formation (Nagasawa et al., 1996). The CXCR4 knockout mice also presented similar developmental abnormalities in fetal hematopoiesis and cardiac septal formation, and further revealed defects in the formation of large blood vessels in the gastrointestinal tract and abnormal cerebellar development (Ma et al., 1998; Tachibana et al., 1998; Zou et al., 1998). Furthermore, fetal liver cells from CXCR4−/− were unable to repopulate fetal liver and bone marrow with lymphoid and myeloid lineage cells in adoptive transfer (Kawabata et al., 1999; Ma et al., 1999), possibly due to defective retention of B lineage and myeloid lineage precursors within fetal liver and bone marrow microenvironment (Ma et al., 1999). Recently, Bowman et al. (2000) examined developing B cells in mice for their responses to a series of chemokines expressed in the bone marrow, i.e., SDF1/CXCL12, thymus expressed chemokine (TECK)/CCL25 (Vicari et al., 1997), B lymphocyte chemoattractant (BLC)/B-cell attracting chemokine 1 (BCA1)/CXCL13 (Gunn et al., 1998a; Legler et al., 1998), secondary lymphoid-tissue chemokine (SLC)/6Ckine/CCL21 (Hedrick and Zlotnik, 1997; Hromas et al., 1997b; Nagira et al., 1997; Tanabe et al., 1997; Yoshida et al., 1998b), and EBI1ligand chemokine (ELC)/macrophage inflammatory protein 3␤/CCL19 (Ross et al., 1997; Yoshida et al., 1997). Chemotactic responses to SDF-1/CXCL12 were observed at all stages of B cell differentiation. Pre- and pro-B cells and cells capable of generating pro-B cell colonies in the presence of IL-7 and flt3 ligand migrated to TECK/CCL25. Pro-B cells but not pre-B cells migrated to BLC/CXCL13. Responses to the CCR7 ligands, SLC/CCL21 and ELC/CCL19, were progressively upregulated during B cell development, making it possible that SLC/CCL21 and ELC/CCL19 are involved in the exit of mature B cells from the bone marrow. Thus, developing B cells undergo dramatic changes in their responses to chemokines and expression of chemokine receptors (Bowman et al., 2000), which is likely to regulate microenvironmental migration and localization of B cells during their development within the bone marrow. B. THYMUS T precursor cells derived from the bone marrow home into the thymic cortex, undergo dynamic processes of maturation within the thymus, and are released into the blood as mature T cells (Shortman and Wu, 1996). In the thymic cortex, T precursor cells first proliferate as double negative thymocyes. These cells differentiate into CD4 and CD8 double positive thymocyes following the rearrangement of T cell receptor genes. Double positive thymocytes are then positively selected for self-MHC reactivity and become CD4 or CD8 single positive thymocytes. These single positive thymocytes migrate into the medulla and are subjected to negative selection through interactions with medullary dendritic

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cells and epithelial cells. Autoreactive T cells are eliminated during this process. Finally, mature self-tolerant and self-MHC-restricted T cells are released into the blood and start recirculation (Anderson et al., 1996; Saito and Watanabe, 1998). Chemokines are likely to be involved in the homing of T precursor cells into the thymus and the guided migration of thymocytes within the thymus during the successive stages of thymocyte differentiation. Quite a few chemokines are constitutively expressed in the thymus. However, the identity of chemokines involved in the thymic homing of T precursor cells still remains unknown (Wilkinson et al., 1999). SDF-1/CXCL12 is ubiquitously expressed in various organs including thymus (Shirozu et al., 1995). SDF-1/CXCL12 was shown to preferentially attract immature double negative and double positive thymocytes over mature single positive thymocytes (Kim et al., 1998a). Consistently, its receptor CXCR4 was shown to be expressed in double negative and double positive thymocytes and downregulated in single positive thymocytes (Zaitseva et al., 1998; Suzuki et al., 1999a). TECK/CCL25 was originally described as a CC chemokine expressed by thymic medullary dendritic cells (Vicari et al., 1997). TECK/CCL25 was also reported to attract double negative and double positive immature thymocytes (Campbell et al., 1999b). Recent studies, however, demonstrated that TECK/ CCL25 was expressed by cortical and medullaly epithelial cells (Wilkinson et al., 1999; Wurbel et al., 2000). Its receptor CCR9 was identified (Youn et al., 1999; Zaballos et al., 1999) and found to be expressed by double positive and single positive thymocytes (Youn et al., 1999; Wurbel et al., 2000). Thus, TECK/CCL25 may be involved in the migration of thymocytes in relatively late stages of maturation. Thymus and activation-regulated chemokine (TARC)/CCL17 (Imai et al., 1996) and macrophage derived chemokine (MDC)/CCL22 (Chang et al., 1997; Godiska et al., 1997; Kodelja et al., 1998; Schaniel et al., 1998), which share CCR4 (Imai et al., 1997a, 1998), are expressed by medullary dendritic cells and medullary epithelial cells, respectively (Chantry et al., 1999; Lieberam and Forster, 1999; Imai et al., unpublished results). CD4+CD8low to CD4-single positive thymocytes were shown to express CCR4 (Chantry et al., 1999; Suzuki et al., 1999a). Furthermore, positively selected CD69+CD3intermediate cells were shown to express CCR4 (Suzuki et al., 1999a). Thus, these chemokines are likely to be involved in the migration of CD4+CD8low to CD4-single positive thymocytes from the cortex into the medulla, a necessary step for negative selection. Recently, epithelial cells expressing MDC/CCL22 were found to localize at the outer wall of Hassal’s corpuscle and to express CD30L, while thymocytes attracted by MDC/CCL22 were positive for CD30 (Annuziato et al., 2000). Since CD30 and CD30L have been implicated in the thymic negative selection (Amakawa et al., 1999; Chiarle et al., 1999), CCR4-expressing CD4+CD8low to CD4-single


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positive thymocytes may be attracted by CD30L-expressing medullary epithelial cells via MDC/CCL22 and may be eliminated through upregulation of CD30 if activated by epithelial cell-expressing autoantigens. SLC/CCL21 and ELC/CCL19 were also detected in the thymus (Nagira et al., 1997; Tanabe et al., 1997; Yoshida et al., 1997). In murine thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained by anti-SLC/CCL21 (Tanabe et al., 1997). Furthermore, ELC/CCL19 was found to be expressed by epithelial cells located around medullary vessels (Annunziato et al., 2000). Notably, ELC/CCL19-expressing epithelial cells were negative for CD30L, while thymocytes attracted by ELC were negative for CD30 (Annunziato et al., 2000). Thus, SLC/CCL21 and/or ELC/CCL19 may be involved in the emigration process of mature CD4- or CD8-single positive mature thymocyes from the thymus. V. Secondary Lymphoid Organs and Chemokines

Mature lymphocytes are released from the primary lymphoid organs into blood to start recirculation between the hematolymphatic vascular systems and secondary lymphoid organs (Butcher and Picker, 1996). The secondary lymphoid organs include spleen, lymph nodes, and Peyer’s patches. These organs have common structural features such as T cell zones with numerous HEVs, Bcell zones with primary follicles and germinal centers, and supporting mesh-like structures formed by stromal cells and reticular cells where dendritic cells and macrophages are lodged (Gretz et al., 1996). Naive T cells enter the secondary lymphoid organs via HEVs (Girard and Springer, 1995) and scan antigenic peptides presented by mature dendritic cells (DCs) in the T cell zone. On the other hand, antigen-loaded and/or cytokine-stimulated immature DCs in the peripheral tissues migrate into the T cell zones via afferent lymphatic vessels to become fully differentiated antigen-presenting cells (APCs)(Butcher and Picker, 1996; Butcher et al., 1999). Naive T cells require two stimulatory signals for full activation; T cell receptor-mediated signals and costimulatory signals via CD28 (Janeway and Bottomly, 1994). Mature DCs express CD80 and CD86, the CD28 ligands for costimulatory signals. When activated by APCs, naive T cells proliferate and differentiate into effector/memory T cells. In contrast to naive T cells, effector/memory T cells are less dependent on the costimulatory signals and preferentially migrate into tissues such as skin and mucosal lamina propria (tertiary lymphoid tissues) where they are more likely to encounter target antigens (Butcher et al., 1999). SLC/CCL21 and ELC/CCL19 are constitutively expressed in the secondary lymphoid organs and share CCR7 (Nagira et al., 1997; Yoshida et al., 1997; Campbell et al., 1998a; Yoshida et al., 1998b; Willimann et al., 1998). Notably, murine but not human SLC/CCL21 also transduces signals via CXCR3

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(Soto et al., 1998; Jenh et al., 1999). SLC/CCL21 and ELC/CCL19 were highly efficient chemoattractants for B cells and various subsets of T cells including CD62Lhigh naive T cells in vitro (Gunn et al., 1998b; Kim et al., 1998b; Nagira et al., 1998; Ngo et al., 1998; Yoshida et al., 1998a; Willimann et al., 1998). SLC/CCL21 and ELC/CCL19 were also the first chemokines shown to be capable of rapidly inducing integrin-mediated firm attachment of naive T cells and B cells under flow conditions (Campbell et al., 1998b; Gunn et al., 1998b; Pachynski et al., 1998; Tangemann et al., 1998). In normal mice, SLC/CCL21 is expressed by HEV and some stromal cells in the parafollicular T cell zones of lymph nodes and Peyer’s patches (Gunn et al., 1998b). Furthermore, lymphatic endothelial cells of multiple organs also express SLC/CCL21 (Gunn et al., 1998b). On the other hand, ELC/CCL19 is mainly expressed by interdigitating DCs and also by some stromal cells in the T cell zone (Ngo et al., 1998). Recently, however, ELC/CCL10 protein was also detected on the luminal side of HEVs (Breitfeld et al., 2000). In mice homozygous for a spontaneous mutation called paucity of lymph node T cell (plt), naive T cells failed to home into lymph nodes or the lymphoid regions of the spleen (Nakano et al., 1997; Gunn et al., 1999). Peripheral tissue DCs were also unable to migrate into the T cell zones of lymph nodes and spleen in the plt mice (Gunn et al., 1999). Notably, the plt gene was mapped to the SLC locus, and the plt mice showed deficient SLC/CCL21 expression and reduced ELC/CCL19 expression in the secondary lymphoid organs (Gunn et al., 1999). Recently, it was demonstrated that the mouse SLC is encoded by two genes, one of which is selectively expressed in the lymphoid organs and deleted in the plt mice (Vassileva et al., 1999). CCR7-knockout mice were also shown to have profound morphological alterations in all secondary lymphoid organs, due to impaired homing of lymphocytes and peripheral tissue DCs (Foerster et al., 1999). These mice also displayed severely delayed antibody responses and practically no contact sensitivity and delayed type hypersensitivity (Foerster et al., 1999). Thus, SLC/CCL21 and probably ELC/CCL19 also play key roles in the homing of recirculating T cells via HEV as well as that of antigen-loaded peripheral tissue DCs via afferent lymphatic vessels. Indeed, ectopic expression of SLC/CCL21 in pancreatic islets induced infiltration of T cells and DCs, which eventually led to development of tissues similar to secondary lymphoid tissues (Fan et al., 2000). ELC/CCL19 may also have a specialized role in a close interactions between mature APCs and T cells within the T cell zone (Ngo et al., 1998). Recently, Warnock et al. (2000) reported that absence of SLC/CCL21 presentation or functional inhibition of CCR7 effectively suppressed rolling T cells, but not rolling B cells, to firmly adhere to HEVs in Peyer’s patches. Thus, B cells may be triggered by a chemokine independent from or in addition to SLC/CCL21 or ELC/CCL19 (Warnock et al., 2000). Furthermore, the authors observed


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substantial variability in immunological staining intensity of SLC/CCL21 in HEVs; the most intensely stained segments were predominantly located within interfollicular T cell areas, whereas HEVs negative for SLC/CCL21 staining were most common in or adjacent to follicles (B cell areas). Thus, vascular specialization may also contribute to differential recruitment of circulating T and B cells into appropriate microenvironments. In this context, Schaerli et al., (2000) recently described that HEVs within the follicle mantle selectively express BLC/CXCL13, a B cell-attracting chemokine (see below). Another important chemokine associated with the secondary lymphoid organs is BLC/CXCL13, which is expressed in the B cell zones of the secondary lymphoid organs and selectively attracts B cells via CXCR5 (Gunn et al., 1998a; Legler et al., 1998). CXCR5 was originally reported as Burkitt’s lymphoma receptor 1 (BLR1) and shown to be expressed by B cells and a fraction of CD4+CD44high memory T cells (Dobner et al., 1992; Foerster et al., 1994). Even before identification of its ligand, BLR1/CXCR5-deficient mice were generated and found to have gross anatomical defects in the secondary lymphoid organs, such as lack of inguinal lymph nodes, impaired development of Peyer’s patches, and defective formation of primary follicles and germinal centers in the spleen (Foerster et al., 1996). When injected into normal mice, BLR1/CXCR5−/− B cells failed to transit from the T cell zone to B cell follicles in the spleen and Peyer’s patches (Foerster et al., 1996). Thus, the ligand for BLR1/CXCR5 appeared to be essential for B cell migration within specific anatomical compartments in the spleen and Peyer’s patches. Subsequently, BLC/CXCL13 was identified as the ligand for BLR1/CXCR5 and shown to be selectively chemotactic for B cells and constitutively expressed by follicular dendritic cells (FDCs) in the B cell zones of the secondary lymphoid organs (Gunn et al., 1998a; Legler et al., 1998). Recently, BLC/CXCL13-knockout mice were generated and shown to have severe defects in the secondary lymphoid organs with poor development of primary follicles most probably due to lack of FDCs (Ansel et al., 2000). Thus, naive B cells are likely to be guided into B cell follicles by BLC/CXCL13 produced by FDCs after homing to the secondary lymphoid organs via HEV (Ansel et al., 2000). Mice with targeted gene disruption of TNF, lymphotoxin (LT)-␣/␤ or their receptors were known to have abnormalities in the secondary lymphoid tissues (these mice lack polarized B cell follicles and have disorganized T cell zones; Matsumoto et al., 1997). These mice were found to be critically defective in BLC/CXCL13 expression by FDCs in the B cell zone (Ngo et al., 1999). BLC/CXCL13 was also shown to induce expression of the membranebound LT␣1␤2 in B cells (Ansel et al., 2000). Furthermore, ectopic expression of BLC/CXCL13 in pancreatic islet cells led to formation of secondary lymphoid tissues through an LT␣1␤2-dependent mechanism (Luther et al., 2000). Taken together, the likely scenario is that FDCs, by producing BLC/CXCL13, attract

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B cells and upregulate their expression of membrane-bound LT␣1␤2, which in turn stimulates FDCs to further develop and produce BLC/CXCL13 (Ansel et al., 2000). Such a positive feedback loop between FDCs and B cells is likely to be important in development and homeostasis of B cell follicles.

VI. Effector/Memory Cells and Chemokines

A. Th1 AND Th2 CELLS Effector/memory T cells of the CD4 lineage are subdivided into Th1 and Th2 types depending on their cytokine profiles. Th1 cells produce cytokines such as IL-2, IFN-␥ and LT-␤, and are responsible for cell-mediated immunity and organ-specific autoimmune diseases. Th2 cells produce cytokines such as IL-4, IL-5, IL-6, and IL-13, and are involved in humoral immunity and allergic diseases (Mosmann and Sad, 1996). Similar polarization is also seen in cytotoxic CD8+ T cells, i.e., Tc1 and Tc2 (Sad et al., 1995). Th1 and Th2 cells have differential migration patterns in accordance with the type of immune responses. For example, Th1 cells accumulate in skin lesions of delayed type hypersensitivity or antigen-induced arthritis, whereas Th2 cells accumulate in tissues with allergic diseases such as atopic dermatitis and asthma. Such differential migration patterns are regulated in part by selective expression of certain cell adhesion molecules. For example, selective expression of PSGL-1, the fucosyl transferase VII-dependent ligand for P- and E-selectins (Knibbs et al., 1998), was shown to account for selective infiltration of Th1 cells into Th1-dominant lesions such as sensitized skin or arthritic joints (Austrup et al., 1997). IL-12, a potent inducer of Th1 polarization, was shown to upregulate ␣6␤1 integrin and CCR1 in human Th1 cells (Colantonio et al., 1999). Chemokines regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, and SDF-1/CXCL12 were shown to activate ␤1 integrins in Th1 cells but not in Th2 cells (Clissi et al., 2000). Besides adhesion molecules, recent studies have revealed differential expression of chemokine receptors on Th1 and Th2 cells. While still needing careful in vivo evaluation, human Th1 cells were shown to selectively express CXCR3 and CCR5, and human Th2 cells express CCR3, CCR4, and CCR8 (Sallusto et al., 1997, 1998a; Bonecchi et al., 1998a; D’Ambrosio et al., 1998; Zingoni et al., 1998; Annunziato et al., 1999; Imai et al., 1999). CXCR5 may also be Th2selective (Foerster et al., 1994; Fynn et al., 1998; Ansel et al., 1999). In the case of murine effector/memory T cells, CXCR3, CCR5, and CCR7 were shown to be Th1-selective, whereas CCR3 and CXCR4 were Th2-selective (Siveke and Hamann, 1998; Randolph et al., 1999). There were, however, discrepancies in Th2-selective expression of CCR4 in mice (Schaniel et al., 1998; Randolph et al.,


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1999; Syrbe et al., 1999; Lloyd et al., 2000). It is also important to note that TCR or cytokine stimulation can strongly modulate chemokine receptor expression in memory/effector T cells (Sallusto et al., 1998a; Loetscher et al., 1998; Annunziato et al., 1999; Sallusto et al., 1999a). For example, CXCR3 and CCR5 were shown to be upregulated in resting T cells by IL-2 (Loetscher et al., 1996; Bleul et al., 1997), making it likely that they are also expressed by some Th0 cells (Sallusto et al., 1998a). TGF-␤, which suppresses Th2 polarization, was shown to inhibit CCR3 but upregulate CCR4 (Sallusto et al., 1998a). CXCR4 was markedly upregulated by IL-4 but downregulated by IFN-␥ (Jourdan et al., 1998; Annunziato et al., 1999). Thus, the expression of selective chemokine receptors in Th1 and Th2 cells can be a subject of highly dynamic regulation by local milieu of cytokines and cellular interactions. CXCR3 is the shared receptor for three CXC chemokines commonly induced by IFN-␥ , i.e., IFN-␥ -inducible protein 10 (IP-10)/CXCL10, monokine induced by IFN-␥ (Mig)/CXCL9, and interferon-inducible T cell ␣-chemoattractant (I-TAC)/CXCL11 (Loetscher et al., 1996; Cole et al., 1998). CCR5 is now known to be the shared receptor for four inflammatory chemokines, RANTES/CCL5, MIP-1␣/CCL3, MIP-1␤/CCL4 and MCP-2/CCL8 (Raport et al., 1996; Gong et al., 1998; Ruffing et al., 1998). CCR5 is also the major coreceptor for macrophage-tropic strains of human immunodeficiency viruses (Berger et al., 1999). In peripheral blood, CXCR3 and CCR5 were both expressed in a fraction of T cells as well as those of B cells and natural killer (NK) cells (Loetscher et al., 1998; Qin et al., 1998). CCR5 was also expressed in monocytes (Raport et al., 1996). Blood T cells expressing CXCR3 and CCR5 were mostly CD45RO+ and expressed high levels of ␤1 integrins, a phenotype resembling T cells infiltrating inflammatory lesions (Qin et al., 1998). Endothelial cells stimulated with IFN-␥ were shown to induce selective diapedesis of Th1 cells by producing RANTES/CCL5 (Kawai et al., 1999). Furthermore, intracellular cytokine staining of stimulated peripheral blood T cells revealed that cells capable of producing IFN-␥ were restricted to CXCR3-expressing memory CD4+ T cells, irrespective of presence or absence of CCR5 expression (Yamamoto et al., 2000). In rheumatoid arthritis, a well-known Th1-type disease, infiltrating T cells in synovial fluids were shown to express CXCR3 at high frequencies and, less frequently, CCR5 (Qin et al., 1998; Suzuki et al., 1999b; Wedderburn et al., 2000). T cells expressing CXCR3 and CCR5 were also increased in the blood, and their ligands were upregulated in demyelinating brain lesions of patients with multiple sclerosis (Balashov et al., 1999). In mice, IP-10/CXCL10 was shown to be essential for host defense against Toxoplasma gondii infection, a model of Th1-type immunity (Khan et al., 2000). There were also many studies using CCR5-knockout mice (Zhou et al., 1998; Huffnagle et al., 1999; Sato et al., 1999b; Dawson et al., 2000; Andres et al., 2000) and at least one demonstrated a shift to a Th2-type T cell activation in a colitis

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model induced by orally administered dextran sulfate (Andres et al., 2000). Collectively, these results support that CXCR3 and less consistently CCR5 play the major roles in Th1-dominant responses. CCR3 is expressed highly selectively on eosinophils and basophils, and shared by many inflammatory CC chemokines such as eotaxin/CCL11, RANTES/CCL5, and MCP-3/CCL7 (Daugherty et al., 1996; Kitaura et al., 1996; Ponath et al., 1996; Heath et al., 1997; Uguccioni et al., 1997). This probably reflects the evolutionary importance of eosinophils in the host defense against parasitic infection. Sallusto et al., (1997) originally noted CCR3 expression on a small fraction (∼1%) of peripheral blood memory T cells, which they demonstrated to be polarized to Th2 phenotype. This finding was quite provocative because of the possible recruitment of eosinophils and Th2 cells by the same chemokines in allergic diseases. However, other researchers found it rather difficult to demonstrate CCR3 on Th2 cells at significant levels (Annunziato et al., 1999;Imai et al., 1999). Thus, CCR3 may be expressed on a subset or at a particular activation state of Th2 cells in vivo. Notably, combined treatment with IL-2 (a Th1 cytokine) and IL-4 (a Th2 cytokine) was shown to induce expression of CCR3 on T cells in vitro (Jinquan et al., 1999). CCR4, the shared receptor for TARC/CCL17 and MDC/CCL22 (Imai et al., 1997a, 1998), was regularly found to be selectively expressed on T cell lines chronically polarized to Th2 (Bonecchi et al., 1998b; D’Ambrosio et al., 1998; Annunziato et al., 1999; Imai et al., 1999; Sallusto et al., 1998a). Furthermore, Imai et al., (1999) demonstrated CCR4 expression on a substantial fraction (∼20%) of CD4+ memory T cells in adult peripheral blood, which generated T cell lines capable of produceing IL-4 and IL-5 at high levels upon stimulation. CCR4 was also selectively expressed on Th2-polarized T cell lines generated in vitro from peripheral blood CD45RA+ naive T cells (Imai et al., 1999). Yamamoto et al., (2000) further extended this observation by carrying out intracellular cytokine staining of peripheral blood T cells after stimulation, and demonstrated that T cells capable of producing Th2 cytokines were restricted to CCR4-expressing CD4+ memory T cells. They also found that CCR4-expressing memory T cells were clearly increased in the blood of patients with atopic dermatitis (Yamamoto et al., 2000). Thus, CCR4 is likely to be the major chemokine receptor expressed on peripheral blood Th2 cells in vivo. This does not, however, exclude the possibilty that some Th0 cells or even Th1 cells could express CCR4. In fact, D’Ambrosio et al. (1998) observed upregulation of CCR4 in Th1 cells upon stimulation with anti-CD3 and anti-CD28. Furthermore, Campbell et al. (1999a) reported that CCR4 was expressed on most CLA+ skin-homing memory T cells and a subset of CLA- systemic memory T cells but not on ␣4␤7+ gut-homing memory T cells, while TARC/CCL17 was produced by microvascular endothelial cells in inflammatory skin lesions. According to this observation, TARC/CCL17 and CCR4 could be important for the emigration of CLA+


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memory T cells, probably containing both Th1 and Th2 cells, via inflamed skin microvascular endothelium. Nevertheless, accumulating evidence strongly supports the important pathogenic roles of CCR4 and its ligands TARC/CCL17 and MDC/CCL22 in Th2-type diseases such as bronchial asthma and atopic dermatitis, in both mice and in humans. For example, enhanced expression of TARC/CCL17 and MDC/CCL22 was demonstrated in atopic skin lesions of NC/Nga mice and human patients (Vestergaard et al., 1999, 2000; Galli et al., 2000). Production of MDC/CCL22 by activated T cells correlated with production of Th2-type cytokines, and serum MDC/CCL22 contents were elevated in patients with atopic dermatitis (Galli et al., 2000). Production of MDC/CCL22 and TARC/CCL17 by peripheral blood monocytes was strongly upregulated by Th2 cytokines such as IL-4 and IL-13 but suppressed by IFN-␥ (Andrew et al., 1998; Bonecchi et al., 1998b; Imai et al., 1999). Blocking MDC/CCL22 or TARC/CCL17 by neutralizing antibodies effectively suppressed lung tissue infiltration of T cells and other leukocytes as well as airway hyperactivity to methacholine in murine asthmatic models (Gonzalo et al., 1999; Kawasaki et al., 2001). Naive mice that were adoptively transferred with in vitro polarized Th2 cells and that received multiple antigenic challenges developed prominent infiltration of T cells and eosinophils and airway hypersensitivity where CCR3 and CCR4 played dominant pathogenic roles, the latter more significantly in the chronic phase (Lloyd et al., 2000). Enhanced expression of TARC/CCL17 was also demonstrated in bronchial epithelium of asthmatic patients (Sekiya et al., 2000). It is also notable that IFN-␥ and TNF-␣ could induce expression of TARC/CCL17 by epidermal keratinocytes and bronchial epithelial cells (Vestergaard et al., 1999, 2000; Sekiya et al., 2000). This may reflect potential crosstalk between Th1 and Th2 responses at the interface of body surfaces. It should also be mentioned, however, that Chvatchko et al. (2000) found no changes in T cell development or Th2-dependent allergic airway inflammation by the targeted disruption of CCR4 in mice. This could be in part due to some developmental compensations in the knockout mice and may point out the necessity of studies using receptor-blocking reagents such as neutralizing antibodies for elucidation of the true pathophysiologic roles of chemokine receptors in vivo. CCR8, the receptor for I-309/CCL1 (Roos et al., 1997; Tiffany et al., 1997; Goya et al., 1998), was also shown to be selectively expressed on polarized Th2 cell lines (D’Ambrosio et al., 1998; Zingoni et al., 1998). However, CCR8 expression in peripheral blood T cells has not been examined yet. CCR8 was also once claimed to be a receptor for TARC/CCL17 (Bernardini et al., 1998), but this finding was later denied (Garlisi et al., 1999). Sallusto et al. (1999b) reported that expression of CCR7 divided human memory T cells into two functionally distinct subsets. CCR7− memory T cells expressed L-selectin at low levels and displayed immediate effector function. In contrast, CCR7+ memory T cells expressed L-selectin at high levels and lacked

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immediate effector functions, but efficiently stimulated DCs and differentiated into CCR7− effector cells upon secondary stimulation. Thus, CCR7− memory T cells, which they termed “effector memory” T cells, are likely to migrate preferentially into inflamed tissues, while CCR7+ memory T cells, which they termed “central memory” T cells, are likely to be lymph node homing, and the expression of CCR7 may reflect step-wise differentiation of memory T cells from naive T cells (Sallusto et al., 1999b). Notably, CCR4 was expressed in both central and effector memory T cells, whereas CCR3 was selectively expressed in effector memory T cells (Sallusto et al., 1999b), further suggesting that CCR3 is expressed by a subset (immediate effector) of Th2 cells. CXCR5, the receptor for BLC/CXCL13 and mainly expressed by B cells (Gunn et al., 1998a; Legler et al., 1998), was also noted to be expressed on a fraction of CD4+ memory T cells in peripheral blood and on the majority of CD4+ T cells in spleen and tonsil (Foerster et al., 1994). Recently, accumulation of antigen-specific T cells in follicles in Th2-promoting immunization conditions was shown to correlate with upregulation of CXCR5 in these T cells (Ansel et al., 1999). Moreover, CXCR5high T cells were found to exhibit vigorous chemotactic responses to BLC/CXCL13 but much reduced responses to the T cell zone chemokines, ELC/CCL19 and SLC/CCL21 (Ansel et al., 1999). Thus, reprogramming of chemokine receptor expression from CCR7 to CXCR5 may navigate T cells from the T cell zone to the B cell compartment in order to carry out B cell help. Indeed, forced expression of CCR7 in murine Th2 cells by retroviral transduction inhibited their participation in B cell help in vivo (Randolph et al., 1999). Furthermore, naive CD4+ T cells stimulated with anti-CD3 and anti-CD28 were shown to be induced to express IL-4 and upregulate CXCR5 upon co-stimulation through OX40 by OX40L expressed on antigen-activated B cells (Flynn et al., 1998). It was also shown that mice rendered deficient in CD28 signaling were unable to upregulate OX40 expression by T cells upon immunization and failed to form germinal centers (Walker et al., 1999). Thus, activated B cells may instruct antigen-stimulated T cells in the T cell zone to differentiate into Th2 cells expressing CXCR5, which then respond to BLC/CXCL13 and migrate into B cell follicles to carry our B cell help. Furthermore, CXCR5+CD4+ memory T cells present in B cell follicles and germinal centers of human tonsils were shown to express high levels of costimulatory molecules such as CD40L and inducible costimulator (ICOS) and to efficiently support production of IgA and IgG by tonsillar B cells in coculture even though these cells were not efficient in production of cytokines (Breitfeld et al., 2000; Schaerli et al., 2000). In addition, high levels of BLC/CXCL15 expression were demonstrated in the mantle zone and follicluar HEVs, which may account for direct recruitment of circulating CXCR5+ memory T cells into the B cell follicles (Breitfeld et al., 2000; Schaerli et al., 2000). Based on these findings, a term follicluar B helper T


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cells (TFH) has been proposed for this type of memory helper T cells (Breitfeld et al., 2000; Schaerli et al., 2000). Lymphotactin/SCM-1/XCL1,2 are the sole members of the C subfamily (Kelner et al., 1994; Kennedy et al., 1995; Mueller et al., 1995; Yoshida et al., 1995, 1996). Its receptor was identified and termed XCR1 (Yoshida et al., 1998c, 1999). Lymphotactin/XCL1 was shown to inhibit anti-CD3-induced proliferation of CD4+ T cells but to enhance that of CD8+ T cells (Cerdan et al., 2000). Furthermore, lymphotactin/XCL1 was shown to inhibit production of Th1-type cytokines but not Th2-type cytokines from CD4+ T cells stimulated with antiCD3 (Cerdan et al., 2000). The latter observation may indicate selective expression of XCR1 on Th1 cells. B. INTESTINAL IMMUNITY AND CHEMOKINES The mucosal surfaces of the oral cavity, pharynx, esophagus, urethra, and vagina are covered by stratified, nonkeratinized or parakeratinized epithelia. The lining of the airways, including nasopharynx, trachea, bronchi, and bronchioles ranges from pseudostratified to simple epithelium. On the other hand, the vast mucosal surfaces of the gastrointestinal tract are lined by a single layer of epithelial cells. Since foreign antigens and microorganisms on mucosal surfaces are separated from the mucosal immune system by epithelial barriers, efficient antigen sampling strategies are crucial for the local immune systems. In stratified epithelia, motile DCs similar to Langerhans cells in the skin (see below) migrate into the intraepithelial spaces, where they uptake antigens to carry back to local mucosa-associated lymphoid tissues or regional lymph nodes. In simple epithelia, on the other hand, M (microfold) cells are present in specialized follicle-associated epithelium (FAE) to transport antigens directly to organized lymphoid tissues underneath (Neutra et al., 1996; Mowat and Viney, 1997). The mucosal immune system can be functionally divided into the inductive site and effector site (Neutra et al., 1996; Mowat and Viney, 1997). Organized lymphoid aggregates present in the wall of the small and large intestines are the primary inductive sites (secondary lymphoid tissues) in the gastrointestinal tract. In the small intestine, these collections are called Peyer’s patches, which are composed of specialized epithelium (FAE) with M cells, subepithelial dome overlying B cell follicles with germinal centers, and interfollicular regions containing HEVs and efferent lymphatics. On the other hand, the main effector sites are the intestinal lamina propria, where effector T cells and B cells home via blood vessels after induction in Peyer’s patches. The intestinal epithelium also contains unique populations of T cells called intraepithelial lymphocytes (IELs) (Neutra et al., 1996; Mowat and Viney, 1997). In contrast to other lymphocyte populations present in the gut, IELs are considered to develop locally from precursors present in the cryptopatches (Oida et al., 2000).

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Recent studies have also revealed the important roles of chemokines in the gastrointestinal immune system. Liver and activation-regulated chemokine (LARC)/MIP-3␣/Exodus/CCL20 (Hieshima et al., 1997b; Hromas et al., 1997b; Ross et al., 1997) was the first chemokine demonstrated to be expressed constitutively in the gastrointestinal tract. In situ hybridization revealed that intestinal follicle-associated epithelium constitutively expresses LARC/CCL20 (Tanaka et al., 1999; Iwasaki and Kelsall, 2000). Its receptor was identified and termed CCR6 (Baba et al., 1997; Greaves et al., 1997; Liao et al., 1997; Power et al., 1997). Originally, immature DCs were found to express CCR6 and to be attracted by LARC/CCL20 (Greaves et al., 1997; Power et al., 1997; Dieu et al., 1998; Dieu-Nosjean et al., 1999). Recently, CCR6 was also shown to be expressed by B cells and most ␣4␤7+ intestine-seeking memory T cells as well as some CLA+ skin-seeking memory T cells in human peripheral blood (Liao et al., 1999). Thus, it is likely that LARC/CCL20 attracts immature DCs, B cells, and ␣4␤7+ memory T cells toward the subepithelial dome of organized lymphoid tissues in the small intestine and colon. Furthermore, by studying anatomical localization of DC subsets in murine Peyer’s patches, Iwasaki and Kelsall (2000) demonstrated that CD11b+ myeloid DCs were present in the subepithelial dome (SED), CD8␣+ lymphoid DCs were present in the T cell-rich interfollicular region, and DCs without expression of CD8␣ or CD11b (double negative) were present in both regions. They further demonstrated that CCR6 was functionally expressed only by CD11b+ myeloid DCs present in the SED (Iwasaki and Kelsall, 2000). CCR6-knockout mice were generated and indeed shown to have selective absence of myeloid DCs expressing CD11c and CD11b from SED of Peyer’s patches (Cook et al., 2000). These mice also displayed a diminished humoral immune response to orally administered antigen and to the enteropathic rotavirus infection (Cook et al., 2000). Notably, CCR6+ mice also had dramatic increases in various subsets of TCR␣␤ T cells within the entire mucosa of the small intestine for unknown reasons (Cook et al., 2000). Besides in the thymus, TECK/CCL25 was also found to be expressed highly selectively in the small intestine (Zabel et al., 1999; Kunkel et al., 2000; Wurbel et al., 2000). Immunohistochemical studies revealed that crypt epithelium and lower villus epithelium in the jejunum and ileum, but not other epithelia of the digestive tract including colon and stomach, was strongly positive for TECK/ CCL25 (Kunkel et al., 2000; Wurbel et al., 2000). Its receptor CCR9 (Youn et al., 1999; Zaballos et al., 1999; Zabel et al., 1999), was found to be expressed by a subset of ␣4␤7+ intestine-seeking memory T cells in human peripheral blood (Zabel et al., 1999). Furthermore, all intestinal lamina propria lymphocytes and IELs, but only a small subset of lymphocytes in the colon, were found to express CCR9 (Kunkel et al., 2000). Thus, TECK/CCL25 and CCR9 appear to play important roles in recruiting effector T cells into small intestine.


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Fractalkine/CX3CL1, a membrane-type chemokine having a combined property of a chemoattractant and a cell adhesion molecule (Imai et al., 1997b; Fong et al., 1998; Haskell et al., 1999; Goda et al., 2000), was also found to be expressed by normal intestinal epithelial cells and upregulated during active Crohn’s disease, whereas a subpopulation of isolated IELs was found to express its receptor CX3CR1 (Muehlhoefer et al., 2000). Thus, fractalkine/CX3CL1 may play a role in both normal and pathologic immune responses of the intestinal mucosa. Recently, another chemokine expressed in the mucosal tissues was identified and termed mucosae-associated epithelial chemokine (MEC)/CCL28 (Pan et al., 2000; Wang et al., 2000). MEC/CCL28 attracted memory T cells via CCR10 and eosinophils via CCR3 (Pan et al., 2000; Wang et al., 2000). Among different tissues, MEC/CCL28 was expressed at high levels in salivary gland, colon, trachea, and mammary gland, but not in small intestine. Consistently, MEC/CCL28 was found to be expressed by bronchial and mammary gland epithelial cell lines and in epithelia from salivary gland and colon (Pan et al., 2000). Thus, TECK/CCL25 and MEC/CCL28 may recruit different subsets of lymphocytes to different segments of the mucosal surfaces in order to mount specialized local immune responses. C. SKIN IMMUNITY AND CHEMOKINES The skin, which is covered with a layer of keratinizing squamous epithelial cells, also has important immunological functions. Unlike the gastrointestinal tract, the skin has no organized lymphoid aggregates or M cells specialized for antigen transfer. Instead, many mobile DCs are present in the epidermal and dermal layers. The epidermal DCs called Langerhans cells and dermal DCs are phenotypically different and typically CD1a+CD11b-CD36-E-cadherin+ Birbeck granule+ factor XIIIa− and CD1a+CD11b+CD36+E-cadherin-Birbeck granulefactor XIIIa+, respectively (Hart, 1997). It is now believed that immature DCs expressing CCR6 are attracted toward the body surfaces by LARC/CCL20 produced by surface lining epidermal cells (Charbonnier et al., 1999). Some CLA+ skin-seeking memory T cells in human peripheral blood were also shown to express CCR6 (Liao et al., 1999) and may be attracted by LARC/CCL20 expressed by epidermal keratinocytes. Upregulation of LARC/CCL20 and recruitment of CCR6-expressing T cells and DCs were indeed shown to be involved in the skin lesions of psoriasis and atopic dermatitis (Dieu-Nosjean et al., 2000; Homey et al., 2000a; Nakayama et al., 2001). Defensins are a family of small cationic antimicrobial peptides found in mammals, insects, and plants (Ganz and Lehrer, 1994). Recently, Yang et al. (1999a) demonstrated that ␤-defensins, which are the members produced by surface lining cells such as skin epidermal cells and trachea-bronchial epithelial cells upon microbial invasion or by proinflammatory stimuli, were also found to be a potent agonist for CCR6 (Yang et al., 1999a). Thus, CCR6 may have a capacity to

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link innate and adaptive immune responses by responding to both ␤-defensins and LARC/CCL20. As mentioned above, most CLA+ skin-seeking memory T cells in human peripheral blood were reported to express CCR4 (Campbell et al., 1999a). Furthermore, microvessels in inflamed skin tissues were found to produce its ligand TARC/CCL17 (Campbell et al., 1999a). Thus, CCR4 and TARC/CCL17 may play important roles in emigration of CLA+ skin-homing memory T cells from microvessels in inflammatory skin lesions. Interleukin 11 receptor ␣-locus chemokine (ILC)/cutaneous T cell attracting chemokine (CTACK)/CCL27 is another chemokine expressed highly selectively in the skin (Baird et al., 1999; Hromas et al., 1999; Ishikawa-Mochizuki et al., 1999; Morales et al., 1999). This chemokine is also the potential host homologue of MC148R (Ishikawa-Mochizuki et al., 1999), which is a viral chemokine encoded by skin-infecting molluscum contagiosus virus (Bugert et al., 1998; Damon et al., 1998; Luttichau et al., 2000). Epidermal keratinocytes were shown to produce ILC/CCL27 (Morales et al., 1999). Its receptor CCR10 was identified (Homey et al., 2000b; Jarmin et al., 2000) and shown to be expressed in T cells and skin-derived Langerhans cells (Homey et al., 2000b). Thus, ILC/CCL27 and CCR10 may play a role in recruitment of memory T cells and Langerhans cells into the skin. However, CCR10 was found to be mainly expressed in the mucosal tissues (Jarmin et al., 2000). Furthermore, its ligand MEC/CCL28 selectively expressed in the mucosal tissues such as salivary gland, mammary gland and colon was identified (see above)(Pan et al., 2000; Wang et al., 2000). Thus, ILC/CCL27 may have another receptor selectively expressed in skin-homing lymphocytes and DCs. VII. Dendritic Cells and Chemokines

During maturation of DCs, there is a dynamic shift in the expression of chemokine receptors. Immature DCs were shown to express CCR1, CCR3, CCR5, and CXCR1, while maturing DCs strongly upregulated CCR7 and CXCR4 (Sallusto et al., 1998b; Ogata et al., 1999; Sato et al., 1999a). As stated in previous sections, CCR6, the receptor for LARC/CCL20 (Baba et al., 1997; Greaves et al., 1997; Liao et al., 1997; Power et al., 1997), was shown to play a major role in trafficking of immature DCs (Dieu et al., 1998; Charbonnier et al., 1999; Dieu-Nosjean et al., 1999, 2000; Tanaka et al., 1999; Yang et al., 1999a; Iwasaki and Kelsall, 2000). Lung DCs and DCs differentiated from CD34+ precursor cells in cord blood by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF were originally found to express CCR6 (Greaves et al., 1997; Power et al., 1997). On the other hand, DCs differentiated from peripheral blood monocytes by GM-CSF and IL-4 failed to express CCR6 (Greaves et al., 1997). Recently, it was shown that DCs differentiated from monocytes by


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GM-CSF and IL-4 in the presence of TGF-␤1 expressed CCR6 (Yang et al., 1999b). This observation is consistent with the critical role of TGF-␤1 in generation of Langerhans-type DCs both in vitro and in vivo (Borkowski et al., 1996; Geissmann et al., 1998; Jaksits et al., 1999; Ogata et al., 1999; Zhang et al., 1999). Since mucosal epithelial cells associated with follicles (follicle-associated epithelium) and skin keratinocytes were shown to express LARC/CCL20 (Charbonnier et al., 1999; Tanaka et al., 1999; Iwasaki and Kelsall, 2000; Nakayama et al., 2001), immature DCs are likely to be attracted to the body surfaces mainly via CCR6. Upon being loaded with antigens, immature DCs start a maturation process, which was shown to be accompanied by downregulation of CCR6 and upregulation of CCR7 (Dieu et al., 1998; Sallusto et al., 1998b; Sozzani et al., 1998; Yanagihara et al., 1998; Dieu-Nosjean et al., 1999). This probably allows maturing DCs to be guided by SLC/CCL21 into draining lymph nodes, where they become mature antigen-presenting cells capable of stimulating naive T cells (Chan et al., 1999; Foerster et al., 1999; Gunn et al., 1999; Kellermann et al., 1999; Saeki et al., 1999). Recently, DCs migrated out of mouse skin ex vivo were shown to express CXCR5 and to accumulate in B cell zones of regional lymph nodes when injected into footpad of mice (Saeki et al., 2000). Thus, some activated skin DCs may upregulate CXCR5 to be guided by BLC/CXCL13 to B cell zones of regional lymph nodes where they may have direct effects on B cells (Saeki et al., 2000). DCs are also important producers of chemokines. TARC/CCL17, MDC/ CCL22, and pulmonary and activation-regulated chemokine (PARC)/dendritic cell-derived chemokine 1 (DC-CK1)/CCL18 were shown to be constitutively expressed by immature DCs and to be upregulated following maturation (Hashimoto et al., 1999; Kanazawa et al., 1999; Lieberam and Forster, 1999; Sallusto et al., 1999c; Schaniel et al., 1999; Tang and Cyster, 1999). Maturing DCs also upregulate expression of fractalkine/CX3CL1 (Kanazawa et al., 1999; Papadopoulos et al., 1999; Schaniel et al., 1999). Recently, another membranetype chemokine CXCL16 was identified and shown to be expressed by mature DCs (Matloubian et al., 2000). Furthermore, maturing DCs, while upregulating CCR7, were also shown to start producing its ligand ELC/CCL19 (Ngo et al., 1998; Sallusto et al., 1999c). Notably, expression of CCR7 by DCs was strikingly resistant to ligand-induced downregulation (Sallusto et al., 1999c). This probably explains how maturing DCs expressing ELC/CCL19 still remain responsive to SLC/CCL21 and can be guided into draining lymph nodes via CCR7. After homing to the T cell zone they probably start attracting CCR7-expressing naive T cells by ELC/CCL19 and/or CCR4-expressing memory T cells by TARC/ CCL17 and MDC/CCL22 (Ngo et al., 1998; Kanazawa et al., 1999; Lieberam and Forster, 1999; Sallusto et al., 1999c; Schaniel et al., 1999; Tang and Cyster, 1999). The roles of fractalkine/CX3CL1 and CXCL16 in mature

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DCs (Kanazawa et al., 1999; Papadopoulos et al., 1999; Schaniel et al., 1999; Matloubian et al., 2000) are currently unknown. But it is rather striking that their respective receptors, CX3CR1 and CXCR6, are commonly expressed in cells such as CD8+ T cells and NK cells (Imai et al., 1997b; Matloubian et al., 2000; Yoneda et al., 2000). Thus, these membrane-type chemokines may have a unique role in interaction of mature DCs with CD8+ T cells and NK cells. However, no immunological abnormalities including DC migration and function have been observed in CX3CR1-knockout mice so far (Aliberti et al., 2000). FDCs were shown to express PARC/CCL18 and BLC/CXCL13 (Adema et al., 1997; Hieshima et al., 1997a; Gunn et al., 1998a; Legler et al., 1998). As discussed above, BLC/CXCL13 attracts B cells and TFH cells toward B cell follicles. On the other hand, PARC/CCL18 was shown to attract naive T cells (CD45RA+) and is thus suggested to be involved in primary immune responses (Adema et al., 1997). Notably, macrophages were also induced to produce PARC/CCL18 by Th2 type cytokines such as IL-4, IL-13 and IL-10 (Kodelja et al., 1998). The sequence analysis of the PARC/CCL18 gene led to a conclusion that the PARC/CCL18 gene was generated by fusion of two MIP-1␣/CCL3-like genes in the human genome and probably not present in the mouse genome (Tasaki et al., 1999). VIII. Concluding Remarks

In this review, we have mostly focused on the novel “immune chemokines” directed to lymphocytes and DCs, because of their prominent importance in the development and function of the immune system. However, we would like to point out that the classical “inflammatory chemokines” mainly attracting neutrophils, monocytes, and eosinophils are also likely to play significant roles in the immune system. For example, Th1/Th2 polarization was strongly affected in the targeted disruption of CCR2 or its ligand monocyte chemoattractant protein 1 (MCP-1)/CCL2 (Boring et al., 1997; Gao et al., 1997; Gu et al., 2000), even though these changes might be secondary to some primary defects. CCR5, which is the shared receptor for the well-known inflammatory chemokines, RANTES/CCL5, MIP-1␣/CCL3, MIP-1␤/CCL4 and MCP-2/CCL8, is also important for Th1 responses (see above). Obviously, innate immunity and adaptive immunity are not clearly separable in our self-defense system but are tightly integrated. Another issue that we would like to point out is the difficulty frequently met when the strategy of targeted gene disruption in mice is employed to study in vivo pathophysiological roles of the chemokines and their receptors. For example, CCR4-knockout mice had no obvious problems in mounting Th2-type responses, even though acute blocking of CCR4 ligands by neutralizing antibodies in vivo had clear therapeutic effects on a murine model of asthma


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(Gonzalo et al., 1999; Kawasaki et al., 2001). While CX3CR1-knockout mice did not show any neurological or immunological abnormalities so far (Aliberti et al., 2000), it is unlikely that fractalkine/CX3CL1 and its receptor CX3CR1 have no biological roles in the brain or the immune system. CCR5 is not considered to be totally dispensable, even though people homozygous for the CCR5-32 allele have no obvious health problems (Berger et al., 1999). These discrepancies are probably due in part to intrinsic redundancy and developmental adaptability of the chemokine system. Considering these problems, the classical strategy employing neutralizing antibodies is once more a useful and even necessary strategy to elucidate diverse pathophysiological roles of chemokines and their receptors in vivo (Yoneyama et al., 1998; Gonzalo et al., 1999; Kawasaki et al., 2001). Yet another problem to be pointed out is the significant species differences in the chemokine system, between mice and humans. This is discussed in the first part of this review. In this context, the immune chemokines are generally more conserved between the species and accordingly may have fewer problems in extrapolating results from animal studies to humans than the inflammatory chemokines. Even though the infiltration of blood leukocytes into sites of infection and injury is essential in the host defense against microbial invasion and tissue injury, infiltrating leukocytes often contribute to the pathologic processes of the underlying diseases by augmenting and prolonging inflammatory conditions. Thus, the intervention therapies against classical “inflammatory chemokines” such as IL-8/CXCL8 and MCP-1/CCL2 have been shown to be highly beneficial in animal models such as ischemia/reperfusion injuries and type II collageninduced arthritis (Harada et al., 1996; Strieter et al., 1996). Likewise, the “immune chemokines” directed to lymphocytes and DCs are likely to be involved in various chronic inflammatory disorders which show prominent lesional infiltration of lymphocytes, such as rheumatoid arthritis, atherosclerosis, atopic dermatitis, asthma, inflammatory bowel diseases, transplant rejection, and various autoimmune diseases. For example, infiltration of CD4+ T cells in the synovial membrane is regarded as having a crucial pathogenic role in the early stages of rheumatoid arthritis (Harris, 1990). The initial lesion of atherosclerosis, the so-called fatty streak, is an aggregation of lipid-laden macrophages and T cells within the intima of the arterial wall, suggesting some immunological reactions during the initial process of atherogenesis (Ross, 1993). In fact, PARC/CCL18 and ELC/CCL19 were detected in human atherosclerotic plaques (Reape et al., 1999). As mentioned above, CD4+ helper T cells are now known to be subdivided into Th1 and Th2 subtypes; Th1 cells are important in cell-mediated immunity and cytotoxicity, whereas Th2 cells are involved in humoral immunity and allergic responses (Mosmann and Sad, 1996). Diseases such as rheumatoid arthritis are considered to be accompanied by selective tissue infiltration of Th1 cells, while allergic diseases such as asthma and atopic dermatitis are likely to be

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associated with lesional accumulation of Th2 cells. Thus, intervention therapy aimed at chemokines directed to various T cell subsets is likely to be therapeutic in a variety of immune-based chronic inflammatory disorders through suppression of pathogenic infiltration of lymphocytes. Indeed, treatment with blocking antibodies against TARC/CCL17 or MDC/CCL22 has been shown to be highly beneficial in murine models of fulminant hepatitis and asthma (Yoneyama et al., 1998; Gonzalo et al., 1999; Kawasaki et al., 2001). Besides in intervention therapy, chemokines may be useful in inducing highly localized accumulation of specific types of leukocytes in target tissues. For example, chemokines or their expression vectors may be delivered into cancer tissues to induce infiltration of specific types of leukocytes. Without, or with, other activation factors, accumulated leukocytes may directly attack cancer cells and may also enhance host immune responses against them. For such purposes, the lymphocyte-directed chemokines are especially useful for targeting selective subsets of lymphocytes and DCs into cancer tissues (Dilloo et al., 1996; Gao et al., 1998; Sharma et al., 2000; Vicari et al., 2000). Obviously, our present knowledge of the roles of the chemokine superfamily in the immune system is still far from complete. To promote our understanding of the in vivo pathophysiological roles of various chemokines, it is important to determine the cells in each lymphoid and other tissues that produce individual chemokines constitutively and/or upon induction, the subsets of lymphocytes and other types of cells in tissues and circulation that express receptors for individual chemokines, and what kinds of innate and adaptive immune responses individual chemokines take parts in. Such studies will lead us to a deeper understanding of the genesis and function of the immune system and also to a new method for modulation of our immune responses in order to treat various chronic inflammatory disorders where excessive and/or aberrant immunological responses are likely to play major pathogenic roles. ACKNOWLEDGMENT This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses CHRISTOPH SCHANIEL,* ANTONIUS G. ROLINK, AND FRITZ MELCHERS Basel Institute for Immunology, CH-4005 Basel, Switzerland

I. Introduction

During many periods of their life, lymphocytes are restless loners. After having been born, raised, and educated in the primary lymphoid organs—T lymphocytes in the thymus, B lymphocytes in the bone marrow—they migrate through blood into the spleen to mature and to show their usefulness in participating in defenses against foreign invaders (such as viruses, bacteria, parasites, foreign material, or cells). Once mature and experienced, they can enter the recirculatory routes between blood and lymph and enter lymph nodes and Peyer’s patches. During other periods of their life, lymphocytes are social. They congregate in special areas of lymphoid organs, awaiting invasions by foreigners. Invasions unite them in collaborative effort to mobilize their defense powers and to inflame and kill the areas of invasion, to kill the invaders themselves and to ingest and destroy them. Migrations of single cells through cell barriers and into special areas of the body, as well as congregations of these single cells into lymphoid structures in organs of the immune system, are both guided combinatorial multistep processes (Butcher et al., 1999; Foxman et al., 1997; Springer, 1994; Melchers et al., 1999). It has become evident that migrations and congregations of the right cell at the right places are controlled by arrest and adhesion of cells to the surface of other cells or to the extracellular matrix and by chemoattraction of one type of cell expressing the right chemokine receptor by other types of cells producing the corresponding chemokine. Whenever cells need to traverse the cell layer to which they are attracted they can do so in a process called transmigration. As one example, blood-borne lymphocytes enter lymphoid organs through small capillaries formed by endothelial cells, called high endothelial venules. Binding of cells to other cell surfaces and transmigration involve selectins and integrins (Hynes, 1992; Shimizu et al., 1999). Both processes are driven by chemokines. A specific combination of selectins, integrins, and chemokine receptors and their corresponding ligands has been termed the “address code” which delivers a cell to the right place. A multitude of selectins, integrins, and chemokines and their ligands/receptors allows specific and flexible programming of cell migrations and congregations. ∗

To whom correspondence should be addressed. E-mail: [email protected]. 111 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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FIG. 1. Phylogenetic tree of chemokines and chemokine receptors. Evolutionary distance between chemokines (A) and chemokine receptors (B) were estimated according to their Clustal W alignment (gap penalty length 9). The location of the branch points is not drawn to scale.

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FIG. 1. (continued)

Chemokines (Fig. 1A), their receptors (Fig. 1B), and the biological activities they display were originally described for cells of the innate immune system, e.g., for monocytes, macrophages, eosinophils, and basophils, as well as for dendritic cells and T lymphocytes before and during responses to invaders. Most of them are characterized only by their expression and chemotactic potential measured in vitro. The in vivo function(s) of only a limited number of chemokines and chemokine receptors has so far been determined by natural mutations or by targeted gene inactivation. This frequent lack of the knowledge of the in vivo function is often also the consequence of a degeneracy of production and activity of many chemokines and their receptors, making the functional assignment for a given chemokine and receptor pair more difficult. One cell can produce more than one chemokine, different types of cells can express either

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the same chemokine or the same chemokine receptor, one chemokine can be recognized by more than one receptor, and one receptor can be activated by more than one chemokine. In this review, we focus mainly on chemokines and chemokine receptors, which regulate attractions and migrations of hematopoietic cells, thereby organizing humoral immune responses in secondary lymphoid organs.

II. Structures of Chemokines and Their Receptors

For an understanding of interactions between chemokines and their receptors, and the signal transduction resulting from these interactions, knowledge of the three-dimensional structure of both the chemokine receptors and their ligands would be of help. Several structures of chemokines have been solved either by nuclear magnetic resonance techniques or by X-ray crystallography (Chung et al., 1995; Clore and Gronenborn, 1995; Crump et al., 1998; Dealwis et al., 1998; Elisseeva et al., 2000; Lubkowski et al., 1997; Mizoue et al., 1999; Ye et al., 2000). The structures of the monomers of most chemokinos are very similar. Differences exist in the conformation of the first disulfide bridge, which is due to the number of amino acid residues separating the two N-terminal conserved, chemokinecharacteristic cysteines. In addition, the three-dimensional orientation of the N-terminal amino acid residues preceding these cysteines is different. These two structural differences appear to be critical for signal transduction and likely also for receptor recognition. Changes in the N-terminal residues of chemokines have been shown to abrogate signal transduction, although ligand binding of the receptor appeared intact (Clark-Lewis et al., 1991; Moser et al., 1993; Proudfoot et al., 1996). No three-dimensional structure of a chemokine receptor has so far been published. However, general structural features are known. The chemokine receptors are integral membrane glycoproteins, which traverse the membrane seven times. They average at 350 amino acids, and belong to the large group of G-protein coupled receptors. Mutagenesis data suggest that interaction with the pertussis toxin-sensitive regulatory G protein is localized to the third cytoplasmic loop and the C-terminal tail (Gosling et al., 1997). Receptor–ligand interaction results in signal transduction involving heterotrimeric G proteins, adenylyl cyclase, phospholipases, protein tyrosine and serine/threonine kinases, phospatidylinositol-3-kinase, and the Rho family of small GTPases, and in the release of intracellular second messengers such as calcium, cAMP, and phosphoinositides (review by Sanchez-Madrid and del Pozo, 1999).


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III. Rules to Understand Receptor–Ligand Interaction and Migration in Vivo

Intravital microscopy is the method for observing cell migrations in vivo. The potency of a given chemokine on a distinct cell population is experimentally easily assayed by in vitro migration analysis. Measurement of intracellular calcium release upon receptor–ligand interaction is another method to assess chemokine responsiveness. Both migration and calcium measurements allow detection of receptor desensitization that normally occurs following receptor–ligand interactions. A cell can regain its sensitivity for a given chemokine, even at lower concentrations when the chemoattractant is removed. This process, however, takes time. The time required for recovery of the chemotactic responsivness depends on the initial concentration of the chemokine and can vary between a few minutes and hours (Chuntharapai and Kim, 1995; Goldman and Goetzl, 1984; Samanta et al., 1990; Sullivan and Zigmond, 1980; Zigmond and Sullivan, 1979; Zigmond et al., 1982). Cell migration would be understood most easily if a given cell were to express only one type of chemokine receptor that, in turn, would recognize only one ligand. Thus, each cell could only follow one gradient and, hence, find one single target site. This, however, would be a very ineffective and unflexible way to organize migrations of cells of the immune system. In reality, a cell of the system should be able to choose between different targets and be ready for a series of interactions. This, in fact, is the case. A variety of cells of the innate and adaptive immune system express a variety of chemokines and chemokine receptors. Several ligands [e.g., macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES; Table I] can bind to the same receptor (CCR5), and several receptors (e.g., CCR1 and CCR5) can recognize the same ligand (MIP-1␣). Moreover, one type of cell often expresses more than one type of chemokine receptor, and the same chemokine receptors are produced by different types of cells. Interaction of the ligand with its receptor(s) results not only in signal transduction but also in desensitization of the receptor itself, rendering the cell unresponsive to the triggering chemokine. This will lead to unresponsiveness to a second chemokine that is recognized by the same receptor. In certain circumstances, cross-desensitization of receptors may occur (Richardson et al., 1998, 2000). In this case, another receptor that does not bind the triggering ligand but is expressed on the same cell is desensitized together with the ligand-recognizing receptor. How can combinatorial stepwise migration through chemoattractant fields containing several different gradients of many distinct chemokines occur in vivo? From in vitro chemotaxis assays, it appears that the chemokine concentration, although not known in vivo, is an important factor. Cells that express the appropriate receptor do not react when there is too little chemokine. High-dose

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TABLE I LETTER CODE OF CHEMOKINES Code (1)

Family

Receptor(s)

CC

CCR1, CCR5

CC CC

CCR5 CCR3, CCR5

(13)

ABCD-2

(14)

TARC MCP-3

Macrophage inflammatory protein 1␣ — Macrophage inflammatory protein 1␤ Regulated on activation normal T cell-expressed & secreted Stroma cell-derived factor 1 Pre-B cell growth-stimulating factor Thymus-expressed chemokine Activated B cell and dendritic cell-derived chemokine 1 Macrophage-derived chemokine Stimulated T cell chemotactic protein 1 Dendritic cell and B cell-derived chemokine Secondary lymphoid tissue chemokine 6 Cysteine chemokine Exodus-2 Thymus-derived chemotactic agent 4 Epstein–Barr virus-induced molecule 1 ligand chemokine Macrophage inflammatory protein 3␤ Exodus-3 — B-lymphocyte chemoattractant B cell-attracting chemokine 1 Macrophage inflammatory protein 3␣ Liver and activation regulated chemokine Exodus-1 Monocyte chemoattractant protein 1 Monocyte chemotactic and activating factor — Dendritic cell chemokine 1 Pulmonary and activation regulated chemokine Alternative macrophage activation-associated chemokine 1 Activated B cell and dendritic cell-derived chemokine 2 Thymus and activation regulated chemokine Monocyte chemotactic protein 3

(15) (16) (17) (18) (19)

MCP-4 MCP-5 IP-10 MIG I-TAC

Monocyte chemotactic protein 4 Monocyte chemotactic protein 5 IFN-␥ inducible protein-10 Monokine induced by IFN-␥ —

(2) (3) (4) (5) (6)

(7)

(8)

(9) (10)

(11)

(12)

MIP-1␣ LD78␣ MIP-1␤ RANTES

Name

SDF-1 PBSF TECK ABCD-1 MDC STCP-1 DC/B-CK SLC 6Ckine — TCA-4 ELC MIP-3␤ — Ck␤-11 BLC BCA-1 MIP-3␣ LARC — MCP-1 MCAF JE DC-CK1 PARC AMAC-1

CXC

CXCR4

CC CC

CCR9 CCR4

CC

CCR7

CC

CCR7

CXC

CXCR5

CC

CCR6

CC

CCR2

CC

unknown

CC

CCR4

CC

CCR1, CCR2, CCR3 CCR2, CCR3 CCR2 CXCR3 CXCR3 CXCR3

CC CC CXC CXC CXC

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TABLE I Continued Code (20) (21) (22)

(23)

MCP-2 — — MPIF-1 Ck␤-8 I-309 TCA-3

Name Monocyte chemotactic protein 2 Eotaxin-1 Eotaxin-2 Myeloid progenitor inhibitory factor 1 — — Thymus-derived chemotactic agent 3

Family

Receptor(s)

CC CC CC

CCR3 CCR3 CCR3

CC

CCR8

Note. Chemokines, their alternative names, and their known receptors are listed chronologically as they appear in the text.

inhibition is observed in chemokine dose response curves of in vitro migration assays. Thus, too high concentrations of a chemokine are bad for continued cell migration. Elegant studies performed by Foxman and colleagues give a possible explanation of how combinatorial multistep navigation may occur in vivo (Foxman et al., 1997, 1999). Migrating cells appear to develop a memory of their chemoattractant environment, ensuring migration through a complex chemoattractant field (Foxman et al., 1999). In such a gradient field, cells, which express the required chemokine receptors, initially move up the steepest local gradient, become less sensitive to that chemoattractant as they migrate closer to its source, and thus, become relatively more responsive to the other chemoattractant source. Thus, they can eventually turn away from the initial source and migrate toward the second one (Fig. 2). Therefore, cells can regain their migratory sensitivity to the first chemoattractant, and at even lower concentrations when the influence of the second chemokine has become desensitizing, so that they migrate toward the first source again (Foxman et al., 1999). Thereby, a cell responsive to two chemokines produced by two spatially separated sources will migrate in a “zig-zag” manner toward the two chemokine-producing target cells (Fig. 2). It can be expected that in reality such “zig-zag” movements of cells are controlled by more than two sources of two separate chemokines. IV. The Generation of Cells Involved in the Humoral Defense against Foreign Invaders

A. POPULATION OF THE BONE MARROW BY MIGRATIONS OF HEMATOPOIETIC STEM CELLS DURING EMBRYOGENESIS The bone marrow is seeded by hematopoietic stem cells, which originate from so-called hemangioblasts. Hematopoietic stem cells give rise to all participants

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FIG. 2. The billiard theory of chemoattraction. “Zig-zag” movement of a cell is shown with two different chemokine receptors toward two spatially separated chemokine sources, producing either one of the two chemokines.

of an immune response. Hence, migrations and attractions of cells are critical steps in the generation of the blood system. The bone marrow itself is colonized by stem cells from the fetal liver which, in turn, is, populated by hematopoietic stem cells, which migrate there from the aorta–gonad–mesonephros region. As a first step of bone marrow colonization, the hematopoietic stem cells loosely attach to and start rolling on the bone marrow endothelium. This rolling is mediated by vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin on the endothelium, and PSGL-1 and ␣4␤1 integrin on the hematopoietic stem cells, as shown by intravital microscopy of bone marrow sinusoids in combination with antibody inhibition studies (Mazo et al., 1998), and by bone marrow transplantation into E- and P-selection double-deficient mice (Frenette et al.,


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1998). However, in none of these studies was the inhibition complete, suggesting that additional interactions are required. Recent studies have revealed a mandatory role for ␤1 integrin in colonization of fetal liver, bone marrow, thymus, and spleen by fetal and adult hematopoietic stem cells (Hirsch et al., 1996; Potocnik et al., 2000). ␤1-deficiency by itself causes embryonic lethality (Fassler and Meyer, 1995; Stephens et al., 1995). However, chimeric mice generated with ␤1 integrin-negative embryonic stem cells clearly demonstrate that ␤1-negative hematopoietic cells cannot migrate to fetal liver, and thus to bone marrow (Hirsch et al., 1996).These cells are instead found in blood islands in the yolk sac. They can properly generate erythroid and myeloid cells detectable by in vitro colony-forming assays. Moreover, ␤1-deficient embryonic stem cells are not deficient in the hemato-lymphopoietic capacities, since they can differentiate in vitro into mature B lymphocytes. This was shown by Potocnik and colleagues who performed a more detailed analysis of the functions of ␤1 integrin in the migratory defect of the aorta– gonad–mesonephros hematopoietic stem cells by using ␤1 integrin-conditional knockout mice (Potocnik et al., 2000). Their studies confirmed that lack of ␤1 integrin does not ablate any hemato-lymphoid differentiation potential of the hematopoietic stem cells. However, the absence of ␤1 integrin on fetal as well as adult hematopoietic stem cells completely abrogates their potential to repopulate, thus to home to, primary sites of hematopoiesis (i.e., fetal liver, bone marrow, thymus, and spleen). ␤1-deficient hematopoietic stem cells instead accumulate in the peripheral blood. Surprisingly, ␣4 integrin seems dispensable for homing to and seeding of hematopoietic organs by hematopoietic stem cells. Thus, a normal homing capacity of ␣4-deficient hematopoietic stem cells into fetal liver, spleen, or bone marrow was seen in the ␣4-deficient mice (Arroyo et al., 1996, 1999). ␣4-deficient bone marrow cells reentered their bone marrow niche. Correspondingly, VCAM-1-deficient mice were found to have no defects in the capacity to populate bone marrow, thymus, spleen, and peripheral lymph nodes with any type of hematopoietic cell (Friedrich et al., 1996). B. MIGRATIONS OF PLURIPOTENT AND COMMITTED PROGENITORS DURING DEVELOPMENT OF DIFFERENT HEMATO-LYMPHOID LINEAGES IN AND BETWEEN PRIMARY LYMPHOID ORGANS A hematopoietic stem cell must at least divide asymmetrically to renew itself, while the other progeny cells can have a varying tendency to differentiate, often migrating to a new microenvironmental niche where they remain in close contact with the same or a different type of stroma (Watt and Hogan, 2000). The progeny cells change their potential, first to an erythroid–myeloid–lymphoid, then to a myeloid–lymphoid, and finally to a lymphoid progenitor (for reviews, see Akashi et al., 2000; Singh, 1996). Monocytes which differentiate from the myeloid–lymphoid progenitors leave the bone marrow via the venous blood,

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change in the heart to arterial blood, and are thereby distributed into lymphoid and nonlymphoid tissues where they can differentiate to granulocytes, dendritic cells, macrophages, or osteoclasts. The lymphoid progenitors still have two choices, either to remain in the bone marrow and enter the B-lineage differentiation pathway or to travel through blood to the thymus, entering first the medullary–cortical junctions and then moving to the cortical regions to differentiate along the T-lymphoid lineage pathway. Interestingly, the absence of ␣4 integrin results in a defect in B cell and T cell development (Arroyo et al., 1996), even though it is not involved in seeding of the hematopoietic organs (see above). In ␣4-deficient RAG-1−/− chimeric mice, B cell development in bone marrow is blocked at an earlier stage than is seen in RAG−/− mice, that is, before the pro-B cell stage (Arroyo et al., 1996, 1999). T cell development is defective due to the inability of thymic progenitors to leave the bone marrow. Nevertheless, early T cell precursors can develop in the fetal liver and home to the fetal thymus. As a consequence, the ␣4-deficient mice have normal numbers of T lymphocytes for the first month after birth. The T cell numbers, however, decrease with age. In the bone marrow, B cell development occurs in a sequence of cellular stages which can be followed phenotypically by cell surface markers, and genetically by the rearrangement status of their immunoglobuline heavy (IgH) and L chain gene loci (Melchers and Rolink, 1999). First, the IgH chain locus is rearranged DH to JH then VH to DHJH, followed by VL to JL rearrangements on the IgL chain loci. Those cells with productive IgH and L chain gene rearrangements express IgM on their surface (sIgM+), if the H and L chains can pair with each other. Immature sIgM+ B cells are screened for their capacity to recognize autoantigens, which are present in the appropriate sites in the bone marrow. High avidity self-reactive B cells are arrested in their further differentiation and die in situ (“negative selection”), while the remaining nonautoreactive immature B cells are allowed to exit the bone marrow via the venous sinuses (Melchers and Rolink, 1999). The immature sIgM+ B cells change in the heart to arterial blood, as do the monocytes. In the thymus, stepwise V(D)J-rearrangements at the T cell receptor (TCR) ␣-, ␤-, ␥ -, and ␦-gene loci result in the generation of ␣/␤ TCR+ and ␥ /␦ TCR+ thymocytes through a sequence of cellular stages which, for the ␣/␤ TCR-expressing cells, are strikingly similar to the corresponding development of sIgM+ B cells in the bone marrow. The ␣/␤ TCR-expressing thymocytes expressing both CD4 and CD8 as coreceptors on the same cell are confronted with processed antigen (in the form of peptides) on the major histocompatibility complex (MHC) class I or II molecules on specialized antigen-presenting cells. CD4+CD8+ thymocytes expressing TCR with high avidity for MHC class I or II/autoantigen peptide complexes are arrested in their further differentiation, and die in situ (“negative selection”). Those cells displaying ␣/␤ TCR with


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intermediate affinities for MHC class I downregulate the expression of the CD4 molecule and mature to CD8 single-positive cells to become killers (“positive selection”). On the other hand, those T cells expressing ␣/␤ TCR with intermediate affinities for MHC class II downregulate the expression of the CD8 molecule and mature to CD4 single-positive cells to become helpers. CD4 and CD8 single-positive T cells migrate to the medulla and leave the thymus via venous blood to move to arterial blood in the heart. C. MIGRATIONS WITHIN A PRIMARY LYMPHOID ORGAN Lymphoid and myeloid cell development in bone marrow and thymus takes place in a microenvironment of stromal cells that includes fibroblasts, preadipocytes, adventitial reticular cells, endothelial cells, dendritic cells, and macrophages. Lymphopoietic progenitors and precursors of the different cell lineages on one side and stromal cells on the other cross talk to each other by chemoattraction, adhesion, and cytokine secretion. It appears that orderly cellular development is not only controlled by close contacts but also by programmed migrations of lymphopoietic cells on stromal cells, which form assembly lines for the stepwise formation of T cells and B cells. A similar assembly line can be expected to exist for the third type of cell involved in a humoral immune response, the antigen-presenting cell. During murine embryonic life, hematopoietic development occurs in fetal liver, before it shifts (after birth) to the bone marrow. This prenatal wave of lymphopoiesis in the fetal mouse liver has allowed two groups to study the defects of targeted disruption of the gene encoding the SDF-1 chemokine receptor CXCR4 in fetal liver before the mutant mice die perinatally (Kawabata et al., 1999; Ma et al., 1999). The CXC chemokine SDF-1 is highly expressed by fetal liver and bone marrow stromal cells (Nagasawa et al., 1994; Shirozu et al., 1995; Tashiro et al., 1993) while its receptor CXCR4 is expressed on hematopoietic progenitors, B cell progenitors, mature T and B lymphocytes, and monocytes (Loetscher et al., 1994). It has been observed previously that CXCR4-deficient and SDF-1-deficient mice show very similar defects in neuron migration, organ vascularization, and hematopoiesis (Ma et al., 1998; Nagasawa et al., 1996; Tachibana et al., 1998; Zou et al., 1998). B lymphopoiesis is severely reduced in fetal liver and bone marrow, myelopoiesis is decreased in fetal liver and virtually absent in bone marrow, while T lymphopoiesis is unaffected. The two groups have now shown that the proper retention, hence the proper filling, of the myeloid and B-lymphoid progenitor and precursor compartments with normal numbers of cells is abolished when the chemokine receptor CXCR4 is defective (Kawabata et al., 1999; Ma et al., 1999). Instead, these progenitors and precursors then appear in increased numbers in the blood, indicating that only the proper retention in the primary organ, but not the production of these cells, is defective. The interaction between SDF-1 and CXCR4 appears to be monogamic, since

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FIG. 3. Trafficking of developing thymocytes through the thymic cortex and medulla. ∗ , SDF-1; TECK; •, ABCD-1; ◦, SLC; ♦, ELC;

alternative chemokines and their receptors cannot compensate their loss. The early defects in myeloid and B-lymphoid development obscure any possible later defects of SDF-1 reactive CXCR4-expressing mature T, B, and myeloid cells in the secondary lymphoid organs. It will be interesting to selectively monitor the effects of targeted CXCR4 inactivation in the mature cell populations. Although SDF-1 is expressed in the thymus and the early CD4+CD8+ thymocytes are reactive to it (Campbell et al., 1999b; Fig. 3], T lymphopoiesis in SDF-1-deficient and CXCR4-deficient mice is normal. This is probably so because thymus-expressed chemokine (TECK) acts on these same CD4+CD8+ thymocytes (Campbell et al., 1999b). TECK is expressed by thymic dendritic cells and thymic epithelial cells of the cortex and, to a lesser extent, by those of the medulla (Vicari et al., 1997; Wurbel et al., 2000). The expression of CCR9, the high-affinity receptor for TECK in thymocytes, nicely correlates with the


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maximal chemotactic activity of TECK on CD4+CD8+ thymocytes (Norment et al., 2000; Wurbel et al., 2000). Migration of the positively selected, so-called transitional CD4+CD8+CD69+ thymocytes from the cortical area to either CD4+ or CD8+ thymocytes of the medulla could, in addition to TECK, be controlled by ABCD-1, recognized by the CCR4 receptor (Campbell et al., 1999b; Chantry et al., 1999). The final migration out of the thymus is probably controlled by secondary lymphoid tissue chemokine (SLC) and EBI1-ligand chemokine (ELC) both recognized by CCR7, since these chemokines attract the CD4+ and CD8+ thymocytes of the thymic medulla, but not the immature CD4+CD8+ thymocytes of the thymic cortex (Campbell et al., 1999b).

V. The Population of Secondary Lymphoid Organs by Lymphoid Cells

A. LYMPHOID ENTRY INTO SECONDARY LYMPHOID ORGANS After T lymphocytes have been made, negatively selected against autoantigens and positively selected for their capacities to recognize peptides of foreign antigen in the context of MHC molecules in the thymus, they migrate through blood into the central artery and enter the spleen through the central arteriole or lymph nodes through high endothelial venules (Fig. 4A). Resting, naive T cells congregate in areas near the arteriole or the high endothelial venules and form the so-called periarteriolar lymphoid sheath (Fig. 4A). As mentioned previously, B lymphocytes are made in the bone marrow. Like T lymphocytes in the thymus, B lymphocytes are purged of autoantigen-reactive cells before they exit into the blood as immature B lymphocytes. They enter the lymphoid organs through the central arteriole and the high endothelial venules, migrate first through the periarteriolar sheath, and finally congregate in the follicular areas and the marginal zone (Fig. 4A). However, the molecular mechanisms regulating the selection and migration of immature B cells to the spleen are incompletely understood. Once the immature B lymphocytes have reached the spleen, the majority, but not all, are recruited into the pool of recirculating, long-lived and antigen-reactive (in the sense of proliferation) mature B cells (Rolink et al., 1998). There is good evidence that this maturation process occurs near the T cell-rich areas of the spleen. In the spleen, the central arteriole opens into a marginal sinus that is surrounded with endothelium and macrophages, which allow entry of lymphocytes into the organ (Tanaka et al., 1996). Next to the marginal sinus is the marginal zone which contains several specialized cells, including marginal zone macrophages, metallophilic macrophages, reticular cells, and the marginal zone B cells. Mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-expressing metallophilic macrophages and sinus-lining nonlymphoid cells are thought to

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FIG. 4. Cell movements during activation of B lymphocytes, dendritic cells, and T lymphocytes during a T cell-dependent, antigen-specific immune response of B lymphocyes. Movements of cells and actions of antigens are shown. Attracting actions of chemokines on chemokine reseptors are drawn with arrows. In (A), the areas of the response are schematically drawn. Antigen can enter from the outside through the epithelial layers of skin and mucosa. Antigens and cells can travel through blood and lymph. Cells enter the secondary lymphoid organs (spleen, lymph nodes) through high endothelial venules (HEV). T cells become aggregated in the periarteriolar lymphoid sheath (PALS, spleen) or the paracortex (lymph nodes), while B lymphocytes congregate in the follicular regions (spleen) or the cortex (lymph nodes).


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Fig. 4 (continued) In (B), antigen enters through the epithelia of skin (top) or mucosa (bottom). The uptake of antigen by immature dendritic cells positioned in the epithelia activates the production of MIP-1␣, MIP-1␤, and MCP-1 in the skin epithelia, and MIP-3␣ and ␤-defensins in the mucosal epithelia. CCR1, CCR2, CCR5, and CCR6 expressed on immature dendritic cells attract more of these cells to the site of antigen invasion in skin, while CCR6 expressed on these cells does the same at the mucosal sites. Uptake and processing of antigen induces maturation and migration of dendritic cells. As they arrive as maturing dendritic cells at the HEV they express CCR7, which allows them to be attracted to HEV, which secrete SLC. Once inside the secondary lymphoid organs, the mature dendritic cells are attracted to reticular stromal cells which express CCR7-specific SLC and previously immigrated dendritic cells which express CCR7-specific ELC.

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Fig. 4 (continued) In (C), resting B cells (small) are shown to be activated by antigen in extrafollicular regions. Once activated (large), they produce TNF␣ and lymphotoxins (LT␣1␤2), which act directly, or indirectly via stromal cells, on follicular dendritic cells (FDC). Signalling via the TNF␣ -, LT␣1␤2-specific LT␤-receptors induces BLC production. BLC attracts activated B cells, which express the BLC-specific CXCR5 receptor, to the FDC. In (D), the activation of resting T cells (small) and the action of activated T cells (large) are shown. Resting T cells expressing CCR7 are initially attracted to reticular stromal cells secreting SLC. As activated, mature, antigen-presenting dendritic cells enter the T cell-rich areas, those T cells are activated which have a T cell receptor that is specific for the MHC-peptide complexes displayed on the surface of the dendritic cells. While they are initially attracted to the dendritic cells via ELC–CCR7 interactions, activated T cells become additionally attracted to the same dendritic cells secreting the CCR4-specific ABCD-1 and ABCD-2 chemokines. Once activated,


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Fig. 4 (continued) T cells divide to form clones of antigen-specific T cells. These antigen-specific activated T cells now have three options to migrate. They can migrate to the sites of antigen invasion in skin or mucosa, attracted by ABCD-1 or ABCD-2 secreted by dendritic cells at sites of invasion. There they can help fight the infection by inflammatory or cytotoxic actions. Alternatively, they can migrate to the follicular regions where activated B cells attract them with ABCD-1 and ABCD-2. This, then, leads to T–B cell collaborations, which allow B cells to proliferate, to mature to immunoglobulinsecreting cells, to switch their immunoglobulin-class expression, to somatically hypermutate the V region parts of their immunoglobulin heavy and light chain genes, and to become memory B cells— all by forming germinal centers at and near the sites of the original collaboration of T cells with B cells. For further detail see the text.

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regulate entry of ␣4␤7 integrin-expressing lymphocytes as well as antigens from the blood into the white pulp (Kraal et al., 1995; Tanaka et al., 1996). Marginal zone macrophages may also play a role in the migration, since depletion of macrophages abrogates the proper localization of lymphocytes in the periarteriolar lymphoid sheath (Kraal et al., 1989). Mice deficient for the transcription factor NKX2.3 lack MAdCAM-1 expression in the spleen, mesenteric lymph nodes, and Peyer’s patches (Pabst et al., 2000). The most significant phenotype is, however, the one which fails to form a marginal zone, resulting in no clear separation of the red pulp from the white pulp. The cells normally found in the marginal zone seem to be missing in the mutant mice. As a further consequence, no defined T cell-rich areas form, and therefore, T and B cells appear mixed. SLC (Gunn et al., 1998b; Hedrick and Zlotnik, 1997; Hromas et al., 1997; Nagira et al., 1997; Tanabe et al., 1997) recognized with high affinity by CCR7 (Campbell et al., 1998a; Yoshida et al., 1998a) is the candidate chemokine mediating homing of naive lymphocytes and dendritic cells through high endothelial venules into secondary lymphoid organs, based on several findings (Fig. 4B and D). SLC is the only chemokine known to be constitutively expressed by endothelial cells of the high endothelial venules (Gunn et al., 1998b; Nagira et al., 1998; Willimann et al., 1998). SLC induces strong chemotaxis of dendritic cells and na¨ıve T cells, and to a lesser extent that of memory T cells and B cells (Campbell et al., 1998a; Chan et al., 1999; Dieu et al., 1998; Gunn et al., 1998b; Hromas et al., 1997; Kellermann et al., 1999; Nagira et al., 1997; Tanabe et al., 1997; Willimann et al., 1998). After T, and possibly B, cells roll on endothelial cells (through interactions of L-selectins on lymphocytes with the heavily glycosylated mucin-like protein CD34 expressed on the endothelium), lymphocytes upregulate, in a SLC-dependent manner, their integrin activity (Gunn et al., 1998b). This causes the lymphocytes to tightly adhere to the endothelial cell layer through binding of the lymphocyte integrin ␣L␤2 (CD11a /CD18) to intercellular adhesion molecule (ICAM)-1, -2, and -3 expressed on high endothelial venules of Peyer’s patches and peripheral lymph nodes or of integrin ␣4␤7 with MAdCAM-1 on high endothelial venules of Peyer’s patches and mesenteric lymph nodes (Campbell et al., 1998a; Gunn et al., 1998b; Pachynski et al., 1998; Stein et al., 2000). The firmly attached lymphocytes then transmigrate through the endothelium into the secondary lymphoid tissue following a chemotactic gradient. Mice homozygous for the autosomal recessive paucity of lymph node T cell (plt) mutation show a defect in migration of na¨ıve T cells to peripheral lymph nodes, Peyer’s patches, or the lymphoid regions of the spleen (Gunn et al., 1999; Nakano et al., 1997, 1998). The plt gene responsible for this homing defect was mapped to mouse chromosome 4 in a region most closely linked to the locus D4Mit237 corresponding to that of human chromosome 9p13 (Nakano et al., 1998). The human chemokines SLC and ELC locate to this region (Nagira et al.,


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1997; Yoshida et al., 1997), suggesting that the homing defect of T cells seen in plt mice might be due to a defect in one or both of these two chemokines. Indeed, the product of plt is one of two SLC chemokines that is expressed specifically in lymphoid organs (Gunn et al., 1999; Vassileva et al., 1999). Besides their differential expression pattern, the two SLCs are distinguished by a single amino acid substitution (serine to leucine) at position 65 (Hedrick and Zlotnik, 1997; Tanabe et al., 1997; Vassileva et al., 1999). In addition to the defect in T cell homing, dendritic cells fail to accumulate in the spleen and lymph node T cell zones of the homozygous plt mice. As a result, a markedly increased (> 300-fold) susceptibility to infection with murine hepatitis virus is observed (Gunn et al., 1999). Surprisingly, B cell homing to and localization within secondary lymphoid organs appears not to be disturbed in plt mice (see also CCR7-deficient mice below), even though immature transitional B cells respond vigorously to SLC (Bowman et al., 2000). In agreement with the observation in plt mice that B cells can enter into secondary lymphoid organs in a SLC-independent way is the finding that B cells do arrest on high endothelial venules in vivo in the absence of SLC (Warnock et al., 2000). All this suggests that, in contrast to dendritic cells and T cells, alternative chemotactic activities regulate entry of B cells into secondary lymphoid organs and their localization within the tissue (see below). Forster and colleagues have recently found a disturbed distribution of T and B cells in secondary lymphoid organs of CCR7-deficient mice (Forster et al., 1999), such that in spleen and lymph nodes the T and B cell-rich areas do not form properly. Instead, the T and B cells are found in lower numbers in the splenic periarteriolar lymphoid sheath and the paracortex of the lymph nodes, while they appear in higher numbers in the blood. Most of the T cells locate to the marginal sinus of the spleen, while the B cells migrate through the T cell-rich areas to the B cell-rich areas without being retained as (im)mature B cells in the periarteriolar lymphoid sheath. The observed defects could be interpreted to indicate that CCR7 expression on T and B cells is required first during cellular development to allow entry of lymphocytes from the blood into secondary lymphoid organs (Fig. 4B). In the case of the T cells, this probably occurs through CCR7 recognition of SLC on high endothelial venules, as demonstrated in the plt mice and the CCR7-deficient mice. The entry mechanism(s) for B cells remain(s) to be clarified. CCR7 is then required once more to recognize either SLC produced by resident stromal cells or ELC produced by recently recruited mature dendritic cells (Dieu et al., 1998; Gunn et al., 1999; Nagira et al., 1997 1998; Ngo et al., 1998; Sallusto et al., 1998; Willimann et al., 1998; Yoshida et al., 1998a,b; Fig. 4B) to allow proper positioning of resting T and B cells into their appropriate microanatomical sites (described in more detail below). SDF-1, produced by stromal cells in secondary lymphoid organs, and recognized by CXCR4, expressed on resting T and B cells, may also play a role in the proper localization of T and B cells. However, SDF-1- and CXCR4-deficient mice organize

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normal microanatomical structures with T and B cell-rich areas in secondary lymphoid organs (Ma et al., 1999). Hence, entry for T cells is probably regulated by SLC/CCR7 interactions alone, whereas B lymphocyte homing to the appropriate B cell-rich areas might be controlled through both SLC/CCR7 and SDF-1/CXCR4 interactions that may be functionally redundant. In agreement with this hypothesis is that SDF-1 triggers rapid arrest of peripheral blood lymphocytes to ICAM-1 under flow conditions (Campbell et al., 1998b). This SLC/CCR7–SDF-1/CXCR4 redundancy might explain the normal B cell homing to and localization within secondary lymphoid tissues in plt mice as well as SDF-1- and CXCR4-deficient mice. However, whether SDF-1 is expressed by high endothelial venules has not been characterized, and its importance at this site remains to be established. The important, and probably nonredundant function of SLC in homing of lymphocytes to secondary lymphoid organs is also evident in transgenic mice, which produce SLC under the control of the rat insulin II promoter (RIPSLC) selectively in the pancreas (Fan et al., 2000). Pancreatic islets of young transgenic mice (4–6 weeks of age) show small focal infiltrates near the centers containing CD4 and CD8 T cells as well as dendritic cells. Limited numbers of scattered B cells are found at the perimeter of the islets not associated with the cluster of T cells and dendritic cells. This is further evidence that B cells respond differently to SLC than do dendritic cells and T cells. In older transgenic animals (6 weeks to 4 months of age), the islets appear dislocated to the borders of much larger infiltrates. Nevertheless, it is evident that the islet tissue remains intact. Furthermore, the infiltrates in older animals resemble normal lymphoid tissue, evident by the development of stromal reticulum characteristically seen in lymph nodes, and by the expression of adhesion molecules on vascular endothelium with the morphology of high endothelial venules. The larger cluster of T cells and dendritic cells merges with B cell follicle-like areas, which are found to contain only a few follicular dendritic cells. No germinal center formation was observed. This study nicely demonstrated that ectopic expression of the chemokine SLC is sufficient to trigger lymphoid neogenesis. VI. Compartmental Homing within Secondary Lymphoid Organs

Once B and T lymphocytes have traversed the endothelium in lymph nodes and Peyer’s patches, or once they have entered the spleen from central arterioles, B cells localize either in the marginal zone (marginal zone B cells) or in the B cellrich areas (primary follicles), and T cells in the T cell-rich areas (paracortex in the lymph nodes and the periarteriolar lymphoid sheath in the spleen). It appears that resting B cells can congregate only in follicular regions if the reticular cells in the follicular region have properly differentiated and, thus, can act to accept the incoming cells. The same is true for the marginal zone.


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A. THE INVOLVEMENT OF MEMBERS OF THE FAMILY OF TUMOR NECROSIS FACTORS AND THEIR RECEPTORS IN THE FORMATION OF A PROPER MICROARCHITECTURE IN SECONDARY LYMPHOID ORGANS Proper lymphocyte compartmentalization guided by a proper formation of the microarchitecture of secondary lymphoid organs is critically dependent on the activity of proteins of the tumor necrosis factor (TNF) family of cytokines and their receptors (for review see Fu and Chaplin, 1999). Lymphotoxin (LT) ␣-deficient mice show profound defects in formation of lymph nodes and complete lack of Peyer’s patches (De Togni et al., 1994). The spleen is present, but shows no normal follicular dendritic cell network. B and T cells cannot segregate properly within the white pulp. There is convincing evidence that these deficiencies result from a lack of LT␣ expression by B cells. Both follicular dendritic cell clusters and germinal centers were restored in lethally irradiated LT␣-deficient animals only when reconstituted with a mixture of bone marrow cells from LT␣-deficient and TCR-deficient mice, but not from LT␣-deficient and BCR-deficient mice (Fu et al., 1998). Deletion of the gene for LT␤ leads to loss of some, but not all, lymph nodes and to a disturbed structure of the B cell-rich areas in the spleen, while the T cellrich periarteriolar lymphoid sheath appears more or less unaltered (Alimzhanov et al., 1997; Koni and Flavell, 1998). LT␤-receptor (LT␤-R), which recognizes heterotrimers of one LT␣ in complex with two LT␤ (LT␣1␤2) and homotrimers of LIGHT, but not LT␣3, LT␣2␤1 or TNF3, also plays a crucial role in the differentiation of reticular cells and in the formation of follicular regions, which are filled with resting B cells (Futterer et al., 1998; Rennert et al., 1998). Thus, LT␤-R-deficient mice do not form follicles. As a consequence, B cells mix with T cells, and the two appear not distinctly separated from each other. On the other hand, when resting B cells from LT␣deficient mice are injected into normal SCID mice they home properly into follicular regions (Gonzalez et al., 1998). From all these experiments, one might conclude either that a continuous supply of newly immigrating, short-lived immature B cells is needed to maintain a full follicular region, or that development and maintenance of follicular dendritic cell clusters is strictly dependent on LT␣-expressing B lymphocytes. TNF also plays a critical but somewhat less distinct role than LT␣ in compartmentalization of secondary lymphoid organs. TNF-deficient mice retain segregated T and B cell-rich zones and show normal distribution of B cells in the marginal zones (Le Hir et al., 1995; Marino et al., 1997; Pasparakis et al., 1996). However, these mice lack primary B cell follicles and a properly organized follicular dendritic cell network, and thus germinal centers. The role of TNF and LT␣ in generation of splenic microarchitecture with B cell-rich follicles, and the movement of B cells within this architecture, was further clarified in bone marrow chimeras by reconstitution of irradiated

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wild-type mice with TNF-deficient or TNF/LT␣-double-deficient mature splenic B cells and vice versa (Cook et al., 1998). Transfer of TNF-deficient or TNF/LT␣double-deficient mature splenic B cells into wild-type recipients resulted in precise migration into the follicles. This may not be a surprise, since these TNFdeficient and TNF/LT␣-double-deficient splenic B cells express wild-type levels of the follicle-homing receptor CXCR5. Even administration of soluble TNFreceptor-IgG fusion protein to wild-type mice did not prevent B cell localization in the follicle, or germinal center formation during an immune response (Cook et al., 1998). Normal splenic B cells transferred into wild-type mice are located in the follicles. Wild-type B cells are found in the vicinity of the follicles when transferred into TNF-deficient mice. Normal splenic B cells transferred into TNF/LT␣-double-deficient mice fail to home to the follicles, presumably due to disruption of the follicular architecture in these mice. Furthermore, wild-type mice reconstituted with LT␣-deficient bone marrow lose follicular dendritic cells and germinal centers with time, but preserve the interface between T and B cell areas (Matsumoto et al., 1996). Thus, the follicular tropism of B cells is dependent on the follicular cell composition, but not on direct TNF or LT␣ signaling. T cell zone tropism remained normal only when antigen-activated B cells were transferred into TNF-deficient recipients, but not into TNF/LT␣-doubledeficient recipients (Cook et al., 1998). In conclusion, TNF, and not LT␣, has an exclusive role in the demarcation between T and B cell zones. It appears that TNF, which most likely is produced by nonhemopoietic precursor cells, is nonredundantly needed for the development of a normal white pulp, which is capable of supporting antigen-dependent responses in it, including primary and secondary follicle (germinal center) formation. This is in agreement with studies by Tkatchuk and colleagues, who reported that TNF-receptor 1 expression on nonhemopoietic cells plays a major role in the direction or promotion of B cell localization within the splenic white pulp (Tkachuk et al., 1998). Mice homozygous for the autosomal recessive alymphoplasia (aly) mutation are characterized by the absence of lymph nodes and Peyer’s patches, the lack of the splenic marginal zone and well-defined follicles as well as distinct thymic cortical and medullary regions (Koike et al., 1996; Miyawaki et al., 1994). B cell responses are elicited, albeit weakly to T cell-independent as well as -dependent antigens (Shinkura et al., 1996). However, isotype switching is defective, and neither hypermutation nor germinal center formation is observed in aly/aly mice. These phenotypes resemble in part the defects observed in LT␣-, LT␤-, or LT␤-R-deficient mice (De Togni et al., 1994; Futterer et al., 1998; Koni and Flavell, 1998). However, the aly gene was mapped to a locus on mouse chromosome 11 that is distinct from those for LT or LT␤-R (Kuramoto et al., 1994). Alymphoplasia in aly/aly mice is caused by a point mutation (guanosine to adenosine) in the gene encoding NF-␬B-inducing kinase, resulting in an amino acid substitution (G855R) in its C-terminal region which interacts with the members


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of the TNF receptor-associated factor family (Song et al., 1997). Members of the TRAF family have been implicated in many important signal transduction pathways, such as signaling via CD40 in B cells (Kehry, 1996). As mentioned above, the defects in aly/aly mice are similar to but not identical to LT␤-R-deficient mice. In LT␤-R-deficient mice, alymphoplasia does not affect B cells, as their numbers are normal in the spleen, whereas aly/aly mice have drastically reduced numbers of B cells (Shinkura et al., 1996). In addition, LT␤-R-deficient mice show no abnormalities in thymic structure, indicating that signaling through a receptor that is different from LT␤-R causes disruption of the thymic architecture in aly/aly mice. While it is clear that members of the TNF family and their receptors play an important role in the morphogenesis and the functions of secondary lymphoid organs, especially in germinal center formation during humoral immune responses, much needs to be learned of the nature of a variety of environmental cells, which interact with hematopoietic and lymphoid cells in these processes of organogenesis. It is also evident that we need to know which signaling pathways are used in these cells, and how these pathways are interconnected from the TNF family receptors to the nucleus. B. CHEMOKINES AND THEIR RECEPTORS REGULATE COMPARTMENTAL TRAFFICKING IN SECONDARY LYMPHOID ORGANS Chemokines also regulate cell movements within the T cell-rich areas. As already briefly mentioned, SLC is not only expressed by lymphoid endothelium, but also by stromal-like cells within the periarteriolar lymphoid sheath of spleen, lymph nodes, and Peyer’s patches (Gunn et al., 1998b; Nagira et al., 1997; Willimann et al., 1998; Fig. 4B). The second ligand for the chemokine receptor CCR7, ELC is also expressed in the T cell-rich areas of secondary lymphoid organs, primarily by recently immigrated mature dendritic cells (Dieu et al., 1998; Ngo et al., 1998; Sallusto et al., 1998a; Yoshida et al., 1997, 1998b; Fig. 4B and D). Both chemokines have been shown to promote migration of T cells and B cells in vitro (Campbell et al., 1998a; Gunn et al., 1998b; Hromas et al., 1997; Kim et al., 1998; Nagira et al., 1997; Ngo et al., 1998; Tanabe et al., 1997; Willimann et al., 1998; Yoshida et al., 1997, 1998a,b). SLC is not expressed in and ELC is markedly reduced in its expression in homozygous plt mice (Gunn et al., 1999). Moreover, expression of both chemokines is strongly dependent on (i.e., induced by) the two cytokines LT␣1␤2 and TNF (Ngo et al., 1999). SLC is also decreased in mice deficient for RelB, a member of the NF-␬B transcription factor family (Tanabe et al., 1997). B cell homing to, and follicular localization in secondary lymphoid organs of plt mice appears normal (Gunn et al., 1999; Nakano et al., 1997, 1998). This indicates that SLC, in contrast to T cells and dendritic cells, could at best play only a redundant role for B cell homing to secondary lymphoid organs. The redundancy could be caused by SDF-1, which

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could substitute for the lack of SLC. Since homozygous plt mice express reduced levels of ELC, but normal levels of B-lymphocyte chemoattractant (BLC), the latter chemokine also could guide B cells through the periarteriolar lymphoid sheath and localize them in the follicular areas (see below). In contrast, dendritic cells and T cells show a drastic defect in migration to and proper localization within secondary lymphoid organs of homozygous plt mice (Gunn et al., 1999; Nakano et al., 1997, 1998). This suggests that for dendritic cells and T cells, SLC is absolutely required for homing to spleen, lymph, nodes, and Peyer’s patches. Moreover, the reduced ELC levels are not enough to allow a proper localization of these cells within the T cell-rich areas. All these findings are in complete agreement with the observation of disturbed distribution of T and B cells in secondary lymphoid organs of mice deficient for the SLC and ELC receptor CCR7 (Forster et al., 1999). The importance of chemokines and their receptors in guiding homeostatic trafficking of lymphocytes to distinct microanatomical niches within secondary lymphoid organs also is evident from studies on the putative chemokine receptor BLR1 (for Burkitt’s lymphoma receptor 1, now called CXCR5) that is expressed on mature and activated B lymphocytes as well as on a small subset of CD4+ T cells (Bowman et al., 2000; Cyster et al., 1999; Forster et al., 1994, 1996; Pevzner et al., 1999). It is not clear at present how much of this CXCR5dependent B cell traffic is a consequence of antigenic stimulation, hence part of the recirculation of long-lived B cells (see below). Targeted disruption of CXCR5 leads to a defect in development of B cell follicles in spleen, Peyer’s patches, and many lymph nodes (Ansel et al., 2000; Forster et al., 1996; Fig. 4C). In these mice, a few IgD+IgM− B cells surround, in a thin rim, the centralized T cell areas. The major part of the B lymphocytes resides in a rather strong marginal zone encompassing the small rim of IgD+ B cells. However, in immunized CXCR5-deficient mice, peanut agglutinin-positive, germinal center-like B cells appear around the central artery within the periarteriolar lymphoid sheath, as it occurs in LT␤-R-deficient mice (Futterer et al., 1998), and not in the normal (secondary) follicles. This defect in migration is not due to a defective follicular dendritic cell network, but rather is intrinsic to the CXCR5-deficient B cells, as they fail to populate the normal follicular areas of wild-type recipients. The unique and specific expression of the gene encoding the homing-receptor CXCR5 to mature resting and activated B cells (as well as a small subset of T helper memory cells) (Bowman et al., 2000; Cyster et al., 1999; Forster et al., 1994, 1996; Pevzner et al., 1999) is controlled by activation of a TATA-less blr1 core promoter and in it by the three essential elements, Oct-2, OBF-1 (Bob1 or OCA-B), and NF-␬B (Wolf et al., 1998). These findings may explain some of the defects observed in the three transcription factor-deficient mice (Corcoran et al., 1993; Caamano et al., 1998; Franzoso et al., 1997, 1998; Kim et al., 1996; Nielsen et al., 1996; Schubart et al., 1996).


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Since CXCR5-deficient animals show defective follicular localization of B cells, the follicular stroma is suggested to be a source of the CXCR5 ligand. The ligand recognized by CXCR5 was identified as BLC (Gunn et al., 1998a; Legler et al., 1998). In situ hybridization analysis showed that BLC is constitutively expressed by cells within the follicular dendritic cell network in lymphoid follicles of secondary lymphoid organs, such as spleen, mesenteric lymph nodes, and Peyer’s patches (Gunn et al., 1998a). There is cross talk between B cells and follicular dendritic cells. Normal development and function of follicular dendritic cells expressing LT␤-R (Murphy et al., 1998) require signaling via LT␣1␤2 and TNF expressed by B cells (Endres et al., 1999). Expression of BLC has been found to be strongly dependent on these cytokines (Ngo et al., 1999), which is in agreement with the production of BLC by cells within the follicular dendritic cell network (Fig. 4C). The importance of the CXCR5 ligand BLC in structuring of microarchitecture in secondary lymphoid organs has been demonstrated by ectopic expression of BLC in the pancreatic islets (Luther et al., 2000). Such ectopic expression leads to lymphoid infiltration into the pancreas. Ectopic expression of SLC in pancreatic islet attracts initially T lymphocytes and dendritic cells, and only at later time points B cells (Fan et al., 2000). On the other hand, ectopic BLC expression recruits mainly B lymphocytes to the pancreatic islets (Luther et al., 2000). In medium-size infiltrates, some T cells and dendritic cells are also found, but the majority of the lymphocytes are B cells, and few high endothelial venules have developed. As the lymphoid infiltrates grow larger, more high endothelial venules develop. Also, endothelial cells and stromal-like cells in the T cell-rich zone are now expressing SLC. This selective expression of SLC in the T cellrich area probably organizes the compartmentalization in T and B cell zones. However, follicular dendritic cells cannot be detected in the B cell-rich regions of these abnormal, artificial lymphoid organizations. The requirement for the BLC-CXCR5 ligand–receptor pair in the development of a proper microarchitecture in secondary lymphoid organs has been demonstrated in BLC-deficient mice (Ansel et al., 2000). These mice have severe defects in development of secondary lymphoid organs. Most lymph nodes either are found at low frequency or are absent. The number of Peyer’s patches is severely reduced, and the ones found are smaller than the Peyer’s patches of wild-type animals. On the other hand, mesenteric lymph nodes appear absolutely normal in BLC-deficient mice. In BLC-deficient mice, B cells fail to organize in follicular regions, and instead appear as a ring of cells at the edge of the T cell-rich periarteriolar lymphoid sheath (Ansel et al., 2000). The boundary between the B cell-rich area and T cellrich zone is poorly demarcated because the B cells are not organized in follicles. As mentioned, formation of primary lymphoid follicles requires a properly functioning follicular dendritic cell network, which, in turn, is dependent on

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membrane LT␣1␤2 and TNF expressed by B cells (Endres et al., 1999). Expression of BLC is strongly dependent on these cytokines (Ngo et al., 1999). Surprisingly, BLC induces recirculating B cells to upregulate LT␣1␤2, thus promoting follicular dendritic cell development and BLC expression. Hence, BLC establishes a positive feedback loop that appears to be important for primary follicle formation (Ansel et al., 2000). Like CXCR5-deficient mice, BLC-deficient mice form germinal centers in spleen and lymph nodes after immunization with a T cell-dependent antigen. These germinal centers are, however, displaced (Ansel et al., 2000). All these findings are in complete agreement with the observations made in the CXCR5-deficient mice (see above). In conclusion, both the BLC-CXCR5 ligand–chemokine receptor and the TNF/LT␣1␤2-LT␤-R ligand–cytokine receptor interactions are mandatory for proper follicular localization of B cells and the development of Peyer’s patches and most lymph nodes (Fig. 4C). Mesenteric lymph nodes are present and show normal architecture in both CXCR5- and BLC-deficient mice (Ansel et al., 2000; Forster et al., 1996), which is in conflict with the phenotype of LT␤-R-deficient mice in which mesenteric lymph nodes are lacking (Futterer et al., 1998). Surprisingly, the CXCR5 ligand BLC is highly expressed in follicles of these lymph nodes, a fact that would also suggest a role for BLC–CXCR5 interactions in follicular organization in mesenteric lymph nodes (Gunn et al., 1998a). Moreover, mesenteric lymph node follicular B cells as well as marginal zone B cells and B1 B cells, which are normally excluded from follicles, express CXCR5 at levels comparable to their splenic counterparts, and display comparable migratory capacities toward BLC in vitro (Bowman et al., 2000). All these observations suggest that follicular localization of B cells might be under complex combinatorial control as a function of organ site and other chemokine receptor–ligand pair(s), so that a contribution of CXCR5/BLC interaction is mandatory in spleen, Peyer’s patches, and many lymph nodes for follicular localization of B cells, but redundant or not needed at all in mesenteric lymph nodes. VII. Cellular Traffic Leading to a Humoral Immune Response: Finding the Right Partner

A. ANTIGEN UPTAKE AND MIGRATION OF IMMATURE DENDRITIC CELLS An immune response to a foreign invader often begins with the uptake of that invader by dendritic cells in the epithelia of skin and mucous membranes with which we face the outside world (reviewed by Banchereau and Steinman, 1998). Immature dendritic cells reside as so-called Langerhans cells in the cutaneous epithelium. Immature dendritic cell populations also exist in peripheral blood. They are probably permanently poised at the interface of blood and epithelial cells of skin and mucous membranes, ready to extravasate upon the alarm evoked


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by invasion of an antigen (Robert et al., 1999). Capture of only small amounts of antigen by immature dendritic cells is the initiating force of humoral immune responses. Immature dendritic cells, including Langerhans cells of the skin, are very efficient in the uptake and processing of antigens. Uptake can occur through one of four possible pathways: (1) macropinocytosis, (2) endocytosis mediated by Fc␥ and Fc⑀ receptors, (3) endocytosis through the mannose receptor and the C-type lectin receptor DEC205, and (4) engulfment of apoptotic bodies through the vitronectin receptor ␣v␤3 (see Banchereau and Steinman, 1998; Bell et al., 1999) for recent general reviews on dendritic cells and their capacities and functions). Once taken up, antigens are processed by proteolytic degradation, loaded as peptides onto MHC class I and II molecules, and deposited on the cell surface as peptide–MHC complexes for presentation to T cells. Immature dendritic cells belong to the monocyte differentiation lineage of hematopoietic cells. They express a variety of chemokine receptors: high levels of CCR1, lower levels of CCR2 and CCR5, and very low levels of CCR4, CCR7, CXCR1, and CXCR4 (Sallusto et al., 1998b). Immature dendritic cells derived from CD34+ hematopoietic progenitors express CCR6 and respond vigorously to MIP-3␣ (Dieu et al., 1998). Immature dendritic cells more directly derived from monocytes also express CCR6 when cultured in the presence of TGF␤, and migrate toward MIP-3␣ (Yang et al., 1999a). Many of the receptors expressed on immature dendritic cells respond to chemokines produced at sites of inflammatory lesions by a variety of different cell types. Hence, they recruit immature dendritic cells to the sites of invading antigen (Fig. 4B). The capacity of MIP-3␣ to interact with CCR6 in the recruitment of immature dendritic cells to sites of invasion of foreign antigen has been demonstrated in vivo (Fushimi et al., 2000). Transplanted tumors, which express MIP-3␣ as a consequence of retroviral infection, attract immature dendritic cells that, in turn, induce cytotoxic T cells to migrate to the site of tumor growth. The mechanism of attraction of the cytotoxic T cells is yet unknown. These sequential migrations, in the end, result in suppression of tumor growth. It has also been postulated that trafficking of Langerhans cells to the skin, with the aim of screening the epidermis for any possible invading antigen, is regulated by the interaction of MIP-3␣ with CCR6 (Charbonnier et al., 1999). The importance of MIP-3␣/CCR6 interactions in special sites of humoral immune responses has been demonstrated in CCR6-deficient mice (Cook et al., 2000; Fig. 4B). In these mice, all primary and secondary lymphoid organs show normal cellularity and relative numbers of the various leukocyte subsets, including dendritic cells. However, CD11c+CD11b+ myeloid-derived dendritic cells are lacking in the subepithelial dome of Peyer’s patches. CCR6 appears dispensable for development of all the major leukocyte populations, including dendritic cells in most lymphoid organs, indicating that other chemokine/chemokine receptor interactions can replace the MIP-3␣/CCR6 interaction. By contrast,

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MIP-3␣/CCR6 interaction appears to be required for the development of the cells structuring the subepithelial dome. CCR6-deficient mice also have twice as many T lymphocytes within the entire mucosa of the small intestine than do wild-type mice, although with no apparent foci of inflammation in the small or large intestine. The cellular changes seen in Peyer’s patches and the mucosa of the small intestine suggests that CCR6-deficent mice might have defects in humoral immune responses in mucosal tissues. Indeed, oral administration of antigens to CCR6-deficent mice resulted in a diminished humoral immune response. Furthermore, CCR6-deficent mice infected with the murine strain of rotavirus, an enteric pathogen, have impaired mucosal production of virus-specific IgA and a slight delay in clearance of the virus (Cook et al., 2000). These defects are mucosa-specific and not due to a general immune defect, as CCR6-deficent mice respond with a normal systemic response to orally or subcutaneously administered antigens. In conclusion, MIP-3␣/CCR6 interactions play a nonredundant role in immune responses in mucosal tissues, probably by localizing CD11c+CD11b+ myeloid-derived dendritic cells to the subepithelial dome of Peyer’s patches (Fig. 4B). On the other hand, the normal immune responses in CCR6-deficent mice to subcutaneously administered antigens brings up the question of a mandatory role of MIP-3␣/CCR6 interactions in controlling the trafficking of Langerhans cells to the skin, as suggested by Charbonnier and collegues (Charbonnier et al., 1999). B. ␤-DEFENSINS: LINKING INNATE AND ADAPTIVE IMMUNITY BY THEIR ANTIMICROBIAL AND CHEMOTACTIC ACTIVITY Defensins are small cationic antimicrobial peptides with three to four intramolecular cysteine disulfide bonds, and are found in mammals, insects, and plants (Broekaert et al., 1995; Fehlbaum et al., 1994; Ganz and Lehrer, 1994; Stolzenberg et al., 1997). They are divided into two groups, designated ␣- and ␤-defensins, based on the position and bonding of their cysteine residues. Unlike ␣-defensins, ␤-defensins are released upon microbial invasion, and are upregulated by stimulation with LPS in epithelial cells of the skin and mucosal surfaces (Diamond et al., 1996; Harder et al., 1997; Stolzenberg et al., 1997). Although ␤-defensins show no primary structural similarity to MIP-3␣, they are recognized by CCR6 and can attract immature dendritic cells and memory T cells in vitro (Yang et al., 1999b). The findings that ␤-defensins are produced by mucosal membranes after microbial infection and can attract dendritic cells of the monocyte lineage in vitro appear compatible with observations in vivo that CCR6 is mandatory for the recruitment of CD11c+CD11b+ myeloid-derived dendritic cells to the subepithelial dome of Peyer’s patches and essential for mucosal immune responses (Cook et al., 2000; Fig. 4B). Thus, ␤-defensins and


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CCR6 appear to play an important role linking innate and adaptive immune responses. This can now be analyzed in detail in the CCR6-deficient mice. C. ACTIVATION AND MIGRATION OF DENDRITIC CELLS Activated by contact with the foreign invader, immature dendritic cells, regardless of their origin (Langerhans cells, hematopoietic progenitor- or monocyte-derived), begin to produce inflammatory chemokines, such as MCP-1 (recognized by CCR2), MIP-1␣ (recognized by CCR1 and CCR5), and MIP-1␤ (recognized by CCR5) (Fig. 4B). These chemokines attract additional immature dendritic cells to migrate to the site of invasion (Sallusto et al., 1998a). They also induce the migration of monocytes, macrophages, and na¨ıve T cells, thereby setting off an inflammatory reaction. At the same time, inflammatory stimuli, such as IL-1, TNF␣, LPS, or doublestranded RNA, which are locally released, induce maturation of immature dendritic cells to mature, professional antigen-presenting cells (Cella et al., 1997; Sallusto and Lanzavecchia, 1994). This sets in motion a rapid, coordinated change in chemokine receptor expression (Sallusto et al., 1998a). Maturing dendritic cells downregulate the receptors that brought them as immature dendritic cells to the site of antigen invasion, and upregulate those chemokine receptors that control their migration from the epidermis, with which we face the outside world, into peripheral lymphoid organs such as the spleen, draining lymph nodes, and Peyer’s patches. There they home to the T cell-rich areas to present to the T lymphocytes their peptide–MHC complexes (Fig. 4B). The main chemokine/receptor pairs involved in this multistep migration have been identified as SLC/CCR7 and ELC/CCR7. Maturing dendritic cells strongly upregulate expression of CCR7 and are, in contrast to immature dendritic cells, attracted toward ELC in vitro (Sallusto et al., 1998a; Fig. 4B). In homozygous plt mice that are deficient for SLC, and in CCR7-deficient mice, dendritic cells cannot home to secondary lymphoid organs (Forster et al., 1999; Gunn et al., 1999). D. ENCOUNTER OF DENDRITIC CELLS WITH T LYMPHOCYTES Once mature dendritic cells have reached the secondary lymphoid organs and express antigenic peptide-MHC complexes stabily on their surface, they have to come together with resting T cells to activate them either as cytotoxic T cells or as T cells giving help to B cells. This confrontation of T cells with antigenpresenting cells is crucial in order to elucidate an immune response. A dominant role for such an attraction of T cells to dendritic cells has been postulated for the CC chemokine ELC that is produced by mature dendritic cells in the T cell zones, and that has been shown to preferentially attract in vitro naive T cells (Kim et al., 1998; Ngo et al., 1998; Fig. 4B). DC-CK1, another CC chemokine (so far only identified in humans), is expressed by dendritic cells in the outer region of the periarteriolar lymphoid sheath. It also attracts naive T cells (Adema

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et al., 1997). DC-CK1 could act in conjunction with ELC to navigate the virgin T cells to the dendritic cells for T cell–dendritic cell encounters. Although the cellular expression pattern of ELC and DC-CK1 in tissues is almost the same, in lung, DC-CK1 is produced at high levels, while ELC is not expressed at all, suggesting slightly distinct roles for the two molecules. Potent attraction of virgin T cells to the antigen-presenting dendritic cells might allow efficient scanning of multiple T cells for the ones with TCRs specific for the antigen. In addition, na¨ıve T cell attraction to dendritic cells may also have a regulatory role in the maintenance of peripheral homeostasis, as T cell survival is dependent on continuous TCR interactions with MHC-(self )peptide complexes (Freitas and Rocha, 1999). Once the T cells are activated, they are likely to be kept in the vicinity of the dendritic cells for continuous antigenic stimulation. This continued attraction might be effected by the recently identified CC chemokines ABCD-1 and ABCD-2, which are produced in high amounts by dendritic cells in secondary lymphoid organs and which attract activated but not resting T cells (Schaniel et al., 1998, 1999; Fig. 4B). This scenario has recently received further support by findings in vivo, showing that Langerhans cells upregulate ABCD-1 as they migrate from the skin into the periarteriolar lymphoid sheath of lymph nodes to become efficient antigen-presenting cells (Tang and Cyster, 1999). E. GENERATION OF HELPER T CELLS OF TYPE 1 AND TYPE 2 The combination of cytokines present during the encounter of dendritic cells with T cells determines the pattern of cytokines produced by the effector T cells (Mosmann and Coffman, 1989; Romagnani, 1994). The effector T cells are subdivided into either polarized helper T cells of type 1 (Th1 cells) or type 2 (Th2 cells). Th1 cells secrete IL-2 and INF-␥ and predominantly control cellmediated immune responses by activating mononuclear phagocytes. Th2 cells produce IL-4, IL-5 and IL-13 and are involved in responses dominated by IgE that is produced by immunoglobulin isotype-switched B cells, eosinophils, and basophils, and are prominent in the pathogenesis of allergic diseases. Several lines of evidence support the idea that chemokines might also play a role in T cell polarization toward Th1 or Th2. The “inflammatory” CC chemokine MCP-1, which attracts monocytes, memory T cells, and natural killer cells in vitro (Allavena et al., 1994; Carr et al., 1994; Maghazachi et al., 1994; Matsushima et al., 1989; Yoshimura et al., 1989) is associated with the development of polarized Th2 responses (Lu et al., 1998; Chensue et al., 1995). Maturing dendritic cells induce expression of MCP-1 (Sallusto et al., 1998a), which, in turn, enhances the production of IL-4 by T cells (Karpus et al., 1997). MCP-1-deficient mice have problems recruiting monocytes and macrophages to inflammatory sites, as tested in several experimental models (Lu et al., 1998). This finding might have implied a role of MCP-1 in Th1-polarized responses. However, recent results indicate instead that MCP-1 is needed for the development of Th2-polarized


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cells and responses (Gu et al., 2000). MCP-1-deficient mice have no defect in homing of na¨ıve T cells. However, these mice are unable to mount Th2 responses. This defect might be explained by the inability of lymph node cells to produce normal amounts of the cytokines IL-4, IL-5, and IL-10, which are important for polarization of T cells toward Th2. MCP-1 must, however, exert its effects on Th2 polarization through receptor other than CCR2, which is so far the only known receptor for MCP-1, since CCR2-deficient mice appear to have a Th1 rather than a Th2 defect (CCR2 recognizes also MCP-3, MCP-4, and MCP-5; (Boring et al., 1997; Warmington et al., 1999). Given their different effector functions, it seems only rational that Th1- and Th2-polarized cells are differentially recruited to sites of inflammation or to peripheral organs (Lichtman and Abbas, 1997). Indeed, it has been found that polarized Th1 and Th2 cells show differential expression of chemokine receptors and, thus, chemotactic responsiveness in vitro (Bonecchi et al., 1998; Loetscher et al., 1998; Sallusto et al., 1997, 1998b; Zingoni et al., 1998). The chemokine receptors CCR5 (→ MIP-1␣, MIP-1␤, RANTES) and CXCR3 (→ IP-10, Mig, I-TAC) are expressed predominantly by Th1-polarized cells, whereas Th2-polarized cells produce mainly CCR3 (→ RANTES, MCP-2, -3, -4, Eotaxin-1, -2), CCR4 (→ ABCD-1, -2) and CCR8 (→ I-309). F. ENCOUNTERS OF B LYMPHOCYTES WITH T LYMPHOCYTES Mature, resting B cells bind native antigen often by recognizing their threedimensional configuration via their B cell receptors. Binding leads to activation if the affinity is above a certain threshold. This may occur in any of several possible locations: in the blood, near high endothelial venules in lymph nodes where antigen has been shown to be drained from the subcapsular sinus, within the marginal zone of the spleen, or even on the surface of follicular dendritic cell within the follicles. However, the way resting B cells are stimulated in vivo by antigen still remains unclear (see Fig. 4C). Activation often occurs in combination with the costimulatory action of mitogens, which often are also inflammatory molecules, such as LPS or double-stranded nucleic acids, and often without the help of T cells. Activation may affect follicular or extrafollicular B cells, maybe at different stages of maturation. Since athymic nu/nu mice have normal B cell-rich follicles in their secondary lymphoid organs, it is likely that T cell-independent B cell activation can be sufficient to organize B cells properly into follicles. Once B lymphocytes are activated through their B cell receptor, all the major participants (dendritic cells, T cells, and B cells) in the humoral immune response have now been activated by foreign antigen. However, the B cells will either have to recruit activated helper T cells to their sites in the follicles, or they will have to migrate to the boundaries between the B cell-rich follicles and the T cell-rich periarteriolar lymphoid sheath, that is, to the sites where dendritic cell–T cell interactions have already occurred.

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Little or no movement of antigen-specific T cells to extrafollicular foci of B cell proliferation has been observed. However, T cells have been seen to move into follicles and expand there, in part to become memory and effector T cells (Garside et al., 1998; Fig. 4D). It has been seen in histological sections of spleen and lymph nodes that cognate interactions of antigen-activated B cells with the primed T cells occur on the follicular side at the boundary of B and T cell-rich zones of the secondary lymphoid organs (Garside et al., 1998). This navigation of antigen-activated B cells to the outer edge of the T cell-rich area is presumably lead by dendritic cell-expressed ELC (Ngo et al., 1998). In addition to promoting migration to the outer region of the T cell zone, B cell receptor engagement induces strong expression of ABCD-1 and ABCD-2 and low levels of MIP-1␣ (Schaniel et al., 1998, 1999). Since dendritic cells also produce ABCD-1 and ABCD-2, the production of these activated T cell-attracting chemokines by B cells should enhance the possibility of an encounter between antigenpresenting B cells and activated antigen-specific T cells (Fig. 4D). If there were a way to downregulate the expression of these chemokines in dendritic cells, it might give the B cells a greater chance to attract the antigen-specific T cells. Once the T cells are in the follicles, they are likely to give help to B lymphocytes, thereby playing an important role in the development of germinal centers and the mantle zone. It is known that they are required for isotype class switching and hypermutation of B cells, and, last but not least, in the establishment of the memory B cell pool (Ahmed and Gray, 1996; Berek and Milstein, 1988; Berek et al., 1991; Klaus and Humphrey, 1977). Both ABCD-1 and ABCD-2 are recognized by CCR4 (Imai et al., 1997, 1998). In addition, ABCD-1 is recognized by at least one other, yet unidentified, receptor (Schaniel et al., 1999; Struyf et al., 1998). CCR4-deficient mice apparently have no defect in mounting humoral immune responses (Chvatchko et al., 2000). Hence, attraction of antigen-primed CCR4-expressing T cells by ABCD-1- and ABCD-2-producing antigen-specific B cells appears to be redundant. This migration is likely to be effected by the yet unknown receptor recognizing ABCD-1, as well as any other chemokine receptor/ligand pair which has not yet been discovered to be active in T cell–B cell interaction. Changes also occur in the tropism of T cells during their activation in the T cell-rich areas that may enhance their intrinsic propensity to migrate to the B cell follicles (Garside et al., 1998). For instance, in vitro antigen-stimulated Th2 cells become unresponsive to SLC and ELC as a result of CCR7 downregulation (Randolph et al., 1999). After adoptive transfer, these Th2 cells, but not Th1 cells migrate to the border of the periarteriolar lymphoid sheath in close proximity to the B cell follicles (Randolph et al., 1999). Forced expression of CCR7 in Th2 cells results in retention of the Th2 cells in the T cell-rich zones and abrogates delivery of B cell help (Randolph et al., 1999). In vivo antigen-activated CD4+ T lymphocytes have been shown to induce expression of CXCR5 and become responsive to BLC in vitro (Ansel et al.,


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1999). This upregulation of CXCR5 on CD4+ T cells appears to depend on OX40/OX40 ligand interaction (Flynn et al., 1998). The failure of mice deficient in signaling via CD28 to develop germinal centers may be due to the inability of T lymphocytes to upregulate OX40. Hence, they are incapable of inducing expression of CXCR5 and, thus, become attracted into B cell follicles (Walker et al., 1999). Moreover, constitutive expression of OX40 ligand on dendritic cells results in significantly increased numbers of CD4+ T cells localized within follicles, and in large germinal centers (Brocker et al., 1999). It should, however, be noted that, on one hand, CXCR5-expressing CD4+ T cells failed to home to follicles after adoptive transfer (Ansel et al., 1999). CXCR5- and BLC-deficient mice, lacking primary follicles, can also develop a microenvironment in the T cell-rich areas of the spleen that allows formation of functional germinal centers (Ansel et al., 2000; Voigt et al., 2000). Furthermore, OX40-deficient mice are neither disturbed in organization of follicular or germinal center structures nor hampered in mounting normal humoral immune responses (Kopf et al., 1999). These findings make two points: First, the defect is forming germinal centers in mice deficient for CD28 signaling appears not to be due to a failure of OX40 induction on T cells, nor to a lack of the B cell folliclehoming receptor CXCR5. Second, CXCR5, upregulated on CD4+ T cells by signaling through OX40, appears not to be mandatory for follicular localization of T cells. This suggests that a series of redundant chemokine receptor–ligand pairs recruit T cells that deliver help to B cells, among them ABCD-1 and ABCD-2 attracting activated T cells (Fig. 4D). In summary, cell migrations that allow effective collaboration between T and B cells should be controlled by the successive responsiveness of a variety of different receptors to a set of specific chemokines. First, the T cells that could deliver help to B cells must be able to become unresponsive to chemokines such as SLC and ELC, and temporarily unresponsive to ABCD-1 and ABCD-2 to allow them to move away from the dendritic cells toward the B cells. Only then they can enter the complex gradient of chemokines that include ABCD-1 and ABCD-2 produced by the activated B cells asking T cell help, BLC secreted by cells of the follicular dendritic cell network, and other (yet unidentified) chemokines (for a possible explanation, see Section III and Fig. 2). VIII. Migration of Effector and Memory T and B Cells

A more rapid and vigorous immune response is initiated when the same antigen stimulates the immune system for a second time. This phenomenon is known as immunological memory. It is the basis for a successful vaccination. When immunological memory is generated, so-called memory cells of the T and B lymphocyte lineage are formed (reviewed in Ahmed and Gray, 1996). Memory cells are characterized by a series of properties that distinguish them from their virgin counterparts. They are long lived, have a lower threshold for activation,

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and appear to need fewer costimulatory signals to differentiate from B cells into plasma cells and from T cells into killer and helper T cells. Most if not all memory B cells are believed to have been generated in a germinal center reaction (reviewed in Liu, 1997; MacLennan, 1994). The vast majority of memory B cells have switched their immunoglobulin isotypes and have somatically hypermutated their IgH and IgL chain variable regions of the immunoglobulin they express (Berek et al., 1991; Coico et al., 1983; Decker et al., 1995; Jacob et al., 1991; Kosco-Vilbois et al., 1997). Immunoglobulin class switching is thought also to occur in germinal centers. When antigenexperienced B cells leave the germinal centers, they assume a new route of circulation and travel by blood to lymph and back in search of foreign antigen. Such memory B cells probably can also settle in special sites, such as bone marrow. Other sites of memory cell accumulation are most closely associated to antigen draining sites within (secondary) lymphoid organs where new antigen enters. Little is known about chemokine receptor–ligand pairs and adhesion molecules, which guide the trafficking and settlement of memory lymphocytes. A. MIGRATIONS OF THE FINAL EFFECTOR CELLS 1. Plasma Cells Antibodies secreted by plasma cells often provide the first line of defense against any invader. Plasma cells are generated either T-independently in extrafolliclar sites or T-dependantly in germinal center reactions from mature B cells. Plasma cells can also be formed from memory B cells that seem to be biased toward this terminal differentiation (Arpin et al., 1997). However, plasma cells differ from memory B cells in many aspects. For instance, plasma cells downregulate surface expression of many typical B cell markers, including surface immunoglubulin, and MHC class I and II molecules (Abney et al., 1978; Halper et al., 1978). This feature indicates that, unlike memory B cells, plasma cells do not contribute to antigen presentation. Most plasma cells generated during immune responses have the unique function of antigen-specific antibody production in the highest amount and the shortest time possible (i.e., 10000 molecules per second) (Helmreich et al., 1961; Hibi and Dosch, 1986). Plasma cells generated in a T cell-independent way normally have a short life and die within a few days (Smith et al., 1996). Some plasma cells produced T celldependently become long lived. One could consider them as antibody-secreting memory cells and, thus, as belonging to the memory B cell pool. These long-lived plasma cells can be detected in the bone marrow for at least one year after the first antigen encounter (Manz et al., 1997; Slifka et al., 1995, 1998). The mechanism that brings these long-lived plasma cells to the bone marrow and keeps them there is not known. One might suggest that SDF-1/CXCR4 interactions


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play a similar role in retention of long-lived plasma cells in the bone marrow as it does for precursor B cells. This, however, awaits to be demonstrated. 2. Memory/Effector T Cells Two clonally expanded T cell populations emerge from a primary immune response: so-called effector T cells, which fight the spread of the foreign antigen, and memory T cells, which protect against subsequent antigen invasions and perform qualitatively different and quantitatively enhanced responses upon secondary antigen encounter (Ahmed and Gray, 1996; Dutton et al., 1998; Zinkernagel et al., 1996). Both effector and memory T cells are likely to recirculate from blood to lymph and back, and are distributed to all lymphoid organs, particularly to epithelial surfaces, such as the skin and the intestine, where antigen might be encountered again. The basis of immunological T cell memory has long been debated. According to one view, the long-lived recirculating memory T cells terminally differentiate to effector cells upon antigen reencounter. Another theory suggests that constant contact with antigen is required to maintain long-term memory (Ahmed and Gray, 1996; Gray, 1993; Mackay, 1993; Sprent, 1994; Sprent and Tough, 1994). Memory/effector T cells mostly travel through peripheral tissues (Mackay et al., 1990). However, some memory T cells must reach the secondary lymphoid organs to mount secondary immune responses upon reencounter of antigen. This implies that different trafficking properties, and thus different expression of chemokine receptor, must exist for distinct subsets of memory T cells. One population will express chemokine receptors guiding their migration to peripheral tissues, whereas the other population expresses chemokine receptors allowing them to enter secondary lymphoid organs. Because CCR7 has been shown to be essential for T lymphocyte trafficking to secondary lymphoid tissues (Forster et al., 1999; Gunn et al., 1999), Sallusto and colleagues thought that this chemokine receptor might identify a subset of peripheral blood memory/effector T cells that is prone to specifically migrate to secondary lymphoid organs. Indeed, the memory/effector T cell population can be subdivided into two classes based on expression of CCR7 (Sallusto et al., 1999). The CCR7-expressing cells were defined as central memory T cells and the CCR7-negative cells as effector memory T cells, based on functional analyses of these T cell subpopulations (Sallusto et al., 1999). The effector memory T cells express several “inflammatory” chemokine receptors and are endowed with various effector functions. They represent an immediately available pool of antigen-primed cells that can be recruited to inflammatory or cytotoxic reactions, thus quickly fighting invasions by foreign antigen. The central memory T cells, on the other hand, that lack inflammatory and cytotoxic (i.e., effector) functions are, however, capable of homing to secondary lymphoid organs due to expression of CCR7. There they can efficiently activate dendritic cells,

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help B cells, and may likely become effector memory T cells (Sallusto et al., 1999). Memory T cells are distributed to all lymphoid organs, as well as to epithelial surfaces like the skin and the intestine, where the chance of first reencounter of antigen is high. Systemic cutaneous memory CD4+ T cells expressing the cutaneous lymphocyte antigen (CLA) are thought to be recruited to the skin by CCR4 and its ligands (Campbell et al., 1999a). Most cutaneous, but not the intestinal, memory T cells express high amounts of CCR4 and respond to the CCR4 ligands ABCD-1 and ABCD-2. ABCD-2 but not ABCD-1 expression has been observed by activated epithelium and on venules of chronically inflamed skin infiltrated by CCR4-expressing cells (Campbell et al., 1999a). Furthermore, ABCD-2 induced integrin-dependent adhesion of skin, but not intestinal, memory T cells to ICAM-1, and caused their rapid arrest. CCR4 and its ligands appear to be responsible for homing of cutaneous, but not the intestinal, memory T cells. What chemokine receptor–ligand pair(s) could recruit intestinal memory T cells? One candidate is CCR9, which recognizes TECK. CCR9 is expressed by ␣4␤7+ intestinal, but not cutaneous, memory T cells (Zabel et al., 1999). TECK has also been found expressed in the small intestine, where TECK-responsive, CCR9-expressing intestinal memory T cells have also been identified (Vicari et al., 1997; Wurbel et al., 2000; Zabel et al., 1999). CCR6/MIP-3␣ might be another chemokine receptor–ligand pair involved in the migration of intestinal memory T cells. Almost all ␣4␤7 memory T cells express CCR6 and respond to MIP-3␣ (Liao et al., 1999), which is expressed in high amounts in Peyer’s patches near the boundary to the lumen of the small intestine (Cook et al., 2000; Tanaka et al., 1999). Na¨ıve T cells do not express CCR6. The suggestion that ␣4␤7 memory T cells are recruited to the intestine via CCR6/MIP-3␣ interaction is supported by findings in CCR6-deficient mice (Cook et al., 2000). As already mentioned, these mice are defective in mounting proper mucosal immune responses. This is thought to be the cause of lack of CD11c+CD11b+ myeloid-derived dendritic cells in the subepithelial dome of Peyer’s patches (Cook et al., 2000). However, these analyses did not specify whether the changes seen in CCR6-deficient mice result exclusively from the loss of function of this single CD11c+CD11b+ dendritic cells population or of other cell types, such as intestinal memory T cells. In summary, memory T cells can be divided into effector and central memory T cells, displaying distinct functions according to their differential capacity to express CCR7. CCR7-negative T cells are capable of rapid recruitment to inflammatory reactions and cytotoxicity, and are the descendant of the central memory T cell (Sallusto et al., 1999). CCR7-positive central memory T cells are prone to home to secondary lymphoid organs where they can give help to B cells. Moreover, cutaneous and intestinal memory T cells can be distinguished from


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each other. CCR4/ABCD-2 interactions appear to control the traffic of cutaneous memory T cells, whereas CCR9/TECK interactions, in combination with CCR6/MIP-3␣ interactions, regulate migration of intestinal memory T cells. IX. Possible Clinical Relevance of the ABCD Chemokines

The possibly relevant roles of chemokines and their recptors in several diseases have gained some intrest since the discovery of differential chemokine and chemokine receptor expresssion profiles in tissues of patients suffering from distinct diseases compared to the same tissues of healthy individuals. Here, we focus on diseases in which a role for the ABCD chemokine has been implicated. A. THE ABCD CHEMOKINES AND ENDOTOXEMIA Fulminant hepatic failure is a clinical syndrome characterized by acute and severe liver failure, often causing death. Fulminant hepatic failure can be mimicked in mice by priming with live or heat-killed bacteria followed by a subsequent challenge with a low dose of lipopolysaccharide (LPS) (Tsuji et al., 1997). This model of endotoxic shock can be pathophysiologically classified into two phases: an early priming phase induced by the bacterium, and a late eliciting phase induced by the LPS administration. In the priming phase, mononuclear cells, including IFN-␥ producing T cells, infiltrate the liver, leading to granuloma formation. In the eliciting phase, inflammation further increases, resulting in severe hepatocellular damage around the granulomas. A crucial role for ABCD-2 was demonstrated, and one for ABCD-1 suggested, in fulminant hepatic failure (Yoneyama et al., 1998). ABCD-1 and ABCD-2 were selectively produced by granulomaforming cells predominantly during the eliciting phase of the disease, and CD4+, CD8+, and NK T cells infiltrated the liver parenchyma after LPS challenge. In vivo injection of anti-ABCD-2 monoclonal antibody just before LPS administration significantly reduced the number of liver-infiltrating CD4+, CD8+, and NK T cells, and thereby drastically increased the survival rate of the treated mice. A role for ABCD-1 in fulminant hepatic failure was suggested by its induced expression during the eliciting phase, and its migratory potential on the cells, on which ABCD-2 act (Yoneyama et al., 1998). Preliminary experiments simulating fulminant hepatic failure using Salmonella typhimurium in combination with LPS in ABCD-1-deficient mice may strengthen this suggestion (Takeyuki Shimizu, Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers, unpublished data) Intraperitoneal injection of either a high dose of LPS or a low dose of LPS together with D-galactosamine results in endotoxic shock. LPS-induced endotoxic shock is an inflammatory model involving platelets, monocytes, and

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macrophages. The implication of ABCD-1 and ABCD-2 in endotoxic shock, as suggested in the fulminant hepatic failure model (see above), is supported by findings made in mice deficient for CCR4, the receptor for ABCD-1 and ABCD-2 (Chvatchko et al., 2000). These mice show significantly decreased mortality on administration of both low and high doses of LPS when compared with wild-type animals. The increased survival in CCR4-deficient mice is accompanied by lower levels of the proinflammatory cytokines IL-1␤ and TNF␣, as well as a significantly decreased number of macrophages and lymphocytes in the peritoneal cavity as compared to wild-type mice (Chvatchko et al., 2000). The mechanism of how ABCD-1 and ABCD-2 and its receptor CCR4 contribute to endotoxic shock remains, however, unclear. B. THE ABCD CHEMOKINES AND SEPSIS Sepsis and sepsis-mediated organ failure still cause the most deaths following medical surgery, despite the development of powerful antibiotic therapy and intensive patient care (Astiz and Rackow, 1998). The terms sepsis, severe sepsis, and septic shock are used to document the severity of clinical response to infection. Sepsis is known as a sign of infection and tissue inflammation. Severe sepsis is the development of hypoperfusion with organ dysfunction, and septic shock is documented as hypoperfusion and persistent hypotension. Mortality ranges from 16% in patients with sepsis to 40–60% in patients with septic shock. A murine septic peritonitis model, cecal ligation and puncture, possesses a number of similarities to clinical sepsis with peritonitis associated with postsurgical or accidental trauma (Fink and Heard, 1990). In this model, intraperitoneal administration of ABCD-1 protected mice from cecal ligation and puncture-induced death (Matsukawa et al., 2000). The mechanism whereby ABCD-1 exerts its protective role in the cecal ligation and puncture model of experimental sepsis appears to be due to its activity on phagocytic macrophages. Survival after cecal ligation and puncture was accompanied by an increased recruitment of macrophages into the peritoneum as well as a greatly decreased amount of viable bacteria recovered from the peritoneum. In vitro, ABCD-1 enhanced bacterial phagocytic and killing activities of macrophages in a dose-dependent manner. This bacterial clearing by peritoneal macrophages is due to increased generation of reactive oxygen products and release of lysosomal enzymes upon activation by ABCD-1. Furthermore, administration of ABCD-1 appears to ameliorate cecal ligation and puncture-induced systemic tissue inflammation as well as tissue injury, in part due to modulation of the levels of inflammatory cytokines and chemokines in specific tissues. C. THE ABCD CHEMOKINES AND ASTHMA Asthma is a chronic disease of the lung airways characterized by bronchospasm, or narrowing of the airways, and inflammatory infiltration of eosinophils,


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basophils, and T lymphocytes into the airways of the lung. An additional finding is airway hyperreactivity. The role of chemokines in recruiting leukocytes to the asthmatic lungs and in delivering signals involved in bronchial hyperresponsivness has been well established. Recently, a role for ABCD-1, but not for ABCD-2, was demonstrated in pulmonary homing of type 2 helper T lymphocytes as well as airway hyperreactivity (Gonzalo et al., 1999; Lloyd et al., 2000). A mouse model was used, showing allergic airway disease based on adoptive transfer of in vitro repeatedly polarized allergen-specific effector Th cells. In this model, ABCD-1 but not ABCD-2 expression was increased in the lungs of allergic mice, and a correlation between ABCD-1 production and the recruitment of Th2 cells to the lung after allergen administration could be established. Injection of polyclonal antiABCD-1 antibody decreased the percentage of allergen-specific Th2 cells, but not of Th1 cells in the lungs by at least 50%. Eosinophilia were also decreased by ABCD-1 blockage. Administration of anti-ABCD-1 antibody also reduced airway hyperreactivity, albeit slightly (Lloyd et al., 2000). The availability of ABCD-1-deficient mice now allows researchers to directly investigate the importance of ABCD-1 in lung infiltration of allergen-specific Th cells in the mouse model of allergic airway hyperreactivity stimulating asthmalike disease syndromes. D. THE ABCD CHEMOKINES AND ATOPIC DERMATITIS Atopic dermatitis is an inflammatory disease of the skin characterized by eczematous skin lesions in specific locations. The pathology of atopic dermatitis differs from that of typical delayed hypersensitivity reactions by an increase in Th2 and not Th1 cells in the lesions. The skin lesions are characterized by infiltrating CLA-expressing Th cells, monocytes/macrophages, granulated mast cells, and eosinophils. An increased amount of dermal dendritic cells and epidermal Langerhans cells has also been observed in the skin lesions of atopic dermatitis. A possible implication of ABCD-1 and ABCD-2 in atopic dermatitis has been observed in the NC/Nga mouse strain by Vestergaard and colleagues (Vestergaard et al., 1999). This strain shows high susceptibility to irradiation and to anaphylactic shock induced by ovalbumin, and also develops eczematous-like lesions in a normal environment, but not under specific pathogen-free conditions (for references, see Vestergaard et al., 1999). ABCD-1 was constitutively produced both in nonlesional and, to a 6-fold higher extent, in lesional skin of mouse (Vestergaard et al., 1999) and man (Galli et al., 2000). The cells expressing ABCD-1 were identified as dermal dendritic cells. In the nonlesional skin, these dermal dendritic cells were located deeper in the epidermis than in the lesional skin. ABCD-2 expression was dramatically increased during the course of atopic dermatitis. Keratinocytes stimulated with TNF-␣, IFN-␥ , or IL-1␤ in

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the basal layer of the epidermis have been identified as the source of ABCD-2 production. In another study, ABCD-2 production was demonstrated in venules associated with lymphocyte homing in chronically inflamed skin with dermatological disorders (Campbell et al., 1999a). ABCD-1 and ABCD-2 were shown to be responsible for vascular chemoattraction of cutaneous, but not intestinal, memory T lymphocytes (Campbell et al., 1999a). In conclusion, the production of ABCD-1 and ABCD-2 in atopic dermatitis-like lesions thus likely explains the chronic infiltration by CLA+ Th2 cells. E. THE ABCD CHEMOKINES AND LYMPHOMAS There is limited information on the expression of chemokines in malignant tissues and their possible roles in the induction, maintenance, or increased severity of the disease. Constitutive expression of ABCD-1 was seen in malignant CD38+ B cells, carrying the bcl-2 translocation, purified from highly invaded lymph nodes from patients with low-grade follicular lymphoma (Ghia et al., 1999). In contrast, several mature B cell lines derived from lymphomas carrying the bcl-2 translocation did not show any ABCD-1 production. Freshly isolated B cells from several patients diagnosed with either B-cell chronic lymphocytic leukemia or B-cell acute lymphocytic leukemia (B-ALL) did not show any ABCD-1 and ABCD-2 expression (Ghia et al., 1999, 2000). However, in the presence of soluble CD40-ligand, ABCD-1 and ABCD-2 were readily detectable in B-ALL already after 1 day of culture. This may be of particular interest, as successful tumor vaccination requires the induction of an antitumor specific immune response as well as the capacity to attract effector cells to the site of tumor growth. It has been demonstrated that B-ALL cells can be modified in vitro by CD40-crosslinking to become efficient antigen-presenting cells, thus inducing the generation and expansion of autologuous antitumor cytotoxic T lymphocytes. Anti-CD40 stimulation of B-ALL cells also leads to production and secretion of high amounts of ABCD-1 and ABCD-2, thereby allowing transendothelial migration of the generated antileukemia specific cytotoxic T cells (Ghia et al., 2000). Hodgkin’s disease is one of the most frequent lymphomas. Hodgkin’s disease is distinguished in four main histological subtypes: lymphocyte predominant, nodular sclerosing, mixed cellular, and lymphocyte depleted (Lukes and Butler, 1966). Recently the chemokine expression pattern in tissues of patients with Hodgkin’s disease of different subtypes was analyzed (Teruya- Feldstein et al., 1999). ABCD-1 was not expressed in higher amounts in Hodgkin’s disease tissues than in tissues from lymphoid hyperplasia. However, within Hodgkin’s disease subtypes, expression of ABCD-1 was present at high levels in nodular sclerosing Hodgkin’s disease, and virtually absent in the other subtypes. Thus, the expression of ABCD-1 and other chemokines in Hodgkin’s disease subtypes may account for the different cell types surrounding the tumorigenic cells.


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F. THE ABCD CHEMOKINES AND HUMAN IMMUNODEFICIENCY VIRUS Human immunodeficiency virus uses chemokine receptors, mainly CXCR4 and CCR5, in conjunction with CD4 to infect healthy cells. The chemokine ligands to these receptors were found to block virus infection. Even though CCR4, the receptor for ABCD-1, is apparently not used by human immunodeficiency virus as coreceptor for infection, N-terminally processed human ABCD-1 showed human immunodeficiency virus suppressor activity independent of the viral phenotype (Pal et al., 1997; Struyf et al., 1998). The most powerful known cause of innate human immunodeficiency virus resistance is CCR532, a mutant allele, coding for a truncated inactive form of CCR5 (Dean et al., 1996; Dragic et al., 1996; Huang et al., 1996; Liu et al., 1996; Michael et al., 1997; Samson et al., 1996; Zimmerman et al., 1997). CX3CR1 that recognizes ABCD-3 is a recently identified human immunodeficiency virus coreceptor too (Combadiere et al., 1998; Reeves et al., 1997; Rucker et al., 1997). CX3CR1 interacts only with a limited number of human immunodeficiency virus envelopes, and ABCD-3 can efficiently block human immunodeficiency virus coreceptor activity of CX3CR1 (Combadiere et al., 1998). That CX3CR1 functions as a human immunodeficiency virus coreceptor suggests that nucleotide polymorphic variations of it may slow or accelerate disease progression. Indeed, rapid progression to acquired immunodeficiency syndrome was observed in human immunodeficiency virus individuals with a structural variant of CX3CR1 (Faure et al., 2000). The human immunodeficiency virus-1 envelope protein gp120 was shown to induce apoptosis in hippocampal neurons, thus perhaps causing directly the acquired immunodeficiency syndrome dementia syndrome (for references, see Meucci et al., 1998). However, in the presence of either ABCD-1 or ABCD-3, human immunodeficiency virus-1 gp120-induced neuronal death was considerably slowed (Meucci et al., 1998). In conclusion, ABCD-1 and ABCD-3 as well as the ABCD-3 receptor CX3CR1 have been implicated in interfering with human immunodeficiency virus infection, progression, or induced cell death. These observations suggest a potential therapeutic utility of agonists of ABCD-1 and ABCD-3 receptors CCR4 and CX3CR1.

X. Future Perspectives

We only begin to understand the role(s) of chemokines and their receptors in organ development and compartmentalization of (secondary) lymphoid tissues, and during humoral immune responses. Signal transduction pathways induced by chemokines binding to their cognate receptors are still not well understood. Chemokine receptors belong to the

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family of G protein-coupled receptors that can be inhibited by pertussis toxin, and several molecules are known to regulate them, including kinases, arrestins, and regulators of G protein-signaling (Bowman et al., 1998) (Ali et al., 1999; Guinamard et al., 1999; Reif and Cyster, 2000). Chemokines and their receptors participate in mechanisms by which different signals from other receptors, such as cytokine or antigen receptors, are linked to distinct ligand-responsiveness on the surfaces of individual cells on the one side, and regulation of gene expression in the nucleus on the other. Signaling through chemokine receptors also changes the cells’ adhesion capacities (Campbell et al., 1998a; Gunn et al., 1998b; Pachynski et al., 1998; Stein et al., 2000). Hence, chemokines and/or adhesion molecules are candidate therapeutic targets for a wide range of inflammatory diseases, including inflammatory bowel disease (Hesterberg et al., 1996), asthma (Das et al., 1995), diabetes (Hanninen et al., 1996), rheumatoid arthritis (Diaz-Gonzalez and Ginsberg, 1996; Diaz-Gonzalez and Sanchez-Madrid, 1998; Postigo et al., 1992), cancer (Ghia et al., 1999; 2000; Teruya-Feldstein et al., 1999), and multiple sclerosis (Swanborg, 1995). Understanding the intracellular links of chemokine-, cytokine-, and adhesion-receptor signaling pathways and their mutual influence on each of these activities may help to find ways of curing some of these diseases in the future. It is obvious that chemokine–chemokine receptor interactions play a vital role in the development of an organ. For hematopoiesis, chemokine receptor– ligand pairs regulate the positioning of cells into special microenvironments in the aorta–gonad–mesonephros, fetal liver, bone marrow, thymus, and spleen for lineage development and differentiation (see, e.g., Kawabata et al., 1999; Ma et al., 1999), inhibit or allow hematopoietic cell differentiation (see, e.g., Gu et al., 2000; Ohneda et al., 2000; Patel et al., 1997), or take part in organ development (see, e.g., Ansel et al., 2000; Fan et al., 2000; Forster et al., 1996, 1999; Luther et al., 2000). Several chemokine receptors act as coreceptors for cell entry of viruses, including the human and simian immunodeficiency viruses (Berger et al., 1999; Horuk, 1999; Littman, 1998; Locati and Murphy, 1999). Research has focused on how viruses may manipulate the host–cell functions as well as modify the host immune response by utilizing chemokine and chemokine receptors. Viruses not only abuse the host’s chemokine receptors for cell entry, they also harbor genetic information for several viral chemokines, which could interact with host receptors, and viral chemokine receptors (Alcami et al., 1998a, b; Dairaghi et al., 1998; Pelchen-Matthews et al., 1999). Understanding the mechanism of entry into cells using the host’s chemokine receptors, and the functions of viral chemokines and chemokine receptors, might in the future allow for a chemokine/receptor-based therapeutic strategy to fight viral infection or spreading. Most of the discoveries are made in vitro. Only a few cases of in vivo observations help to clarify the role of chemokines and their receptors in cell trafficking.


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It is already clear from the in vivo effects of ectopic transgenic expression of chemokines and their receptors, as done for SLC and BLC (Fan et al., 2000; Luther et al., 2000), and from studies of mice in which genes encoding these molecules have been inactivated by natural mutations or by targeted deletion (Ansel et al., 2000; Cook et al., 2000; Forster et al., 1996, 1999; Gunn et al., 1999; Nakano et al., 1997, 1998) that these molecules play central roles in the organization of cell traffic, tissue generation, and organogenesis. For the future, it will be necessary to modify multiple chemokines or their receptors to understand redundancy in chemotaxis. From our own experiments, it seems that ABCD-2 (or even other unidentified chemokines) can compensate for the loss of ABCD-1 in mice (Takeyuki Shimizu, Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers, unpublished). Therefore, we need to study chemotaxis and humoral immune responses in mice in which the genes encoding ABCD-1, ABCD-2, and possibly ABCD-3 are inactivated, with the hope that this will help us to understand the relevant functions of each chemokine receptor–ligand pair in nature. ACKNOWLEDGMENT The Basel Institute for Immunology was founded and supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.

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Factors and Forces Controlling V(D)J Recombination DAVID G. T. HESSLEIN* AND DAVID G. SCHATZ†,‡ *Department of Cell Biology and †Section of Immunobiology, Howard Hughes Medical Institute,Yale

University School of Medicine, New Haven, Connecticut 06520-8011

I. Introduction

All jawed vertebrate species examined to date rely on a combination of innate and adaptive immune systems to combat infectious organisms. The adaptive immune system consists of B and T lymphocytes that express on their surface clonotypic antigen receptors known as immunoglobulins (Igs) and T cell receptors (TCRs), respectively. Collectively, these receptor molecules recognize a huge array of different antigens with impressive specificity, and exhibit extensive diversity prior to antigen encounter. Three different strategies have evolved to generate this preimmune diversity for Ig genes: gene conversion, somatic hypermutation, and combinatorial gene assembly (for review, see Weill and Reynaud, 1996). The first relies on the transfer of short tracts of sequence information from an array of pseudogene sequence donors to a target receptor gene; the second relies on the introduction of mutations (typically point mutations) into the target gene; and the third relies on imprecise assembly of the receptor gene from different combinations of gene segments using a site-specific recombination reaction known as V(D)J recombination. Regardless of the strategy used to generate diversity, B and T lymphocytes must use V(D)J recombination to assemble antigen receptor genes because most Ig and TCR loci do not exist in a functional configuration in the germline. Instead, the genetic information that will encode the variable (antigen-binding) portion of each polypeptide is broken up into discontinuous V (variable), J (joining), and in some cases, D (diversity) gene segments. V(D)J recombination either generates diversity directly (as in primates and rodents) or establishes the functional target gene for the other diversification processes (as for certain loci in chicken, sheep, and rabbits). V(D)J recombination is a crucial part of early lymphocyte development, and anything that disrupts the recombination process prevents expression of antigen receptors, blocks the production of mature lymphocytes, and results in immunodeficiency. Tight regulation of V(D)J recombination is important not only for proper lymphocyte development, but also to ensure the integrity of the genome. The reaction involves the generation and subsequent repair of DNA double-strand ‡To whom correspondence should be addressed. Tel: 203-737-2255; Fax: 203-737-1764; E-mail: [email protected].

169 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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breaks. Such disruptions of chromosomal integrity bring with them the risk of chromosomal translocations. There is now strong evidence indicating that errors in V(D)J recombination are an underlying cause of translocations and lymphoid malignancies (Danska and Guidos, 1997; Kirsch and Lista, 1997; Liao and Van Dyke, 1999; Vanasse et al., 1999). Regulation of V(D)J recombination is imposed at three distinct levels: (i) cell type-specific expression of the recombination machinery, (ii) tight control of the ability of the machinery to gain access to its substrates within chromatin, and (iii) restrictions imposed by the fact that each recombination event requires the productive interaction of proteins bound to two, noncontiguous DNA substrates. The first of these issues relates to the regulation of expression of the recombination activating genes, RAG1 and RAG2, and is discussed only briefly here. The second issue is the “accessibility problem,” and pertains to the regulation of the initial step in the recombination reaction: binding by the RAG1 and RAG2 proteins to an appropriate target DNA sequence. The third issue, the “two substrate problem,” comes into play only after the DNA substrates have been made accessible, and operates at two levels: (i) two DNA substrates must “find” each other for recombination to occur, and (ii) not all pairs of substrates, even when in close proximity, can recombine efficiently with one another. The accessibility and two substrate problems are the focus of this review.

II. Basic Features of V(D)J Recombination

A. STRUCTURE OF ANTIGEN RECEPTOR LOCI The natural substrates for V(D)J recombination are the seven antigen receptor loci: the Ig heavy (IgH) and light chain (Ig␬ and Ig␭) loci, and the TCR␣, ␤, ␥ , and ␦ chain loci. The organization of the gene segments and constant region exons varies among the different loci (Fig. 1), and the structure of a given locus can vary dramatically between different species (for reviews, see Lewis, 1994; Litman et al., 1999; Marchalonis et al., 1998). Considering only the antigen receptor loci of primates and rodents, for which the most is known, a number of common features emerge. Each locus consists of one or more exons encoding the constant region of the polypeptide, upstream of which are the gene segments that are rearranged to assemble the exon that encodes the antigen binding (variable) portion of the protein. The variable exon is assembled from V, D, and J gene segments for the heavy chain, ␤, and ␦ loci, and V and J segments for the ␬, ␭, ␣, and ␥ loci. The V, D, and J gene segments are typically each found in a single, multimember cluster of like gene segments (exceptions include the ␭, ␤, and ␥ loci, which contain two or more (D)-J-C clusters). All of the loci span a considerable linear distance along a chromosome, and some rearrangements join DNA ends which are over a megabase apart. While most loci occupy their own unique region of a particular chromosome, the TCR␣ and ␦ loci are intermingled (Fig. 1). V␣ and V␦ gene

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FIG. 1. Schematic representation of the murine antigen receptor loci. Gene segments are depicted as white rectangles, but pseudogenes have been omitted. Enhancers and promoters are represented by gray circles and rectangles, respectively, and 12-RSSs and 23-RSSs as white and black triangles, respectively. Constant regions are depicted as single rectangles, with no attempt made to indicate individual exons. Most loci have been adapted from (Hempel et al., 1998; Lewis, 1994). The two gray rectangles within the Ig␬ locus represent the two start sites for J␬ sterile transcripts (one of which is the KI/KII element)(Martin and van Ness, 1990). The Ig␭ locus was adapted from (Hagman et al., 1990; Lewis, 1994; Lieber, 1991). The BEAD element in the TCR␣/␦ locus has been described only in humans (Zhong and Krangel, 1997). The TCR␥ locus was adapted from Lewis, 1994, and Vernooij et al., 1993. The HsA element upstream of the V␥ 2 gene segment is described in Baker et al. (1999). Not to scale.

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segments are interspersed (and to some extent, interchangeable), and the D-J-C␦ region lies between the V␣/␦ cluster and the J-C␣ region. As a consequence, V-to-J␣ rearrangements delete the D-J-C␦ region from the chromosome. The V(D)J recombination reaction begins with recognition of conserved DNA motifs that flank each coding segment. These motifs, known as recombination signal sequences (RSSs), consist of well-conserved heptamer (consensus 5′ -CACAGTG-3′ ) and nonamer (consensus 5′ -ACAAAAACC-3′ ) elements separated by a spacer region whose sequence is poorly conserved but whose length is either 12 ± 1 or 23 ± 1 bp (referred to as the 12-RSS and 23-RSS, respectively). Efficient recombination occurs only between a 12-RSS and a 23-RSS, a restriction known as the 12/23 rule (Early et al., 1980; Sakano et al., 1980). This substantially limits the recombination events that can occur. For example, because all V or J gene segments in a given locus are flanked by an RSS of the same type, V-to-V and J-to-J recombination events are prohibited. In the Ig heavy chain locus, V and J gene segments are flanked by 23-RSSs, while D gene segments are flanked on both sides by 12-RSSs (Figs. 1 and 2A) and thus only V-to-D and D-to-J recombination events are allowed. In the TCR␤ and ␦ loci, V and J gene segments are flanked by 23- and 12-RSSs, respectively, while D gene segments have a 5′ 12-RSS and a 3′ 23-RSS (Fig. 1). Thus, V-to-J and D-to-D recombination events are allowed, and the latter is a common feature of the ␦ locus (Davis and Bjorkman, 1988). B. THE DEVELOPMENTAL PATTERN OF RECOMBINATION EVENTS All antigen receptor loci use the same RSS and the same basic recombination machinery. Nonetheless, the different loci rearrange in distinct patterns that are lineage restricted and developmentally orchestrated (for reviews, see Blackwell and Alt, 1988; Sleckman et al., 1998a). Most Ig gene rearrangements occur exclusively in developing B cells, while most TCR gene rearrangements occur exclusively in developing T cells. IgH recombination precedes that of the light chain loci, while TCR␤ locus recombination precedes that of TCR␣. In the IgH and TCR␤ loci, D-to-J rearrangements occur before V-to-DJ events, and both of these loci exhibit allelic exclusion, wherein productive rearrangement of one allele suppresses rearrangement of the other allele. How lineage-, stage-, and allele-specific patterns of recombination can be achieved by a common recombination machinery and RSS is the fundamental issue in the regulation of V(D)J recombination. C. ANTIGEN RECEPTOR TRANSCRIPTIONAL CONTROL ELEMENTS AND STERILE TRANSCRIPTS All Ig and TCR loci contain enhancer and promoter elements that are involved in producing two distinct types of RNA transcripts (Figs. 1 and 2B): transcripts of the fully assembled genes (which direct production of the antigen receptor


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FIG. 2. Schematic of the Ig heavy chain locus. (A) The germline variable region of the human IgH locus is depicted with the size of the gene clusters and their intervening distances indicated above. Gene segments are represented as rectangles and 12-RSSs and 23-RSSs are represented as white and black triangles, respectively. (B) Schematic representation of the gene rearrangements and sterile transcripts in the murine IgH locus. Promoters are represented by white ovals, the intronic enhancer (Ei␮) as a gray circle, and sterile transcripts as thin arrows. Not to scale.

polypeptide), and “sterile transcripts,” which are transcripts of unrearranged or partially rearranged portions of a locus and, with the exception noted below, are not thought to encode a biologically relevant polypeptide. Sterile transcription correlates closely with V(D)J recombination and may play an important role in regulating the reaction (see below). The known transcriptional enhancer elements in antigen receptor loci are found in proximity to the constant region(s) (Figs. 1 and 2B). In the heavy chain locus, one enhancer (the intronic enhancer) lies in the intron between the JH gene segments and C␮, while a second (the 3′ -enhancer) lies at the extreme 3′ end of the locus, after the last of the constant regions. Two enhancers are also found in the Ig␬ locus, where they are located upstream and downstream of the single constant region exon, and in the ␣/␦ locus, where they lie in the J-C␦ intron and downstream of C␣. The one enhancer identified in the TCR␤ locus lies downstream of the second J-C␤ cluster, while the Ig␭ and TCR␥ loci each contain multiple enhancer elements (Fig. 1).

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Numerous promoter elements exist in the IgH locus. One is found upstream of each V and D gene segment, and a transcription start site is also found within the intronic enhancer (Fig. 2B). Early in B cell development, prior to gene rearrangement, the intronic enhancer and the promoter upstream of DQ52 (PDQ52) become active and generate the I␮ and ␮0 germline transcripts, respectively (Fig. 2B). After D-to-J rearrangement, the promoter upstream of the rearranged D is active, leading to production of the D␮ transcript (Fig. 2B). Depending on the reading frame of the rearrangement, this can result in the production of a truncated ␮ protein and arrest of development of the precursor B cell (Gu et al., 1991; Reth and Alt, 1984). Finally, at the stage of V-to-DJ rearrangement, germline transcripts can be detected coming from the promoters upstream of multiple V gene segments (Yancopoulos and Alt, 1985) (Fig. 2B). D. BIOCHEMISTRY OF V(D)J RECOMBINATION The basic V(D)J recombination reaction can be thought of as occurring in two phases. The first involves the recognition of two sites on a chromosome and the introduction of double-strand breaks at these sites, while the second involves resolution of the four broken DNA ends to form two new junctions (Fig. 3) (for reviews, see Fugmann et al., 2000; Gellert, 1996; Lewis, 1994; Oettinger, 1999). 1. DNA Recognition and Cleavage The cleavage phase of V(D)J recombination is performed by the RAG1 and RAG2 proteins, and has been the focus of numerous biochemical and mechanistic studies (for review, see Fugmann et al., 2000). The reaction is thought to begin with recognition of the 12- and 23-RSS by the RAG proteins to form the 12-SC and 23-SC, respectively (Fig. 3). Formation of these complexes requires a divalent metal ion (Hiom and Gellert, 1997), contacts between RAG1 and the nonamer (Difilippantonio et al., 1996; Spanopoulou et al., 1996), and protein–DNA contacts with the heptamer and coding flank (Akamatsu and Oettinger, 1998; Eastman et al., 1999; Hiom and Gellert, 1997; Mo et al., 1999; Nagawa et al., 1998; Swanson and Desiderio, 1998). The two bound RSSs are then brought into close proximity to form a synaptic complex referred to as the paired complex (Fig. 3), in which DNA cleavage is completed (Eastman et al., 1996; van Gent et al., 1996b). Cleavage at each RSS occurs in two steps (McBlane et al., 1995). A nick is first introduced 5′ of the heptamer at its border with the coding flank to yield a 5′ phosphate and a 3′ -hydroxyl group. The 3′ hydroxyl on the coding flank then directly attacks the opposite strand of the DNA (van Gent et al., 1996a), resulting in formation of a blunt signal end and a covalently sealed, “hairpin” coding end. Several features of the first phase of the reaction are worth noting. First, the RAG proteins are capable of forming a stable complex with a single RSS, within


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FIG. 3. Schematic model of the protein–DNA complexes in V(D)J recombination. The 12-RSS and 23-RSS are represented as white and black triangles, respectively, coding segments as rectangles, and proteins as shaded ovals. Several aspects of the reaction are not depicted, including nicking adjacent to RSSs (which may occur before or after synapsis), asymmetric opening of the hairpin coding ends to generate P-nucleotides, and nucleotide addition by TdT.

which nicking can occur efficiently; however, the second step of cleavage (hairpin formation) is strongly dependent on synapsis and formation of the paired complex. This restricts formation of DNA double-strand breaks to a situation in which the recombining partners are in close proximity and the ends can be rejoined efficiently. In addition, this raises the possibility that the RAG proteins have bound and nicked adjacent to numerous RSSs in any given cell without having performed double-strand cleavage. Second, both the synapsis and hairpin formation steps of the reaction appear to be more efficient with a 12/23 pair of RSSs than with a 12 /12 or 23/23 pair (Hiom and Gellert, 1998; West and Lieber, 1998). Third, the high mobility group proteins, HMG1 and HMG2, stabilize the

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23-SC and the paired complex, and strongly stimulate cleavage in vitro (Hiom and Gellert, 1998; Sawchuk et al., 1997; van Gent et al., 1997). Overexpression of these proteins also enhances V(D)J recombination of episomal substrates in transfected cells (Aidinis et al., 1999). Thus it is likely that these proteins, or some other nonspecific DNA binding proteins, assist in the first phase of the reaction. And fourth, much of what is currently understood of the first phase of V(D)J recombination comes from in vitro studies using purified, truncated forms of the RAG proteins and naked (nonnucleosome) DNA templates, and it has recently become clear that wrapping the RSS around a nucleosome dramatically inhibits cleavage (Golding et al., 1999; Kwon et al., 1998; McBlane and Boyes, 2000). One should therefore expect that RSS recognition and synapsis in vivo are considerably more complicated than the above biochemical discussion suggests. Most in vitro studies of V(D)J recombination have made use of truncated “core” forms of the RAG proteins. These core proteins were defined as the minimal regions needed for recombination activity on artificial substrates (Cuomo and Oettinger, 1994; Sadofsky et al., 1993, 1994; Silver et al., 1993) and are useful due to their solubility (McBlane et al., 1995; van Gent et al., 1995). The non-core regions of the RAG proteins have been shown to enhance the efficiency of V(D)J recombination of artificial substrates and endogenous antigen receptor loci (Kirch et al., 1998; McMahan et al., 1997; Roman et al., 1997; Steen et al., 1999). Interestingly, the non-core (C-terminal) region of RAG2 has some sequence similarity to the plant-homeodomain finger-like motif (PHD) domain (Callebaut and Mornon, 1998). Although the function of the PHD domain is unknown, it has been found in multiple proteins involved in chromatin modification and remodeling such as the trithorax group proteins (for review, see Aasland et al., 1995). 2. End Processing and Joining The mechanism of the second phase of V(D)J recombination is not well understood (for reviews, see Critchlow and Jackson, 1998; Lieber, 1999; Smider and Chu, 1997). After cleavage, the four free ends are thought to be held together in a cleaved signal complex (Fig. 3) (Hiom and Gellert, 1998), within which some or all of the processing of coding ends may occur. These processing steps include opening of the hairpin coding ends by the action of an endonuclease, and the deletion and addition of a small number of nucleotides (Lewis, 1994). The coding ends are then joined to form the coding joint, leaving behind the unjoined and unprocessed signal ends, which eventually are ligated to form the signal joint (Fig. 3). Random nucleotide loss and addition can generate tremendous diversity in coding joints (and hence in the encoded antigen receptor polypeptide), but have the consequence that approximately two-thirds of all antigen receptor genes are assembled out of frame. Therefore, a significant fraction of


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developing lymphocytes die because of a failure to express antigen receptor. It is worth noting that, depending on the relative orientation of the two RSSs, V(D)J recombination can result in either deletion (as depicted in Fig. 3) or inversion of the DNA between the two sites of cleavage. While deletion is most common, approximately half of Ig␬ locus rearrangements occur by inversion, and the TCR␤ and ␦ loci also undergo inversion during certain V-to-DJ rearrangements (Lewis, 1994). Five ubiquitously expressed proteins are required for the second phase of V(D)J recombination, and all five are involved in the repair of DNA doublestrand breaks by nonhomologous end joining. They include XRCC4, DNA Ligase IV, and the three components of the DNA-dependent protein kinase (DNAPK), Ku70, Ku80, and the catalytic subunit DNAPKcs (for reviews, see Lieber, 1999; Smith and Jackson, 1999). Ku70 and Ku80 form a heterodimer that binds DNA ends, stimulates ligation (Nick McElhinny et al., 2000; Ramsden and Gellert, 1998), and helps recruit DNAPKcs to DNA (Gottlieb and Jackson, 1993). DNAPKcs is a huge (465 kD) serine/threonine protein kinase whose role in V(D)J recombination is not understood. Its kinase activity is strongly stimulated by DNA ends (Gottlieb and Jackson, 1993) and is important for V(D)J recombination (Kurimasa et al., 1999). XRCC4 and DNA Ligase IV form a heterodimer thought to perform the ligation step of both coding and signal joint formation (Critchlow et al., 1997; Grawunder et al., 1997). RAG1 and RAG2 have been suggested to perform several postcleavage functions in V(D)J recombination, including holding the four cleaved ends together in a cleaved signal complex (Hiom and Gellert, 1998), endonucleolytic opening of the hairpin coding ends (Besmer et al., 1998; Shockett and Schatz, 1999), and the endonucleolytic removal of single-stranded 3′ “flaps” from coding ends (Santagata et al., 1999). The Mre11/Rad50/Nbs1 complex has also been proposed to open the hairpin coding ends (Paull and Gellert, 1998, 1999), but as yet the identity of the factor(s) responsible for this activity in vivo is unknown. Finally, the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT), is responsible for the addition of non-templated nucleotides to coding ends (Gilfillan et al., 1993; Komori et al., 1993). E. REGULATION OF EXPRESSION OF RAG1 AND RAG2 Coexpression of RAG1 and RAG2 is limited to cells of the B and T lymphocyte lineages (for review, see Nagaoka et al., 2000), and hence V(D)J recombination is similarly restricted (Kawaichi et al., 1991). Within the two lymphocyte lineages, high level RAG expression is limited to the early stages of development during which the preimmune repertoire of antigen receptors is generated. In B cell development, RAG expression is first detected in pro-B cells in which the Ig heavy chain locus is being rearranged (Li et al., 1993). Productive heavy chain gene rearrangement and expression leads to a phase of rapid proliferation and

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downregulation of RAG expression. When proliferation ceases, the cells become small pre-B cells, upregulate RAG expression again, and carry out Ig light chain gene rearrangement (Grawunder et al., 1995; Monroe et al., 1999b; Yu et al., 1999a). Successful light chain rearrangement and expression of Ig on the surface of the cell permits maturation of the B cell and triggers termination of RAG expression (Monroe et al., 1999b; Yu et al., 1999b). An exception to this arises when the immature B cell expresses surface Ig that is self-reactive, in which case RAG expression continues at high levels and further light chain rearrangement can occur (a process known as receptor editing) (Gay et al., 1993; Tiegs et al., 1993). A very similar pattern of RAG expression is seen during T cell development. A first wave of expression occurs in double negative cells (DN; so called due to the absence of CD4 and CD8 expression) that rearrange the TCR␤, ␥ , and ␦ loci (Wilson et al., 1994). Cells of the ␣␤ lineage with an in-frame ␤ rearrangement proliferate, transiently downregulate RAG expression, and develop into double positive cells (DP; so called due to expression of both CD4 and CD8) (Hoffman et al., 1996). These cells upregulate RAG expression and rearrange the TCR␣ locus until either the cell dies or it produces a TCR that can interact appropriately with self major histocompatibility molecules, at which point the cell terminates RAG expression and eventually exits the thymus as a mature T cell (for review, see Nagaoka et al., 2000). For the most part, mature B and T cells do not express RAG1 or RAG2, although some interesting exceptions exist in situations where lymphocytes are subject to chronic stimulation through their antigen receptors (McMahan and Fink, 1998; Qin et al., 1999; for review, see Nagaoka et al., 2000). RAG1 and RAG2 are unusual in that they lie immediately adjacent to one another in the vertebrate genome and are convergently transcribed (Oettinger et al., 1990). High level, lymphocyte-specific transcription appears to be dictated by enhancer elements that lie upstream of RAG2. Distinct long-range elements regulate RAG expression in the B and T cell lineages (Monroe et al., 1999a; Yu et al., 1999a). Little is known about the transcription factors that regulate tissuespecific RAG expression, although BSAP and GATA-3 have been implicated as important for transcription from the murine RAG2 promoter in B and T lineage cells, respectively (Kishi et al., 2000; Lauring and Schlissel, 1999). RAG2 expression is regulated transcriptionally and post-transcriptionally. The RAG2 protein has been estimated to have a half-life of over 200 min in the G0 /G1 phases of the cell cycle. As cells enter S phase, RAG2 is phosphorylated on threonine 490 by cyclin A/cyclin dependent kinase-2, and the half-life drops to approximately 10 min where it stays throughout the S, G2, and M phases of the cell cycle (Lee and Desiderio, 1999; Lin and Desiderio, 1993, 1994). Consistent with this, signal ends, which are thought to be a marker of ongoing DNA cleavage by the RAG proteins, are found almost exclusively in cells in


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G0 /G1 (Li et al., 1996b; Schlissel et al., 1993). Thus, phosphorylation of RAG2 by a cyclin dependent kinase is thought to constrain V(D)J recombination to the G0 /G1 phases of the cell cycle. III. Forces Controlling Chromatin Structure and Accessibility

The mechanisms controlling developmentally regulated transcription of the vertebrate ␤-globin locus have been intensively studied, and many of the same features that correlate with transcription of the various globin genes also correlate with active V(D)J recombination. These features include DNase sensitivity, histone acetylation, demethylation, and the important roles played by cis-acting transcriptional regulatory sequences and the factors that bind them. The physical attributes of the two types of loci are also very similar. There are multiple genes within each locus. The activity of each locus (recombination and /or transcription) starts at one end of the locus and progresses to the other end in a developmentally regulated manner. Of particular relevance is the observation that antigen receptor gene segments are often transcribed at the same time they become competent to undergo V(D)J recombination. The existence of such sterile transcripts together with the other similarities between the ␤-globin and antigen receptor loci suggest that, at minimum, recombinationally accessible loci will also be transcriptionally competent. These considerations also suggest that an understanding of mechanisms controlling transcription of ␤-globin genes should provide insights into the accessibility problem in V(D)J recombination. Below, we discuss basic aspects of nuclear organization to provide a context within which to consider the in vivo complexity of V(D)J recombination. We then discuss a number of forces implicated in regulating chromatin structure, and compare their roles in V(D)J recombination and ␤-globin locus transcription. A. NUCLEAR STRUCTURE AND COMPLEXITY An obvious problem presents itself when one confronts the packing of 46 eukaryotic chromosomes, each 1.7 to 8.5 cm long, into a nucleus with a diameter of 3 to 10 ␮m. The solution is to wrap and organize the DNA into protein / DNA complexes known as chromatin. The basic unit of chromatin, the nucleosome, has DNA wound 1.75 times (146 bp) around a complex of histone proteins (Kornberg and Lorch, 1999) (Fig. 4). In turn, nucleosomes together with histone H1 proteins and linker DNA, are wrapped around one another in a solenoid structure known as the 30-nm fiber (Wolffe, 1992). This fiber is folded into higher-order structures until the packing observed in interphase chromosomes is achieved (for review, see Belmont et al., 1999). Chromatin packing can prevent access to the DNA by protein factors, and is inherently refractory to V(D)J recombination, transcription, and replication. While the extent of chromatin unfolding that is needed for these processes to

FIG. 4. V(D)J recombination in the context of nuclear structure. At the top left is a schematic representation of the interphase nucleus with chromosomes depicted as shaded irregular shapes. Successively higher resolution views are depicted schematically, as indicated by the dashed lines. The 30-nm fiber is represented as a series of tightly packed gray circles, each of which represents a nucleosome. Small black circles depict anchor sites, or MARs. At the bottom, 12- and 23-RSSs (white and black rectangle, respectively) are each depicted wrapped around a single nucleosome (represented as a cylinder). It is not known if the RSS must be removed from the nucleosome entirely for RAG-dependent binding, synapsis, and cleavage. How synapsis occurs within the complex environment of the nucleus is not known.


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occur is unknown, 30-nm fibers have been visualized in actively transcribed loci, indicating that DNA can be accessible in such a structure (Andersson et al., 1982; Bjorkroth et al., 1988; for discussions, see Bulger and Groudine, 1999; Swedlow et al., 1993). Nonlymphoid cells made to express the RAG proteins do not recombine their endogenous antigen receptor loci, while in developing lymphocytes, these same loci are actively recombined and display numerous features of open, active chromatin. This, in addition to the lineage and temporal regulation of V(D)J recombination noted above, indicates that chromatin must unfold to some degree for the recombinase to gain access to individual RSSs (Fig. 4). There are regions within the nucleus that have regulatory roles in gene expression and replication (for reviews, see Lamond and Earnshaw, 1998; Misteli and Spector, 1998). For example, interchromatin granules are believed to function as warehouses for splicing factors when they are not in use (Misteli and Spector, 1998). Within 5 hr of the inhibition of RNA polymerase II by ␣-amanitin, splicing factors relocate from sites of transcription into interchromatin granules (Huang and Spector, 1996). Similarly, the addition of ␣-amanitin and other RNA polymerase II inhibitors causes the redistribution of chromatin domains (Haaf and Ward, 1996). These and other observations demonstrate the dynamic nature of nuclear organization and the dramatic impact that changes in gene expression can have on it. Two types of chromatin can be distinguished based on staining characteristics. Darkly stained chromatin (due to its high level of compaction) is known as heterochromatin and is composed of transcriptionally silent DNA. Lighter staining chromatin, known as euchromatin, has the capacity for gene expression, but only approximately 10% is actively transcribed at any one time (Alberts et al., 1994). Interphase chromosomes localize to specific regions in the nucleus, known as chromosomal territories, and heterochromatin and euchromatin are separated into distinct subregions (Lamond and Earnshaw, 1998). Acetylation of histones in a locus has been shown to correlate with transcription and movement of the locus away from heterochromatin, but transit of a locus away from heterochromatic regions does not result in obligatory gene expression (Schubeler et al., 2000). The movement of genes into and out of the vicinity of heterochromatin represents one level at which gene expression is regulated. Transcriptionally active genes have been shown to lie near the surface of chromosome territories (Dietzel et al., 1999; Verschure et al., 1999; Wansink et al., 1996). The combined methods of FISH and BrUTP-labeling, for example, allowed for the visualization of sites of transcription next to but not within chromosomal subdomains in single optical sections (Verschure et al., 1999). The location of actively transcribed genes at the boundary of chromosome territories places them near splicing domains and facilitates the transport of the resultant

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mRNA out of the nucleus. There is some disagreement about whether inactive genes can also be found at the edge of chromosomal territories (for more on this debate, see Belmont et al., 1999; Lamond and Earnshaw, 1998; Verschure et al., 1999, and references therein). Overall, there is a strong correlation between expression and nuclear context of individual genes, and this may have implications for the regulation of V(D)J recombination. As yet, nothing is known about the nuclear location of antigen receptor loci actively undergoing recombination. B. ORGANIZATION AND EXPRESSION OF ␤-GLOBIN GENES The human ␤-globin locus spans approximately 70 kb and contains five genes, each with its own promoter, arranged in the order: ⑀, G␥ , A␥ , ␦, and ␤. The spatial arrangement of the genes parallels their order of expression in erythroid cells during development. The ⑀ gene is expressed early in embryogenesis, and is subsequently downregulated as the ␥ genes are activated. In adults, all genes are silent except for ␦ and ␤, with ␦ expressed at much lower levels (Bulger and Groudine, 1999; Engel and Tanimoto, 2000). At the 5′ end of the locus, upstream of the ⑀ gene, lies a region of DNA important for transcriptional control of all ␤-globin genes. This region was originally defined using human disease models in which chromosomal deletions resulted in silencing of the entire locus. The smallest of these genetic lesions, the naturally occurring Hispanic deletion, includes a region that contains multiple erythroid specific DNase hypersensitive sites. This collection of sites was shown to be essential for position-independent, copy number-dependent expression of the ␤-globin gene in transgenic miniloci, allowing this area to be operationally defined as a locus control region (LCR) (Grosveld, 1987). The ability of the LCR to exert pleiotropic and long-range effects makes its mode of action an appealing model for how enhancer elements might control rearrangement and expression of antigen receptor genes. C. CIS-ACTING ELEMENTS AS ASSEMBLY PLATFORMS Some, and perhaps many, of the cis-acting transcriptional control elements in antigen receptor gene loci have a dual function of regulating gene transcription and gene rearrangement. This has been shown most clearly through transgenic and knockout approaches where deletion of enhancers or promoters found in antigen receptor loci have (with a few exceptions) resulted in reduced or undetectable levels of V(D)J recombination. These phenotypes and their implications will be discussed below. The cis-acting elements of antigen receptor loci, like those of the ␤-globin locus, contain binding sites for ubiquitous and lineage specific transcription factors. The importance of transcriptional regulatory elements for V(D)J recombination suggests that these factors (or a subset of them) regulate the rearrangement process. It seems likely, therefore, that the central role of the cis-acting elements


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is to serve as platforms upon which complexes of transcription factors assemble. These factors could work through and /or have been implicated in multiple mechanisms, such as mediating enhancer and promoter contact, recruitment of histone acetylases and deacetylases, recruitment of demethylases, recruitment of nucleosome remodeling complexes, and activation of transcription that may assist in making loci more accessible. It is plausible that factor binding initiates and stabilizes the conversion from an inaccessible to an accessible locus. The numerous transcription factors that regulate the ␤-globin locus are not discussed here (for review, see Orkin, 1995). Some of the factors implicated in the control of IgH V(D)J recombination will be discussed in detail below. D. METHYLATION The one known covalent modification of the vertebrate genome is methylation of the 5 position of cytosine in the dinucleotide 5′ -CpG-3′ . Cytosine methylation is not randomly distributed in the genome, but instead correlates with inactive or repressed genes and chromosomal regions (for reviews, see Eden and Cedar, 1994; Razin, 1998). Thus, heterochromatin, retrotransposons, endogenous retroviruses, the inactivated X chromosome, and silent, imprinted genes are hypermethylated. In general, methylation of tissue-specific genes is inversely correlated with expression, while CpG-rich islands in the promoter regions of housekeeping genes are maintained in an unmethylated state. In addition, there is a strong correlation between demethylation of antigen receptor loci and V(D)J recombination. A large body of evidence indicates that the principle mechanism by which methylation exerts its repressive effects is by influencing chromatin structure (for reviews, see Bird and Wolffe, 1999; Kass et al., 1997b). Introduction of in vitro methylated DNA into cells leads to formation of a chromatin configuration that is refractory to transcription (Keshet et al., 1986) and to V(D)J recombination (Hsieh and Lieber, 1992). Formation of the repressed state clearly requires both methylation and chromatin assembly (Buschhausen et al., 1987; Hsieh and Lieber, 1992; Kass et al., 1997a). These and other studies demonstrate that, under a variety of experimental conditions, methylation can be a direct cause of the formation of an inaccessible, highly repressed chromatin structure. The molecular link between methylation and silent chromatin appears to be a family of methyl cytosine binding proteins (MeCPs) (for reviews, see Bird and Wolffe, 1999; Kass et al., 1997b; Razin, 1998). How the binding of MeCPs to DNA generates a repressive chromatin configuration has not been fully elucidated. MeCPs might directly occlude binding sites on the DNA or directly alter chromatin structure. For example, MeCP2 is able to displace histone H1 from the nucleosome, perhaps leading to an altered, and highly compacted, chromatin structure (Nan et al., 1997). Perhaps the most important component of MeCP function is recruitment of other proteins to the DNA, including transcriptional

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corepressors, chromatin remodeling factors, and histone deacetylases (for reviews, see Bird and Wolffe, 1999; Kass et al., 1997b; Razin, 1998). Thus, methylation could trigger a cascade of events leading to transcriptional repression as well as remodeling and covalent modification of the nucleosome. Methylation patterns are propagated to both daughter DNA duplexes during DNA replication by a maintenance DNA methyltransferase (Holliday, 1987). The covalent nature of the modification and its ability to be stably maintained through many cell divisions makes methylation an ideal tool for the stable maintenance of the repressed state, as is needed in terminally differentiated cells. In addition, methylation patterns can be changed by de novo methylation and demethylation, processes which are not well understood (for reviews, see Bergman and Mostoslavsky, 1998; Bird and Wolffe, 1999). Disruption of the gene encoding the maintenance methyltransferase has demonstrated that methylation is crucial for genomic imprinting and X-chromosome inactivation (Jaenisch, 1997). It remains unclear, however, whether during normal development, methylation/demethylation play an early, causal role in the chain of events leading to an inactive/active chromatin structure, or whether they act primarily to reinforce and maintain the inactive/active state that has been imposed by other mechanisms. A particularly striking discordance between methylation and transcription comes from the studies of Arabidopsis thaliana. Plants mutated in the MOM gene reactivate transcription of several previously silent, hypermethylated loci, but these loci remain heavily methylated and the methylation pattern persists through multiple generations (Amedeo et al., 2000). Interestingly, MOM is predicted to encode a protein with some sequence similarity to chromatin remodeling factors of the SWI2 /SNF2 family. The vertebrate ␤-globin locus was one of the early model systems used to demonstrate a link between transcriptionally active genes and hypomethylation (for review, see Karlsson and Nienhuis, 1985). Using purified erythroid cell populations, a striking correlation was observed between ⑀- and ␥ -globin gene expression and hypomethylation of sites in the vicinity of the expressed genes. For the ␦- and ␤-globin genes, however, the correlation was much weaker (Mavilio et al., 1983). This typifies findings from other studies: some sites exhibit tissuespecific patterns of methylation that correlate with gene activity, while others exhibit a uniform methylation pattern in expressing and nonexpressing tissues. There is evidence for the ␤-globin locus that methylation is an effect, rather than a cause, of the chromatin changes that underlie gene inactivation. In somatic cell hybrids that switch from ␥ to ␤-globin gene expression in culture, inactivation of ␥ gene expression precedes methylation of the ␥ locus (Enver et al., 1988). Given the correlation between V(D)J recombination and sterile transcription (discussed below) and between transcription and hypomethylation, it is perhaps not surprising that actively recombining loci tend to be hypomethylated


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(reviewed in Bergman and Mostoslavsky, 1998; Mostoslavsky and Bergman, 1997). In several experimental models, a strong connection has been established between methylation and the inability to undergo V(D)J recombination. Using an artificial recombination transgene, it was discovered that different mouse strains methylate the transgene to different degrees, and the degree of methylation correlated tightly with an inability to undergo V(D)J recombination (Engler et al., 1993). As noted above, plasmids methylated in vitro are extremely poor substrates for V(D)J recombination when transfected into cells; however, in this system, methylation inhibits recombination only if the substrate is able to undergo replication (Hsieh and Lieber, 1992). The clearest correlation between methylation status and V(D)J recombination potential for endogenous antigen receptor genes has been established in studies of the Ig␬ locus. In most tissues and in early B lineage cells (prior to the onset of ␬ rearrangement), the ␬ locus is methylated, while in mature B cells (after the completion of ␬ rearrangement), the ␬ locus is unmethylated (Goodhardt et al., 1993; Mostoslavsky et al., 1998). Demethylation and rearrangement of the ␬ locus initiate in pre-B cells, and strikingly, in ␬+ bone marrow B cells, one ␬ allele is hypomethylated and rearranged, while the other allele is hypermethylated and unrearranged (Mostoslavsky et al., 1998). These results strongly suggest that one ␬ allele is chosen (perhaps at random) to undergo recombination first, and that whatever events lead to accessibility for recombination also result in demethylation. It has been proposed that demethylation actually directs the process of locus opening, and thus plays a critical causal role in ␬ recombination (Mostoslavsky et al., 1998). The results also suggest an attractive model for how allelic exclusion is maintained at the ␬ locus. The ␬ intronic enhancer, together with its flanking matrix attachment region (MAR), is sufficient to direct demethylation of transfected ␬ miniloci in B cells (Lichtenstein et al., 1994), and binding of NF-␬B to the enhancer appears to be critical for this activity (Demengeot et al., 1995; Kirillov et al., 1996). There are, however, examples in which methylated loci undergo recombination, and hypomethylated loci fail to do so, raising doubts as to whether methylation directly controls locus accessibility. In RAG2-deficient DN thymocytes, most of the sites examined in the V␤ gene cluster were hypermethylated despite the fact that the V␤ gene segments gave rise to sterile transcripts and were presumably accessible for recombination (Senoo and Shinkai, 1998). Similarly, in RAG2-deficient DP thymocytes, high levels of J␣ sterile transcripts were detected, but the J␣ locus was heavily methylated at all sites examined (Villey et al., 1997). One targeted mutation of the Ig heavy chain intronic enhancer blocked rearrangement but not demethylation of the locus (Chen et al., 1993). Finally, in a recombination minilocus containing an inducible promoter, inducing transcription activated V(D)J recombination despite the fact that the locus remained methylated (Sikes et al., 1999). Overall, as for the

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␤-globin locus, it is reasonable to think that methylation and demethylation are often a consequence, rather than a cause, of the structural changes that underlie alterations in accessibility, and that changes in methylation status serve an important function in the stable maintenance of an open or closed chromatin configuration. E. NUCLEASE SENSITIVITY Measuring sensitivity to nuclease digestion is a common method for detecting structural changes within chromatin. The most frequently used nuclease is DNase I, although restriction enzymes are also used. Two types of DNase sensitivity have been defined: (1) hypersensitivity and (2) general sensitivity (see Carey and Smale, 2000). Hypersensitive sites represent highly accessible regions of DNA where nucleosomes have been removed. These sites are often found in promoters and enhancers where chromatin remodeling has occurred as a result of transcription factor binding. Higher levels of general nuclease sensitivity are considered indicative of “open” gene loci, and are thought to correspond to a decrease in chromatin compaction. Heterochromatin shows low general sensitivity to DNase, whereas regions active for transcription or V(D)J recombination exhibit high levels of general sensitivity. In vitro reconstitution of chromatin showed that histone acetylation (see below) can increase DNase sensitivity (Krajewski and Becker, 1998). The structural changes of chromatin that allow for DNase access may be similar to those required for transcription and V(D)J recombination. DNase hypersensitive sites originally aided in the identification of the ␤-globin LCR (for review, see Engel and Tanimoto, 2000), and have been detected in various antigen receptor gene enhancers. The ␤-globin LCR has been shown to exhibit its distinctive hypersensitive sites in hematopoietic precursor cells, which do not express globin genes (Jimenez et al., 1992). Similarly, hypersensitive sites within antigen receptor gene enhancers and promoters have also been shown to appear prior to locus activation. Cis-acting elements within the TCR␣ locus were shown to be DNase hypersensitive in DP thymocytes (which recombine the ␣ locus) and in DN thymocytes (which have yet to initiate rearrangement of the locus) (Hernández-Munain et al., 1999). The IgH intronic enhancer has hypersensitive sites in both lymphoid and hematopoietic precursor cells (Ford et al., 1988, 1992). The existence of hypersensitive sites in elements at development stages where the element is not functional shows that DNase hypersensitivity is not indicative of element-based activity, but rather is suggestive of the potential for such activity. In contrast to measurements of nuclease hypersensitivity, other assays such as in vivo footprinting can reveal stage-specific changes in the occupancy of transcriptional control elements that reflect the activity of the element and the locus it controls (Hernández-Munain et al., 1999; Shaffer et al., 1997; Spicuglia et al., 2000).


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Increased general nuclease sensitivity is one of the first indications of locus opening for both the ␤-globin and antigen receptor loci. For the ␤-globin locus, the Hispanic deletion results in a complete absence of transcription and no general DNase sensitivity. In contrast, when only the LCR is deleted, transcription is again severely reduced but general DNase sensitivity is similar to that seen in the wild-type locus (Bender et al., 2000; Epner et al., 1998; Reik et al., 1998). Structural changes of the ␤-globin locus that allow for general sensitivity seem to be a necessary first step for ␤-globin transcriptional regulation, but are clearly not sufficient to support transcription. Analysis of the wild-type locus has shown that distinct areas of the locus exhibit increased levels of general sensitivity above that found throughout the rest of the locus. These areas correlate with regions that give rise to intergenic transcripts (Gribnau et al., 2000). This suggests that there is a second level of “local” locus opening that correlates with and may be dependent upon transcription. Analysis of pro- and pre-B cell lines showed that general nuclease sensitivity is found at Ig loci capable of undergoing V(D)J recombination (Persiani and Selsing, 1989). The introduction of miniloci into lymphoid cell lines also allowed for a correlation between general DNase sensitivity and active recombination (Ferrier et al., 1989; Yancopoulos et al., 1986). In some cases, however, miniloci exhibited increased general sensitivity but no recombination (Blackwell et al., 1986). Therefore, the chromatin changes that underlie increased general nuclease sensitivity are necessary but not sufficient for V(D)J recombination and ␤-globin locus transcription. F. NUCLEOSOME MODIFICATION AND REMODELING Acetylation of histones is tightly associated with accessibility and transcriptional activation. The acetylation of lysines in the N-termini of the core histones alters the charge of the tails and reduces their interactions with the nucleosomal DNA, linker DNA, and neighboring nucleosomes. This modification facilitates factor access to DNA, loosens the compaction of chromatin within the 30-nm fiber, and helps to unfold higher-order chromosome structure. The addition and removal of these acetyl groups is performed by histone acetyltransferases (HAT) and histone deacetylases (HDAC), respectively. These enzymes associate with transcription factors and help mediate their effects on transcription in vitro and in vivo (for reviews, see Kornberg and Lorch, 1999; Wolffe and Hayes, 1999). Immunoprecipitation of the chicken ␤-globin locus with antibodies to acetylated histones demonstrated that chromatin acetylation occurs in both transcriptionally active and inactive globin genes (Hebbes et al., 1992). Similar studies showed histone acetylation in both genic and intergenic regions that correlated precisely with general DNase sensitivity (Hebbes et al., 1994). The ␤-globin locus with the Hispanic deletion showed no histone acetylation and no gene expression.

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In contrast, the LCR deleted locus exhibited an intermediate phenotype in which no gene expression was detected, but histone acetylation was found throughout the locus. Wild-type loci exhibited even higher levels of acetylation around the gene being actively transcribed (Schubeler et al., 2000). These studies suggest that locus-wide histone acetylation may be a prerequisite for gene expression at the ␤-globin locus. It is interesting to note the close correlation between histone acetylation and general DNase sensitivity in the ␤-globin locus, especially in light of data showing that in vitro acetylated chromatin exhibits general DNase sensitivity (Krajewski and Becker, 1998). A positive feedback mechanism may exist in which locus acetylation allows transcription to begin, which in turn results in more histone acetylation and further chromatin opening. The association of histone acetyltransferases with RNA polymerase II is consistent with such a mechanism (for review, see Kornberg and Lorch, 1999) and provides a link between transcription and locus opening. Recent experiments with the TCR␣/␦ locus have shown a link between V(D)J recombination and locus acetylation. In human TCR␦ miniloci, the ␦ enhancer is required for the VD-to-J but not initial V-to-D recombination (Lauzurica and Krangel, 1994). Immunoprecipitation of acetylated chromatin showed that chromatin associated with the V gene segments is acetylated in both wild-type and enhancer-deleted TCR␦ miniloci. In contrast, high level acetylation of chromatin associated with J gene segments, like rearrangements involving these segments, is enhancer dependent (McMurry and Krangel, 2000). Mutation of the CBF/PEBP2 binding site in the ␦ enhancer, which is critical for minilocus VD-to-J recombination (Lauzurica et al., 1997), dramatically reduced acetylation of the J gene segments. Examination of the endogenous TCR␣/␦ locus in thymocytes also revealed a pattern of hyperacetylation that mirrored gene segment recombination potential. Overall, the results demonstrate a tight correlation between histone acetylation and the initiation of V(D)J recombination, suggesting a causal link between the two processes (McMurry and Krangel, 2000). In addition to acetylation, other histone modifications such as phosphorylation, ADP-ribosylation, and methylation have been suggested to affect chromatin structure (for review, see Wolffe and Hayes, 1999) and may influence V(D)J recombination. DNA wrapped around the nucleosome is bound poorly by many DNA binding proteins, and the same appears to be true for the RAG proteins. RAG1 and RAG2 alone cannot cleave mononucleosome–RSS DNA complexes in vitro (Golding et al., 1999; Kwon et al., 1998; McBlane and Boyes, 2000). One study found that HMG protein could stimulate RAG-mediated cleavage of certain nucleosomal substrates, and that the rotational and translational positioning of the DNA on the nucleosome could also modulate DNA cleavage (Kwon et al., 1998). A second study obtained conflicting results, with addition of HMG protein and rotational and translational positioning having no affect on cleavage


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(Golding et al., 1999). The effect of histone acetylation on in vitro cleavage of mononucleosomal substrates by the RAG proteins is also controversial. Two studies found that acetylated substrates remain refractory to RAG-dependent cleavage (Golding et al., 1999; McBlane and Boyes, 2000), with proteolytic removal of the histone tails being the only manipulation that allowed for cleavage (Golding et al., 1999). Other experiments, however, find that acetylation of core histones increases RAG-mediated cleavage, with HMG protein further stimulating the reaction (M. A. Oettinger, personal communication). These studies potentially have broad implications for V(D)J recombination, and the discrepancies must be resolved before the mechanisms regulating local accessibility can be understood. At one extreme, it is possible that histone acetylation (with attendant unfolding of higher-order chromatin structure) and RSS rotational /translational positioning are sufficient to allow the RAG proteins access to the RSS. At the other extreme, it is possible that RSSs must be rendered nucleosome free, or at the very least lifted off of the nucleosome, to allow binding and cleavage to occur (Fig. 4, bottom). The phasing and /or removal of nucleosomes in the vicinity of cis-acting elements has been shown to occur at well-studied promoters such as the yeast PHO5 promoter and the mouse mammary tumor virus (MMTV) promoter (see Carey and Smale, 2000). Such perturbations of nucleosome positioning due to factor binding of promoters flanking many antigen receptor gene segments may allow for RSS access. There are examples, however, where gene clusters have only one known promoter element. The J gene clusters in both the IgH and TCR␤ loci do not have cis-acting elements directly upstream of each gene segment. These gene segments may rely on promoters upstream of the nearest D gene segment for regulation of accessibility (Fig. 2B). The role of promoter-based nucleosome remodeling in the regulation of RSS accessibility has been investigated using an artificial episomal recombination substrate with a RSS embedded within a MMTV promoter. Stimulation of cells with dexamethasone, which causes movement of a nucleosome in the MMTV promoter off of the RSS, allowed for detectable recombination. Before remodeling, no rearrangements were detected. Chromatin remodeling and the resulting induction of rearrangement were shown to occur independently of transcription (Cherry and Baltimore, 1999). These observations indicate a possible mechanism for promoter-based accessibility without the need for transcription. Three known protein complexes perform remodeling of nucleosomes: DNA polymerase, RNA polymerase, and SWI/SNF complexes. SWI/SNF complexes have been the subject of intense study, but they have yet to be implicated in V(D)J recombination. Both SWI/SNF and histone modifying complexes are associated with transcription initiation and elongation (for reviews, see Felsenfeld, 1996; Kornberg and Lorch, 1999; Wolffe and Hayes, 1999). These associations

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hold intriguing implications for the regulation of V(D)J recombination, although in no case has nucleosome positioning been determined relative to RSSs in endogenous antigen receptor gene loci. Hence it is not known to what extent remodeling occurs or is required. G. STERILE TRANSCRIPTS Sterile or intergenic transcripts (also referred to as germline transcripts) have been noted in both antigen receptor and ␤-globin loci, and their appearance correlates well with locus activity (transcription in the ␤-globin locus or recombination in the antigen receptor loci). Intergenic transcripts are found in noncoding regions of the ␤-globin locus, and it was hypothesized that these transcripts may be involved in locus opening (Ashe et al., 1997). Further analysis based on intergenic transcription allows the human ␤-globin locus to be divided into three subdomains, one encompassing the LCR, a second surrounding the embryonic and fetal (5′ ) genes, and a third containing the adult (3′ ) genes. There is a strong correlation within the gene clusters between the appearance of coding and intergenic transcripts. Increased general DNase sensitivity was found in intergenic transcript regions, and coincides with the appearance of both coding and intergenic transcripts. The start site for the intergenic transcripts of the adult globin genes was mapped and located to a region that when deleted resulted in elimination of both the intergenic transcript and general DNase sensitivity, and a dramatic reduction in the levels of coding transcripts of the adult genes (Gribnau et al., 2000). This region may contain a regulatory element important for regional ␤-globin control, and the link between intergenic transcripts and nuclease accessibility is intriguing. Possible correlations between intergenic transcripts and other parameters of locus opening, such as hyperacetylation, have not been examined. Analysis of both fractionated developing lymphocytes and immortalized cell lines has shown that sterile transcripts and V(D)J recombination are correlated both temporally and spatially (Fondell and Marcu, 1992; Goldman et al., 1993; Li et al., 1996a; Schlissel et al., 1991b; Yancopoulos and Alt, 1985). The disappearance of some of these transcripts at a later stage of development may be due to developmentally related transcriptional regulation, or may be because the act of recombination removes the start site of these transcripts from the genome (e.g., ␮0 and D␮ in Fig. 2B) (Li et al., 1996a; Schlissel et al., 1991b; Schlissel and Morrow, 1994; Senoo and Shinkai, 1998). Ig␬ sterile transcripts and rearrangements both increase upon treatment of a pre-B cell line with lipopolysaccharide (LPS) (Schlissel and Baltimore, 1989). Overexpression of various transcription factors in lymphoid and non-lymphoid cell lines also resulted in parallel upregulation of germline transcription and rearrangement (Romanow et al., 2000; Schlissel et al., 1991a). The parallel increase of transcription and rearrangement by exogenous stimuli reinforces the


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link between transcription and accessibility. This link has been further strengthened by studies showing that deletion of cis-acting regulatory elements from endogenous antigen receptor loci results in decreases in V(D)J recombination and sterile transcription. Experiments with antigen receptor miniloci have also been useful in establishing the connection between transcription and rearrangement. Transcription of a selectable marker gene adjacent to a heavy chain minilocus yielded increased V(D)J recombination (Blackwell et al., 1986). In TCR␤ minilocus transgenes, both transcription and rearrangement are enhancer dependent (Ferrier et al., 1990; Sikes et al., 1999). The compact size of the chicken Ig␭ locus allowed for the creation of transgenic mice containing the entire endogenous locus. Mutagenesis of the ␭ enhancer and V gene promoter demonstrated a correlation between transcription and rearrangement (Lauster et al., 1993). The analysis of miniloci has also uncovered evidence that transcription and rearrangement are not invariably correlated. Abelson murine leukemia virus(AMuLV) transformed pre-B cell lines from mice containing a TCR␤ minilocus transgene contained V␤ sterile transcripts but no V-to-DJ␤ rearrangements, indicating that transcription is not sufficient for recombination (Okada et al., 1994). In another study with TCR␤ miniloci, mutation of the V␤ promoter eliminated sterile transcription but did not result in a decrease in rearrangement, implying that transcription is not necessary for recombination (Alvarez et al., 1995). Several lines of evidence show that active transcription through an RSS is not needed for recombination to occur at that RSS. Several AMuLV-transformed RAG-deficient cell lines were demonstrated to contain sterile transcripts originating from distinct VH gene families. Upon RAG protein expression in these cells, rearrangements involving transcribed and nontranscribed VH genes were detected (Angelinduclos and Calame, 1998). An episomal construct in which nucleosome remodeling could be separated from transcription was used to test the necessity of transcription for RSS accessibility. Recombination was shown to occur after nucleosome remodeling but in the absence of transcription (Cherry and Baltimore, 1999). While a particular RSS may not need to be transcribed at the same time it undergoes recombination, transcription in the general region of the RSS or through the RSS at some earlier time may still be necessary. In summary, the correlation between sterile transcription and V(D)J recombination is very strong. Despite the wealth of correlative evidence, however, it remains unproven that sterile transcription is a causal precursor of accessibility. The available data from studies of the endogenous antigen receptor loci can be explained by a model in which germline transcription is a byproduct of other forces that operate to create locus accessibility. The same arguments can be applied to the role of intergenic transcripts in establishing an open, transcriptionally active, chromatin configuration in the ␤-globin locus.

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H. MODELS FOR TRANSCRIPTIONAL REGULATION OF THE ␤-GLOBIN LOCUS Genetic manipulation of the ␤-globin locus and the resulting effects on transcription, DNase sensitivity, histone acetylation, etc., have led to the development of two main models to explain how this complex locus is regulated (for reviews, see Bulger and Groudine, 1999; Engel and Tanimoto, 2000; Fraser and Grosveld, 1998; Orkin, 1995). These models focus on how the LCR and its flanking regions function to regulate expression and locus opening. The looping, or competitive, model claims that the Hispanic deletion element (containing the LCR) functions by directly contacting the promoter of the gene within the ␤-globin locus that is to be expressed. Transcript initiation has been shown to occur at only one promoter at any specific time in a given cell (Gribnau et al., 1998). This model explains that single promoter initiation is a result of competition of the promoters for contact with the LCR. Such direct contact would obviously require looping of the intervening chromatin, but as yet there is no direct evidence for such looping. Transgenes containing the LCR and either a single human fetal (␥ ) or adult (␤) globin gene expressed the genes throughout development with no developmental specificity; however, transgenes containing the LCR and both genes restored normal developmental expression, indicating that gene competition is important for properly regulated expression (Behringer et al., 1990; Enver et al., 1990). Analysis of transgenes containing an additional copy of the ␤-globin gene and promoter supported similar conclusions. When the extra copy was placed close to the LCR (replacing the embryonic ⑀-globin gene), it was expressed 10- to 100-fold more efficiently than the copy of the ␤-globin gene that resided in its normal location far downstream in the transgene. Transcription of the extra copy was detected at an early stage of development, when only embryonic globin genes are normally expressed. When the extra ␤-globin gene was inserted just upstream of the normal copy, expression from the two copies was roughly equivalent. Importantly, the total level of transgenic ␤-globin gene transcription remained constant regardless of the position of the extra copy, and was approximately equal to the amount of transcription derived from the endogenous ␤-globin gene (Dillon et al., 1997). Inversion of the ␤-globin gene cluster with respect to the LCR in a transgenic construct resulted in severe perturbations of gene expression. ␤-globin was expressed at all developmental stages, while the embryonic gene (⑀) was not expressed at all. Transcription of the two human ␥ -globin genes was also reduced, presumably due to competition with the adult globin genes for the LCR. In the wild-type locus, the transcript levels of the two ␥ -globin genes are different. In the inverted ␤-globin gene cluster transgene, the expression levels of these two genes were reversed, indicative of competition (Tanimoto et al., 1999). These results argue that proximity to the LCR is an important determinant of gene transcription in the ␤-globin locus.


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The linking model proposes that direct contact between regulatory elements does not have to occur. The Hispanic deletion regulatory region and other unknown chromatin elements are proposed to serve as platforms for propagating open chromatin structures throughout the locus. The chromatin would be opened and maintained in this configuration by protein complexes that extend linearly along the DNA. Such complexes would therefore link the LCR with the promoters. Distinct gene promoters would then serve to recruit developmental and gene-specific factors for individual gene expression. Analyses of transgenes containing the human LCR and individual genes such as the embryonic globin gene (⑀) and the fetal globin genes (␥ ) have demonstrated that correct developmental expression of these genes occurs in absence of any other globin gene (Dillon and Grosveld, 1991; Lloyd et al., 1992; Shih et al., 1990). These data appear to conflict with other studies (Behringer et al., 1990; Enver et al., 1990). Presumably, differences in the transgenic constructs, and hence in exactly which cis-acting regulatory elements were included, explain the discrepancies. Competition can be explained by the linking model, for example by invoking boundary elements that prevent the propagation of accessibility. The globin gene promoters have been proposed to act in this capacity and prevent downstream locus opening (Bulger and Groudine, 1999). Either of the models, or a combination of them, could explain the modulation of chromatin structure that underlies the cellular control of antigen receptor gene rearrangement. The looping model provides an attractive mechanism for a cis-acting element to influence distant elements without requiring that it perturb the chromatin structure of the intervening DNA. For example, a direct interaction between the IgH intronic enhancer and a VH promoter could selectively open the chromatin structure surrounding the RSS of this V gene segment and bring the RSS into close proximity of the DJ gene segment. The linking model provides an attractive means by which structural alterations in chromatin can be propagated from an element to encompass an entire domain. For example, TCR␤ locus accessibility could start at the enhancer and move upstream through the D gene segments to the V gene segments, allowing for progressive RSS access. IV. Cis-Acting Elements and the Assembly of Antigen Receptor Loci

Many lines of evidence indicate that the cis-acting transcriptional control elements of antigen receptor loci are important for regulating V(D)J recombination. Studies of mice in which these elements have been mutated or deleted, either in the endogenous context or in transgenes, have been particularly informative. Here, we discuss the events that occur during assembly of the Ig heavy chain gene, with a focus on the role of various cis-acting control elements. Parallels with other antigen receptor loci are described, but this section is not an exhaustive

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analysis of cis-acting elements (for excellent recent reviews, see Hempel et al., 1998; Krangel et al., 1998; Sleckman et al., 1996, 1998a). A. IGH J GENE SEGMENT RECOMBINATION Deletion of the IgH intronic enhancer demonstrated a role for this cis-acting element in IgH recombination (Figs. 1 and 2B). Mice lacking this enhancer have been generated both by deletion of the element (deletion mice) (Sakai et al., 1999; Serwe and Sablitzky, 1993) and by its replacement with a neomycin gene expression cassette (neo replacement mice) (Chen et al., 1993; Sakai et al., 1999). Neo replacement mice exhibited a dramatic reduction in D-to-JH rearrangements (Chen et al., 1993; Sakai et al., 1999), while B cells from deletion mice showed only a small decrease in D-to-JH rearrangements (Sakai et al., 1999; Serwe and Sablitzky, 1993). This minimal effect seen in the deletion mice indicates that there are additional cis-acting elements that can activate D-to-JH rearrangement. In the neo replacement mice, insertion of a neomycin gene and its promoter may have interfered with the function of these other elements. The heavy chain gene intronic enhancer is flanked by matrix attachment regions (MARs), sequences that associate with a poorly defined insoluble protein fraction thought to comprise a structural scaffold for the nucleus. Transgenic models for studying transcriptional regulation have shown that the presence of MARs flanking the IgH intronic enhancer extends enhancer-dependent accessibility to 1 kb away from the element (Jenuwein et al., 1997). However, examination of mice lacking these MARs (made through RAG-2-deficient blastocyst complementation) demonstrated that they were unnecessary for normal recombination and expression of the heavy chain gene. This discrepancy in the requirement for MARs for accessibility could be accounted for by the differences between transgenic mice and targeted-knockout mice. In addition, the discrepancy may relate to the fact that the transgenic construct was transmitted through the germline, while the construct used for the complementation experiments only passed through embryonic stem cells (Sakai et al., 1999). A second transcriptional enhancer (3′ enhancer), located downstream of the last of the IgH constant regions, was speculated to play a role in regulating heavy chain gene recombination (Fig. 1) (Cogne et al., 1994; Pettersson et al., 1990). Deletion of this element, however, demonstrated it to be critical for switch recombination but not V(D)J recombination (Cogne et al., 1994). The possibility of redundancy with other elements, such as the IgH intronic enhancer, cannot be ruled out and leaves open a role for the 3′ enhancer in heavy chain gene rearrangements. PDQ52 is a specialized IgH D gene cis-acting element found approximately 500 bp upstream of the JH gene segments in the mouse (Dirkes et al., 1994; Kottmann et al., 1992) (Figs. 1 and 2B). This element was shown to have the capacity to operate as a promoter or an enhancer independent of the heavy chain


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gene intronic enhancer (Kottmann et al., 1994). Multiple lineage-specific proteins have been predicted or shown to bind the PDQ52 element (Kottmann et al., 1992; Thompson et al., 1995). The ␮0 sterile transcript initiates from this promoter in early B cell development before recombination and disappears after or during D-to-JH rearrangement (Kottmann et al., 1994; Li et al., 1996a; Schlissel et al., 1991b; Thompson et al., 1995). The passage of the ␮0 transcript through the DQ52 and JH genes may affect the accessibility of these genes. It has been theorized that this element regulates accessibility and serves as an interaction target for the IgH intronic enhancer to prevent out-of-order rearrangements. Any rearrangement not involving DQ52 would delete PDQ52 from the locus and allow further rearrangements (Kottmann et al., 1994). Another possibility is that PDQ52 assists with the propagation of accessibility from the heavy chain gene intronic enhancer to the upstream DH genes. The ability of PDQ52 to operate independently of the IgH intronic enhancer raises the possibility of redundancy and may help explain the extensive D-to-JH recombination that takes place in heavy chain intronic enhancer deleted mice. There are also promoters found in front of the other DH gene segments (Fig. 2B). They were initially identified due to their activation once the D-to-JH step had occurred (Alessandrini and Desiderio, 1991). While there is little evidence of transcripts from these promoters prior to rearrangement, lineagespecific factors may bind to these elements before recombination. These promoters were shown to be dependent on the IgH intronic enhancer for activity (Alessandrini and Desiderio, 1991), but it is equally possible that the PDQ52 element can perform this function; thus, these promoters may also be involved in D-to-JH targeting. B. J GENE SEGMENT RECOMBINATION IN OTHER ANTIGEN RECEPTOR LOCI All antigen receptor loci have enhancers downstream of the recombining portion of the locus (Fig. 1). Many of these enhancer elements have been deleted genetically, resulting in the unifying conclusion that such enhancers regulate D-to-J or V-to-J recombination. Examination of TCR␤ enhancer-deleted mice demonstrated this element to be essential for D-to-J␤ recombination (Bories et al., 1996; Bouvier et al., 1996). The nearly complete block of the D-to-J␤ step in these mice differs from the mild reduction of D-to-JH rearrangements seen upon deletion of the IgH intronic enhancer. Deletion of a MAR close to the TCR␤ enhancer had no effect on V(D)J recombination (Chattopadhyay et al., 1998a,b). This phenotype is very similar to that shown with the deletion of the IgH intronic enhancer MARs. Much like production of the ␮0 transcript from the IgH PDQ52 element, the TCR␤ locus has a sterile transcript originating from a promoter upstream of the D␤1 gene segment. This cis-acting element, termed PD␤1 (Fig. 1), was

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discovered using DNaseI hypersensitivity assays (Chattopadhyay et al., 1998b) and by delineating the start site of the sterile transcript (Sikes et al., 1998). Analysis of mice lacking PD␤1 demonstrated that it was important for D␤1-to-J␤ rearrangement. Rearrangements involving the D␤2 gene segment were not affected, and this may indicate the existence of a similar control element in front of D␤2 (Whitehurst et al., 1999). The levels of the PD␤1 sterile transcript were drastically reduced in the TCR␤ enhancer-deleted mouse, indicating possible promoter /enhancer communication or interaction between these two elements (Bories et al., 1996). PD␤1 activity was shown to be dependent on the TCR␤ enhancer through the use of miniloci in a cell culture system. Deletion of PD␤1 eliminated recombination, and its replacement with an inducible, enhancerindependent promoter allowed for the reconstitution of D-to-J recombination in miniloci lacking the TCR␤ enhancer (Sikes et al., 1999). Analysis of TCR␣ enhancer-deleted mice showed that this element is important for V-to-J␣ recombination, as only minimal rearrangements were detected in the absence of the enhancer (Sleckman et al., 1997). An LCR exists directly downstream of the TCR␣ enhancer (Fig. 1) (Diaz et al., 1994), but its deletion had no effect on V(D)J recombination (Hong et al., 1997). TCR␣ J gene sterile transcripts have been shown to start from an unexplored area between J␣50 and J␣40 (Villey et al., 1997) and from a well-characterized promoter known as the TEA element, located upstream of the most 5′ J␣ gene segment (Fig. 1) (de Chasseval and de Villartay, 1993; de Villartay et al., 1987). Deletion of the TCR␣ enhancer resulted in a severe reduction of sterile transcripts in the J gene cluster (Sleckman et al., 1997); however, occupancy at the TEA element was still detected (Hernández-Munain et al., 1999). The TCR␣ enhancer and the TEA element have been shown to be inactive even when occupied, although subtle changes detected through in vivo footprinting correlate well with element activity (Hernández-Munain et al., 1999; Spicuglia et al., 2000). Deletion of the TEA element showed it to be important for recombination and sterile transcription of the most 5′ J␣ gene segments, but rearrangement to other J␣ gene segments proceeded normally (Villey et al., 1996). This suggests the involvement of other elements in regulating TCR␣ recombination in conjunction with the TCR␣ enhancer. TCR␦ is the third antigen receptor locus generated through the rearrangement of V, D, and J gene segments. Transgenic miniloci have been used to show that the TCR␦ enhancer regulates the D-to-J step by controlling accessibility of the J␦ gene segments (Lauzurica and Krangel, 1994; McMurry et al., 1997). These same TCR␦ miniloci have been used to demonstrate the importance of MARs flanking the TCR␦ enhancer. Deletion of these MARs allowed for TCR␦ V-to-D recombination but little VD-to-J␦ recombination in single-copy transgenic mice. In multicopy transgenic mice, however, deletion of the MARs had no effect on recombination (Zhong et al., 1999). Deletion of the TCR␦ enhancer


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from the mouse germline reduced but did not eliminate D-to-J␦ recombination. Simultaneous deletion of the endogenous TCR␦ and ␣ enhancers still allowed for residual ␦ rearrangements, indicating that additional elements can contribute to the control of TCR␦ recombination (Monroe et al., 1999c). Sterile transcripts of the Ig␬ J gene segments have two start sites within the intervening sequence between the V and J gene clusters (Fig. 1), one within the KI /KII region (Weaver and Baltimore, 1987) and the other further upstream (Martin and van Ness, 1990), and references therein). Deletion of the upstream start site affected both sterile transcription and recombination (Cocea et al., 1999), while deletion of the KI /KII region only affected recombination. Deletion of both of these sequences showed a greater effect on Ig␬ recombination than either single deletion alone (Cocea et al., 1999). The KI /KII region provides the first example of a DNA sequence that affects recombination but not sterile transcription (Ferradini et al., 1996). Like the TCR␣ enhancer, the Ig␬ intronic enhancer exhibits factor occupancy prior to activation of transcription or recombination of the locus. In contrast, the Ig␬ 3′ enhancer exhibits occupancy of critical factor binding sites in a developmentally appropriate manner that correlates with activation of the locus (Shaffer et al., 1997). Mice harboring deletions of either Ig␬ enhancer showed reduced but detectable V-to-J␬ rearrangements. This suggests redundancy between these two enhancers and/or that there are other important cis-acting elements regulating Ig␬ rearrangement (Gorman et al., 1996; Xu et al., 1996). Deletion of a MAR adjacent to the Ig␬ intronic enhancer results in a slight enhancement of V-to-J␬ recombination in pro-B cells (in which light chain gene recombination normally occurs at a low frequency) (Yi et al., 1999). MARs have now been deleted from enhancers in TCR␤, TCR␦, IgH, and Ig␬ endogenous loci or miniloci, with disparate results. It is not possible at this point to draw any general conclusions concerning the role of MARs in the regulation of V(D)J recombination. C. IGH V-TO-DJ RECOMBINATION After the first IgH recombination event, D␮ germline transcripts arise from cryptic promoter elements upstream of all the DH genes (Fig. 2B). It is thought that these promoters become active due to their new proximity to the IgH intronic enhancer (Alessandrini and Desiderio, 1991). The predominance of transcripts coming only from the promoter of a rearranged DJH gene and not from unrearranged DH genes may target, or indicate a targeting mechanism for, the V-to-DJH step. Deletion of the IgH intronic enhancer dramatically reduced V-to-DJH recombination (Sakai et al., 1999; Serwe and Sablitzky, 1993). It is reasonable to think that this enhancer regulates DH gene segment RSS accessibility. If this hypothesis is correct, deletion of the IgH intronic enhancer would eliminate

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transcription (Alessandrini and Desiderio, 1991) and possibly other structural changes at the promoter of the DJH gene. In most cases, PDQ52 would be deleted from the locus, and would not be able to assist in accessibility. It is also possible that the IgH intronic enhancer directly regulates VH gene accessibility, but this idea has not been tested experimentally. Sterile transcripts coming from VH gene promoters appear at the same time as the occurrence of V-to-DJH rearrangement (Fig. 2B). These transcripts disappear after a functional heavy chain is made and expressed (Schlissel et al., 1991b; Yancopoulos and Alt, 1985). It is plausible that VH gene promoters play a role in regulating V-to-DJH rearrangement, but there is no direct evidence for this as yet. Precedence for such a regulatory role for V gene promoters and flanking regions comes from studies of other antigen receptor loci (discussed in the next section). It is also conceivable that the IgH intronic enhancer has no direct effect on the V-to-DJH step. A reduction of D-to-JH rearrangements due to the deletion of the intronic enhancer would result in fewer DJH targets for the V-to-DJH step (Sakai et al., 1999; Serwe and Sablitzky, 1993).If the V-to-DJH step is inherently inefficient even in wild-type cells, the combination of fewer targets and an inefficient reaction could result in a drastic reduction in V-to-DJH rearrangements. There may be additional cis-acting elements in the IgH locus that regulate V(D)J recombination. Interesting areas of the locus to examine are the VH gene region and the intervening region between the VH and the DH gene segments. If such elements are found, they will probably have an effect on the V-to-DJH recombination step. D. V-TO-DJ RECOMBINATION IN OTHER ANTIGEN RECEPTOR LOCI Deletion of the TCR␦ enhancer from the mouse germline resulted in reduced but detectable levels of all types of TCR␦ rearrangements (Monroe et al., 1999c). This result differs from the those obtained with TCR␦ transgenic miniloci, in which deletion of the ␦ enhancer dramatically reduced D-to-J␦ rearrangement but had no effect on V-to-D␦ recombination (Lauzurica and Krangel, 1994). It seems likely that the ␦ enhancer plays a role in all rearrangements of this locus. Deletion of the TCR␤ enhancer eliminates V-to-DJ␤ rearrangement, but because there is virtually no D-to-J␤ rearrangement in these mice, this result is difficult to interpret (Bories et al., 1996; Bouvier et al., 1996). PD␤1 was suggested to regulate TCR␤ V-to-DJ recombination in addition to D-to-J recombination (Whitehurst et al., 1999). The dependence of this promoter on the TCR␤ enhancer for recombination activity (Sikes et al., 1999) may explain part of the phenotype seen in the enhancer deletion (Bories et al., 1996; Bouvier et al., 1996). Again, these results suggest mechanistic parallels with the regulation of IgH locus rearrangement, where the intronic enhancer and promoter elements upstream of D gene segments may collaborate in regulating both D-to-J and V-to-DJ rearrangement.


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The strongest evidence linking V gene promoter regions to the regulation of V(D)J recombination comes from an analysis of TCR␥ transgenic miniloci (Baker et al., 1998). Miniloci containing elements of the C␥ 1 locus were created that recapitulated the preferential rearrangement of V␥ 3 in fetal thymocytes and V␥ 2 in adult thymocytes. Swapping of the regions spanning the V␥ 3 and V␥ 2 promoters switched the rearrangement preference in adult thymocytes such that now V␥ 3 rearranged more frequently than V␥ 2. Interestingly, the promoter region swap did not switch the rearrangement preference in fetal thymocytes, suggesting that these promoter regions regulate adult but not fetal rearrangements at this locus. Evidence also hints at a regulatory role for V␭ promoters. Naturally occurring mutations found in murine Ig␭ gene promoters are associated with reduced recombination to those particular V genes (Stiernholm and Berinstein, 1995), and deletion of the complete V promoter in Ig␭ miniloci severely affected rearrangements (Lauster et al., 1993). E. ORDERED REARRANGEMENT AND ALLELIC EXCLUSION In the IgH and TCR␤ loci, D-to-J rearrangement occurs on both alleles prior to V-to-DJ rearrangement. How this order is imposed is not understood, but several observations indicate that it is the V-to-DJ step that is tightly regulated. First, D-to-JH rearrangement is not lineage restricted, but occurs efficiently in both B and T cell precursors (Kurosawa et al., 1981). In contrast, V-to-DJ rearrangements of both loci are strictly limited to the appropriate lymphocyte lineage. Second, as discussed below, disruption of the function of several different factors or signaling pathways ( Pax5/BSAP, Ig␤, IL7 receptor), or removal of the C-terminal domain of RAG2 (Kirch et al., 1998), specifically interferes with the V-to-DJ step of heavy chain gene rearrangement. Third, allelic exclusion is imposed at the step of V-to-DJ recombination, with Ig␮ or TCR␤ transgenes preferentially inhibiting this step of the assembly process (for reviews, see Schatz et al., 1992; von Boehmer, 1990). Several models can be envisioned for ordered rearrangement of the IgH and TCR␤ loci. First, D and J gene segments may become accessible at an earlier developmental stage than do V gene segments. According to this scenario, D-to-J rearrangements would have already occurred on both alleles by the time V gene segments become accessible. One way to have D and J, but not V, gene segments accessible at an early stage of development would be the existence of a boundary element between the D and V gene clusters. Such an element, similar to the BEAD-1 element found in the TCR␣/␦ locus (Zhong and Krangel, 1997)(Fig. 1), could prevent the IgH and TCR␤ enhancers from acting prematurely on the V gene segments. Alternatively, V, D, and J gene segments might become accessible at the same time, but D-to-J rearrangements occur first because they are strongly favored kinetically. It is not clear how such a kinetic preference would be established. The 3′ most VH gene segment is only 20 kb from the 5′ most

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DH gene segment (Fig. 2A), and one V␤ gene segment (V␤14) is closely linked to the D-J-C␤ cluster (Fig. 1). A third possibility is that V-to-DJ recombination requires prior D-to-J recombination, for example, because joining of D-to-J deletes inhibitory sequences. This model seems unlikely, given recent results, discussed below, demonstrating that V-to-D␤ rearrangement occurs efficiently when D-to-J joining is blocked by mutation of the RSS 3′ of D␤1 (Bassing et al., 2000; Sleckman et al., 2000). Allelic exclusion, whereby a functional gene product is produced by only one of the alleles of a given gene, is most tightly enforced at the IgH and TCR␤ loci. Allelic exclusion is violated at the TCR␣ (Padovan et al., 1993), the TCR␥ (Davodeau et al., 1993), and the TCR␦ (Sleckman et al., 1998b) loci. Ig light chain loci show evidence of multiple in-frame rearrangements in individual cells, although functional allelic exclusion may still be achieved by the failure of certain light chains to form a productive Ig molecule (Yamagami et al., 1999). For the IgH and TCR␤ loci, allelic exclusion appears to be regulated predominantly at the level of gene rearrangement rather than at the level of expression or function. While numerous hypotheses have been put forward to account for allelic exclusion, it seems likely that some version of the regulated, or feedback, model is correct (for review, see Blackwell and Alt, 1988). According to this model, expression of a heavy or ␤ chain protein would be involved in signaling to prevent further rearrangement of the other allele. If one accepts this general type of model, at least two major conceptual challenges remain. How is one allele chosen to rearrange first (or, in other words, what prevents both alleles from rearranging simultaneously)? Second, how are accessible gene segments and loci made inaccessible again? For the first of these questions, one possible answer is that rearrangement is inherently inefficient (Schatz et al., 1992), thus ensuring that the probability of both alleles undergoing V-to-DJ recombination at the same time is very low. An alternative, suggested by experiments that examined methylation of the Ig␬ locus, is that one of the two alleles is chosen stochastically to be accessible (and demethylated) well before the other (Mostoslavsky et al., 1998). To what extent either (or both) of these models applies to the initiation of IgH and TCR␤ V-to-DJ rearrangement is unclear. The driving force behind the question of how accessible loci are made inaccessible is the notion that IgH and TCR␤ V-to-DJ recombination must be prevented while Ig light chain and TCR␣ rearrangements, respectively, are occurring. By analogy with stochastic activation of Ig␬ alleles, one way to achieve this would be to ensure that the VH/V␤ gene segments of one allele remain inaccessible until recombination has been attempted, and has failed, on the other allele. At this point, such a model cannot be ruled out. This raises the question of whether an accessible chromosomal region is ever subsequently made inaccessible. The strongest evidence for this comes from studies of recombination intermediates (signal ends and coding ends) in populations of developing B cells (Constantinescu and Schlissel, 1997), and through the use of an in vitro


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system for performing RAG protein-mediated DNA cleavage in intact nuclei (Stanhope-Baker et al., 1996). Both studies suggest that there is a retargeting of the RAG proteins from the heavy chain locus to the light chain locus during B cell development, and that suppression of V-to-DJH rearrangement is accomplished by making the VH gene segments inaccessible. Attention is focused on the VH and V␤ gene segments because current evidence indicates that the D-J regions of the heavy and ␤ chain loci remain accessible during the stage of Ig light chain and TCR␣ gene rearrangement (Stanhope-Baker et al., 1996; Whitehurst et al., 1999). How an accessible locus might be rendered inaccessible is unknown. Clearly, it need not be accomplished by simply reversing the steps that make a locus accessible, although it seems likely that similar forces will be involved. Methylation is a possible early event, given the ability of methylated residues to attract MeCP proteins, which in turn can recruit histone deacetylases. As yet, however, there is no evidence that an unmethylated antigen receptor locus becomes methylated in the course of lymphocyte development. V. The Factors

Multiple transcription factors and signaling molecules have been implicated in regulating IgH V(D)J recombination. Some of the transcription factors discussed below have been shown to interact functionally with enhancer and/or promoter elements within the locus, and it is likely that this is how they exert some or all of their influence on the recombination process. Some of the factors have been linked to the regulation of V(D)J recombination through the analysis of mice with targeted mutations in the genes for these factors. The phenotypes of such knockout mice must be interpreted cautiously because of possible pleiotropic effects on gene expression and lymphocyte development. For example, a defect in recombination of a particular locus could be due to a failure to make the locus accessible, a failure of the cells to express RAG1/2, or a failure to develop to the stage at which this particular locus normally rearranges. In addition, the existence of factors with a redundant function can obscure a significant role in regulating V(D)J recombination. The following discussion focuses on transcription factors and signaling proteins thought to regulate IgH V(D)J recombination. Other antigen receptor loci are not covered in a systematic manner, nor are the roles played by the factors in controlling gene expression and lymphocyte development. (For general reviews of the transcription factors, see Calame and Ghosh, 1995; Ernst and Smale, 1995; Henderson and Calame, 1998; Reya and Grosschedl, 1998.) A. E2A The E2A gene is alternatively spliced to encode two, ubiquitously expressed, basic helix-loop-helix proteins, E47 and E12. These proteins, along with HEB

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and E2-2, are members of a family of transcription factors that bind a motif known as the E-box and play important roles in lymphocyte development (for reviews, see Bain and Murre, 1998; Henderson and Calame, 1998). E47 and E12 bind multiple sites within the IgH intronic enhancer and other antigen receptor gene control elements and appear to be important for both transcription and V(D)J recombination. The evidence linking E47 and E12 to the regulation of V(D)J recombination is perhaps stronger than for any other factor. E2A-deficient mice exhibit defects in T cell development (see below) and a complete block very early in B cell development (Bain et al., 1994; Zhuang et al., 1994). In these mice, few cells committed to the B cell lineage can be detected, levels of the I␮ and ␮0 sterile transcripts are reduced, and D-to-JH and other Ig gene rearrangements are undetectable. Interestingly, appropriately regulated HEB expression can rescue B cell development in E2A-deficient mice, demonstrating that these proteins can exhibit functional redundancy (Zhuang et al., 1998). A large number of experiments have implicated E47 and E12 in the regulation of V(D)J recombination. Overexpression of E47 in a murine pre-T cell line increased I␮ sterile transcripts 80 fold (to levels 60% of those of a pro-B cell line) and D-to-J rearrangements 150 fold (to 10% of the levels of a pro-B cell line). No effects on ␮0 transcription and V-to-DJ recombination were observed (Schlissel et al., 1991a). Similarly, expression of E47 in a murine fibroblast cell line triggered I␮ germline transcripts at 5–10% of the levels found in a pro-B cell line (Choi et al., 1996). As seen in Fig. 2B, I␮ starts downstream of the J gene cluster and thus does not traverse any recombining gene segments. Nonetheless, it is thought to give some measure of locus opening. Expression of RAG1, RAG2 and either E47 or E12 in a human embryonic kidney cell line resulted in endogenous D-to-JH rearrangement and relatively high levels of V-to-J␬ rearrangement (Romanow et al., 2000). Curiously, in the absence of E12/E47 overexpression, this cell line (a retroviral packaging cell line) exhibited low levels of V␬ sterile transcripts and V-to-J␬ rearrangement (when transfected with RAG1/2 expression vectors). Hence, the ␬ locus exhibits a basal level accessibility in these nonlymphoid cells, with a clear upregulation of ␬ recombination and sterile transcription occurring upon overexpression of E12 or E47 (Romanow et al., 2000). Additional support for a connection between E2A gene products and accessibility comes from the study of minilocus transgenes containing the heavy chain intronic enhancer. Mutation or deletion of E-box motifs in the enhancer resulted in a significant reduction in V(D)J recombination (Fernex et al., 1995). E2A gene products have also been implicated in the control of TCR␥ and ␦ gene rearrangements (Bain et al., 1999). In E2A-deficient mice, ␥ ␦ T cell development is relatively normal in the fetus but significantly impaired in the adult. When adult thymocytes from E2A−/− mice were examined, they were found to contain 10- to 50-fold higher levels of rearrangements of V␥ 3 and V␦1


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gene segments than in E2A+/− mice (these two V gene segments are rearranged almost exclusively during fetal development in wild-type mice). In addition, rearrangement of various V␥ and V␦ gene segments normally found in abundance in wild-type adult ␥ ␦ T cells were substantially reduced in the thymus of E2A−/− mice. Furthermore, rearrangements of certain ␥ and ␦ V gene segments exhibited a dependence on E2A gene dosage, with rearrangement levels varying considerably between E2A+/+ and E2A+/− mice. Hence, a deficiency in E2A gene products substantially perturbs normal patterns of TCR␥ and ␦ gene rearrangements, and it appears that E2A encoded factors play both positive and negative roles in the recombination of these loci (Bain et al., 1999). It is worth noting that deficiencies in E2A or HEB result in defects in early stages of ␣␤ T cell development (Bain et al., 1997; Barndt et al., 1999). E2A gene products have been linked to chromatin remodeling. E47 and E12 have been shown to interact and activate transcription in conjunction with the histone acetyltransferases p300/CBP (Eckner et al., 1996; Ogryzko et al., 1996; Qiu et al., 1998). In addition, one of the activation domains of the E2A encoded proteins has also been shown to be able to interact with the SAGA histone acetyltransferase complex (Massari et al., 1999). These interactions point to histone modification and remodeling as a mechanism by which E47 and E12 may influence accessibility. Id proteins antagonize E-box binding protein function through dimerization and inhibition of DNA binding (Benezra et al., 1990), and perturb both T cell and B cell development (for review, see Bain and Murre, 1998). Id expression inversely correlates with I␮ expression and has been shown to inhibit IgH intronic enhancer function in B cell lines (Wilson et al., 1991). Expression of a B cell-specific Id1 transgene in mice impaired B cell development and severely inhibited RAG expression and thus V(D)J recombination (Sun, 1994). Expression of this transgene also inhibited I␮ and ␮0 expression and hence had effects on B cell development that closely mimicked those observed in E2A-deficient mice. B. PU.1 AND ETS FAMILY FACTORS PU.1, a member of the Ets family of transcription factors (for reviews, see Henderson and Calame, 1998; Simon, 1998) is expressed in the myeloid lineage, throughout B cell development, and in early DN thymocytes but not in later stages of T cell development (Anderson et al., 1999; Klemsz et al., 1990; Nelsen et al., 1993; Rivera et al., 1993). Analysis of PU.1-deficient mice demonstrated this protein to be an important regulator of the development of multiple hematopoietic lineages (McKercher et al., 1996; Scott et al., 1994). The two PU.1 knockout mice, generated in separate laboratories, have slightly different phenotypes, with one lacking B cells entirely (Scott et al., 1994), and the other exhibiting abnormal B cell development but containing some B220+ cells

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expressing RAG1, RAG2, and ␮0 (only in fetal liver) (McKercher et al., 1996). PU.1 binds to the ␮B site of the IgH intronic enhancer and, synergistically with other transcription factors, activates transcription from this enhancer (Ernst and Smale, 1995; Nelsen et al., 1993; Nikolajczyk et al., 1999; Rivera et al., 1993). Other Ets family members, such as Ets-1, ERG-3, FLI-1, ERP, and ELF-1, are capable of binding the ␮A site in the intronic enhancer, and together with other factors, of activating transcription. These proteins are all expressed in early B lineage cell lines, implying that they may be regulators of IgH intronic enhancer function (Akbarali et al., 1996; Anderson et al., 1999; Dang et al., 1998; Erman et al., 1998; Libermann and Baltimore, 1993; Lopez et al., 1994; Nelsen et al., 1993; Nikolajczyk et al., 1999; Rivera et al., 1993). Ets-1-deficient mice show defects in T cell activation and survival and late B cell differentiation but no defects in early B cell development (Bories et al., 1995; Muthusamy et al., 1995), perhaps due to redundancy with other Ets family members. A recent provocative study has implicated PU.1 and Ets-1 in activating IgH sterile transcripts (Nikolajczyk et al., 1999). Expression of exogenous PU.1 in pre-T or fibroblast cell lines, each expressing Ets-1 but not PU.1, triggered I␮ sterile transcription and increased sensitivity of the endogenous IgH enhancer to digestion with restriction enzymes. PU.1 and Ets-1 also bind to the enhancer in plasmids that had been assembled into chromatin in vitro, and again, binding of the transcription factor resulted in increased accessibility to restriction enzyme digestion (Nikolajczyk et al., 1999). Therefore, like E2A gene products, these Ets family factors have the ability to bind to the IgH enhancer in chromatin and induce transcription and local changes in chromatin structure. These results are consistent with the idea that PU.1 and other Ets family proteins help regulate IgH locus V(D)J recombination, but this remains to be tested directly. C. ␮E3 BINDING PROTEINS ␮E3 sites are located within the IgH intronic enhancer and VH gene promoters, and have been shown to be important for IgH transcription (for reviews, see Calame and Ghosh, 1995; Henderson and Calame, 1998). TFE3, TFEB, and USF are ubiquitously expressed proteins that bind the ␮E3 site within the intronic enhancer and some of the VH gene promoters (Beckmann et al., 1990; Carr and Sharp, 1990; Roman et al., 1992). While TFE3 and TFEB activate transcription, USF is a negative regulator of IgH transcription (Artandi et al., 1994; Carter et al., 1997). The strong effect of dominant negative TFE3 but not USF proteins shows that TFE3 (and/or possibly TFEB) can be a potent regulator of IgH transcription (Carter et al., 1997). These proteins multimerize, and it has been suggested that they mediate IgH promoter–enhancer interactions (Artandi et al., 1994; Fisher et al., 1991; Roman et al., 1992). Such a bridging function could play a role in V(D)J recombination by bringing VH gene segments into close proximity of the DJH gene segment. Inactivation of TFE3 in


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ES cells and analysis by RAG2-deficient blastocyst complementation results in mice with defects in B cell activation but normal bone marrow B cell development and V(D)J recombination (Merrell et al., 1997). Redundancy with other related proteins, such as TFEB, could help to explain this phenotype. When the IgH intronic enhancer ␮E3 site and an E2A factor binding site were simultaneously mutated, the resulting transgenic recombination minilocus failed to undergo V(D)J recombination (Fernex et al., 1994). It is unclear which of the two sites (or perhaps both) were critical for rearrangement in cells of the B lineage, and hence the connection between this ␮E3 site and the regulation of IgH recombination remains tenuous. It is possible that TFE3 or related proteins do not actually bind the IgH intronic enhancer. Comparison of the human and murine intronic enhancers shows striking sequence similarity except in the vicinity of ␮E3 site. Instead, a binding site for CBF (also known as AML1 and PEBP2) is found in this position in both the human and mouse enhancer. CBF is expressed in early B cell lines and can activate both human and murine IgH intronic enhancer-based transcription (Erman et al., 1998). The CBF−/− mouse dies during embryogenesis. It develops sufficiently to conclude that CBF is a regulator of hematopoiesis, but not enough to allow study of the effect of this gene on B cell development (Wang et al., 1996a,b). Given the existing data and the number of transcription factors suggested to bind in this region of the IgH intronic enhancer, it is difficult to connect any of the factors, or the binding sites themselves (in the enhancer or the VH promoters) to the regulation of V(D)J recombination. D. OCTAMER BINDING PROTEINS The octamer motif has been found in IgH V region promoters and the intronic enhancer as well as in other elements in Ig loci. Many studies have shown that these sites are essential for transcriptional regulation of IgH and other B cellspecific genes (for reviews, see Calame and Ghosh, 1995; Ernst and Smale, 1995; Henderson and Calame, 1998; Matthias, 1998). There is also some suggestion of a connection between a functional octamer site and efficient V(D)J recombination (Lauster et al., 1993; Stiernholm and Berinstein, 1995). Two Pou-domain proteins, Oct1 and Oct2, bind to and activate transcription from Ig octamer sites. Oct1 is expressed in most cell types, while Oct2 is expressed primarily in B lineage cells (Matthias, 1998; Schlissel et al., 1991b; Staudt, 1991). An additional B cell-specific factor, known as OcaB, was identified by virtue of its ability to stimulate Oct1- and Oct2-dependent Ig-specific transcription (Luo et al., 1992). Oct2- and OcaB-deficient mice have similar B cell phenotypes in which antibody production and late-stage B cell differentiation are affected, but Ig V(D)J recombination and transcription are normal (Corcoran et al., 1993; Kim et al., 1996; Schubart et al., 1996). Given the potential for redundancy between Oct1 and Oct2 and the fact that IgH and IgL transcription was normal in these mice,

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it remains uncertain whether the octamer site or the proteins that bind it play any role in regulating V-to-DJH rearrangement. E. EBF Early B cell factor (EBF) is a transcription factor expressed in pro-B, pre-B, and mature B cells, but not in plasma cells or other hematopoietic cells (Hagman et al., 1993). To date, very little is known about EBF binding to IgH transcriptional control elements. Two putative EBF binding sites are located within the IgH intronic enhancer, and transcription of a reporter vector containing this enhancer was decreased by cotransfection with an EBF expression construct (Akerblad et al., 1996). There is as yet no direct evidence that EBF interacts with any IgH regulatory element. In EBF−/− mice, B cell development is blocked at a very early stage (Lin and Grosschedl, 1995) prior to the onset of most IgH D-to-J recombination (fraction A in the nomenclature of Hardy et al., 1991). The early pro-B cells that do exist in these mice express both the I␮ and ␮0 sterile transcripts, but little if any RAG1. Not surprisingly, only trace levels of D-to-J rearrangement could be detected in these cells (Lin and Grosschedl, 1995). The simplest interpretation of these results is that in the absence of EBF, IgH D and J gene segments are transcribed and hence accessible, but fail to recombine due to a lack of RAG expression and/or because of the developmental arrest. While these results raise the possibility that EBF is not necessary for D-JH accessibility, overexpression of EBF together with the RAG proteins is sufficient to trigger D-to-JH (and V-to-J␭) recombination in a human embryonic kidney cell line (Romanow et al., 2000). Whether this is due to direct binding of EBF to the IgH locus or is a result of indirect effects of EBF overexpression remains unknown. F. PAX5/BSAP The BSAP protein is encoded by the PAX5 gene, and is a member of a large family of transcription factors that bind DNA through a characteristic paired domain (Adams et al., 1992; Czerny et al., 1993). PAX5 is expressed in developing and mature B cells but not in plasma cells and other hematopoietic cells (Barberis et al., 1990; Li et al., 1996a; for review, see Busslinger and Nutt, 1998; Morrison et al., 1998). In Pax5-deficient mice, bone marrow B cell development is blocked at an early stage (Urbanek et al., 1994). The arrest occurs in fraction B (nomenclature of Hardy et al., 1991), within which D-to-JH and some V-to-DJH recombination normally takes place. Strikingly, the Pax5-deficient pro-B cells exhibited normal levels of D-to-JH rearrangements but a ∼50-fold reduction in V-to-DJH rearrangements compared to wild-type pro-B cells (Nutt et al., 1997). The underlying basis for this provocative phenotype is unknown. Pax5deficient pro-B cells express I␮, but VH, ␮o, and D␮ sterile transcripts were not examined (Nutt et al., 1997), and hence nothing is known about VH gene


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segment accessibility in these cells. The interpretation of these results is made more difficult by the finding that Pax5-deficient pro-B cells are not actually committed to the B cell lineage. Despite the fact that they express genes and markers appropriate for the B cell lineage, these cells can differentiate into many of the other hematopoietic lineages if cultured under appropriate conditions (Nutt et al., 1999; Rolink et al., 1999). Thus, while BSAP may exert direct control over IgH gene segment accessibility, it is equally possible that its effect on V(D)J recombination is an indirect consequence of its role in regulating development. G. INTERLEUKIN-7 RECEPTOR The IL-7 receptor (IL-7R) is made up of two proteins: the unique ␣ subunit and the common ␥ chain which is shared by many interleukin receptors. The IL-7R and its downstream signaling pathway regulates differentiation and proliferation during both T and B cell development. Deficiencies in IL-7 or IL-7R␣ perturb V(D)J recombination in ␥ ␦ and ␣␤ T cells and in B cells (for reviews, see Candeias et al., 1997a; Haks et al., 1999; Hofmeister et al., 1999). In IL-7R␣-deficient mice, D-to-JH rearrangement occurs normally, but V-to-DJH rearrangement exhibits an intriguing defect: proximal VH gene families (those closest to the D-J-C␮ portion of the locus) undergo rearrangement, while more distal V gene families do not (Corcoran et al., 1998). Sterile transcripts could be detected from a proximal VH gene segment (V81X) but not from the most distal VH gene family (J558), suggesting a correlation between recombination and sterile transcription. Intriguingly, bone marrow cells from IL-7R␣−/− mice express lower levels of Pax5 than do IL-7R␣+/− controls. This, together with the defect in V-to-DJH recombination in Pax5-deficient mice, suggests the possibility that IL-7R signaling modulates accessibility of the VH gene cluster by influencing BSAP levels (Corcoran et al., 1998). A parallel mechanism has been proposed for IL-7R control of TCR␥ locus recombination. IL-7R␣−/− mice have severely reduced levels of TCR␥ rearrangements and sterile transcripts, suggesting that accessibility of this locus is impaired (Candeias et al., 1997b; Durum et al., 1998; Maki et al., 1996). Direct RAG1/RAG2 cleavage assays using nuclei purified from thymocytes showed a reduction in cleavage in IL-7R␣−/− nuclei compared to wild-type nuclei, demonstrating reduced TCR␥ locus accessibility in the mutant cells (Schlissel et al., 2000). The addition of the histone deacetylase inhibitor, trichostatin A, overcame the block in TCR␥ gene recombination, suggesting a role for histone acetylation in controlling accessibility (Durum et al., 1998). The transcription factor Stat5 has been implicated in regulating TCR␥ locus accessibility via IL-7R signaling (Ye et al., 1999). It is tempting to speculate that Stat5 and BSAP, each acting downstream of IL-7R signals, play similar roles in regulating TCR␥ and IgH locus recombination, respectively.

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H. IG␤ Ig␤ and Ig␣ are integral membrane proteins that are components of the BCell Receptor (BCR) and pre-BCR. They are critical for signaling through these receptors and hence for the proper function and development of B cells. The pre-BCR, which along with Ig␤ and Ig␣ also contains the ␮ heavy chain and surrogate light chains, plays a critical role in the pro-B to pre-B cell transition and hence in the transition from Ig heavy to light chain gene rearrangement (for reviews see Papavasiliou et al., 1997; Roth and DeFranco, 1996). Analysis of Ig␤-deficient mice not only confirmed the importance of this molecule for the pro-B to pre-B cell transition, but revealed a surprising effect on IgH rearrangement: D-to-JH rearrangement was normal but V-to-DJH rearrangement was significantly reduced compared to wild-type mice (Gong and Nussenzweig, 1996). Despite this, Ig␤-deficient pro-B cells expressed normal levels of sterile transcripts from all regions of the heavy chain locus. This result suggests that Ig␤ (and possibly Ig␣) plays a role in regulating the initiation of V-to-DJH recombination, although how it might do so is a mystery. Obviously, Ig␤ must carry out this function in cells that do not yet express a ␮ heavy chain protein. Recent studies indicate that this is plausible. In RAG2-deficient pro-B cells (and hence in the absence of a heavy chain protein), the Ig␤/Ig␣ heterodimer can reach the cell surface, and upon crosslinking, induce the pro-B to pre-B cell transition (Nagata et al., 1997). This crosslinking results in cessation of V-to-DJH rearrangement and VH sterile transcription and an increase in ␬ locus sterile transcription and recombination (Maki et al., 2000). It is currently unknown if there are mechanistic similarities underlying the defect in V-to-DJH rearrangement in Ig␤-deficient mice and the related defects in Pax5 and IL-7R␣-deficient mice. I. OTHER FACTORS, OTHER LOCI It is likely that other transcription factors and signaling molecules play a role in regulating IgH V(D)J recombination. Examples of such factors are the Ikaros transcription factor family and Sox-4. While the Ikaros family has not yet been directly implicated in antigen receptor recombination, members of this family are regulators of lymphoid development and have been shown to associate with histone remodeling and modification complexes (for reviews, see Cortes et al., 1999; Nichogiannopoulou et al., 1998). Additionally, the Ikaros protein localizes to transcriptionally silent lymphoid genes in areas of heterochromatin within murine lymphoid cells (Brown et al., 1997). Hence, the Ikaros protein family appears to regulate complex nuclear events, particularly those associated with gene silencing, and it is possible that these factors assist in regulating antigen receptor gene accessibility. The Sox-4 transcription factor is a member of the SOX subfamily of the HMG box-containing protein family (for review, see Schilham and Clevers, 1998).


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Sox-4 is expressed in pro/pre-B cells and in the T cell lineage (van de Wetering et al., 1993). The Sox-4-deficient mouse showed a severe but incomplete block in B cell development at the pro-B cell stage, but Ig gene rearrangements were not examined (Schilham et al., 1996). It would be interesting to determine whether Sox-4 plays a role in regulating V(D)J recombination. It is worth noting that there are other known elements within the heavy chain locus that remain uncharacterized in terms of transcription factor binding and potential function in regulating V(D)J recombination. The DH gene cryptic promoters and the PDQ52 element are examples. Functionally important binding sites have not been identified within these regions. Most of the factors described above are also expressed during Ig light chain gene recombination. Many have functional binding sites within light chain enhancers and promoters (for review, see Calame and Ghosh, 1995), and these cis-acting elements are important for endogenous light chain recombination (for reviews, see Hempel et al., 1998; Sleckman et al., 1996). For reviews of the regulation of TCR gene V(D)J recombination, see Krangel et al. (1998) and Sleckman et al. (1998a). Of particular interest regarding TCR gene rearrangement are the factors cMyb and CBF/PEPB2, which have been implicated in regulating TCR␦ locus recombination by binding to the ␦ enhancer (Hernández-Munain et al., 1996; Lauzurica et al., 1997), and a large nucleoprotein complex that assembles on and regulates the function of the TCR␣ enhancer (see Spicuglia et al., 2000) and references therein; reviewed in Grosschedl, 1995).

VI. The Two Substrate Problem

A. SYNAPSIS OF ENDOGENOUS RSSS As noted in the introduction, V(D)J recombination is inherently a two substrate reaction. This would appear to be enforced at two levels. First, doublestrand DNA cleavage by the RAG proteins to generate coding and signal ends is strongly stimulated by formation of a synaptic complex containing two RSSs (the paired complex, Fig. 3). Second, while double-strand DNA cleavage at a single RSS may occur at a low level in vivo (Steen et al., 1996), it seems unlikely that such events would lead to a useful outcome, because in such cases the recombining partners would need to find each other after one, or perhaps both, had undergone cleavage. The RAG proteins bind quite poorly to individual signal ends (Agrawal and Schatz, 1997; Hiom and Gellert, 1998), and hence cleavage at a single RSS might yield a relatively unstable complex with considerable potential to engage in aberrant rejoining events such as chromosomal translocations. Nuclear organization and chromatin structure could be expected to present important constraints on the synapsis of two RSSs. Antigen receptor loci can

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be a megabase in length, making synapsis a daunting task (Figs. 2A and 4). The folding of DNA in chromatin may overcome part of this distance problem. The nucleosome provides a seven-fold compaction of DNA, and formation of the 30-nm fiber results in a further seven-fold compaction (Wolffe, 1992). Thus, two RSSs separated by 100 kb could be (in theory) up to 30 ␮m apart in naked DNA but only 600 nm apart in the 30-nm fiber. The organization of chromatin in loops undoubtedly reduces these distances even further (Fig. 4). Such structural features that reduce inter-RSS distance may be particularly important since the nucleus is densely packed with chromatin and other cellular machinery, making bulk movement of chromatin domains an energetically unfavorable process. Recent photobleaching studies indicate that proteins have a high mobility within the nucleus (recovery from bleaching occurred within seconds). In contrast, no movement of a histone-GFP fusion protein was detected in 4 hr of observation, demonstrating the low mobility of chromatin (Kanda et al., 1998; Phair and Misteli, 2000). In cases where the recombining partners are relatively closely spaced on the DNA (as is the case for many D-to-J rearrangement events), one could imagine that random collisions mediated by diffusion suffice to bring the two elements together. For RSSs separated by many hundreds of kilobases, it seems likely that other forces facilitate synapsis. This may be as simple as the organization of chromatin into domains (Fig. 4). According to such a model, the higher-order chromatin structure would ensure that even the most distant V gene segments of a given antigen receptor locus are sufficiently close to the (D)J gene segments for relatively efficient synapsis. Alternatively, one could imagine that antigen receptor loci have developed specialized mechanisms to facilitate, and regulate, synapsis of distant elements. A particularly appealing scheme is suggested by the looping model for LCRmediated regulation of the ␤-globin locus. By analogy to the proposed direct interaction of the ␤-globin LCR with globin gene promoters, one could imagine that antigen receptor gene enhancers interact directly with promoters upstream of D or V gene segments, thereby bringing these elements into close proximity with (D)J gene segments. The ␤-globin LCR is proposed to interact with only one promoter at a time to explain the fact that individual erythrocytes initiate expression from only one gene from the ␤-globin gene cluster at a time. Similarly, antigen receptor gene enhancers might interact with a single promoter at a time, thereby helping to restrict the number of V(D)J recombination events that take place at one time. Looping could be assisted by factors able to form a physical bridge between two distant DNA sites, such as members of the structural maintenance of chromosomes (SMC) family (Jessberger et al., 1998), or sequence-specific DNA binding factors such as members of the helix-loop-helix (HLH) family of transcription factors (Kadesch, 1992). Interestingly, HLH family proteins have binding sites in both the heavy chain enhancer and V gene


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promoters (Ernst and Smale, 1995), and one member of the family, TFE3, has been shown to be able to mediate enhancer–promoter interactions (Artandi et al., 1994). Finally, as noted above, transcriptionally active loci tend to colocalize in subdomains of the nucleus, and hence the process of germline transcription may help move potential recombination partners close together. It is worth noting that a fundamental aspect of the process of synapsis remains unknown. In Fig. 3, synapsis is depicted as occurring between two RSSs, both of which are already bound by the RAG proteins. Because the stoichiometry of the paired complex has yet to be determined, it is equally possible that the reaction follows a different pathway in which the RAG proteins initially bind stably to only one of the two RSSs, and then capture a second, free RSS. It is not clear which pathway is used in vitro or in vivo, or that the two pathways need to be mutually exclusive. This issue may have implications for RAG protein occupancy of endogenous RSSs. For example, if the latter pathway is followed, then only a limited subset of RSSs need be stably loaded with RAG1 and RAG2 (for example, only the J segment RSSs during D-to-J rearrangement, and only the DJ segment RSS during V-to-DJ rearrangement). As yet, nothing is known about endogenous RSS occupancy by the RAG proteins. B. RESTRICTIONS ON PRODUCTIVE RSS INTERACTIONS What determines the efficiency with which V(D)J recombination occurs between two accessible RSSs that are in close proximity? One important determinant is clearly the sequence of the RSS itself. Few endogenous RSSs match the consensus sequence at all positions of the heptamer and nonamer (Ramsden et al., 1994), and the vast majority of variants work less well than the consensus sequence with artificial test substrates (Akamatsu et al., 1994; Hesse et al., 1989); one exception was noted by (Larijani et al., 1999). Considerable evidence supports the idea that sequence variation in the heptamer and nonamer has a significant effect on the efficiency with which endogenous gene segments undergo V(D)J recombination (Connor et al., 1995; Gauss and Lieber, 1992; Larijani et al., 1999; Nadel et al., 1998b; Ramsden and Wu, 1991; VanDyk et al., 1996). In addition, a loose consensus sequence for the spacer region has been described (Ramsden et al., 1994), and sequence variation within the spacer may influence the efficiency with which endogenous gene segments are recombined (Fanning et al., 1996; Larijani et al., 1999; Nadel et al., 1998a). Finally, it is clear that the sequence of the coding nucleotides immediately flanking the heptamer can dramatically influence the efficiency with which V(D)J recombination occurs in test substrates (Boubnov et al., 1993, 1995; Ezekiel et al., 1995, 1997; Gerstein and Lieber, 1993). While variations in the sequences of the heptamer, nonamer, and spacer may (but do not necessarily) act by influencing RSS recognition by the RAG proteins, alterations in the coding flank appear to act by influencing a postbinding step of the reaction, namely DNA nicking (Yu and Lieber, 1999).

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In addition to the sequence variations that influence how efficiently an individual RSS will be recombined, the 12/23 rule demonstrates that constraints can operate at the level of pairs of RSSs. There is, however, increasing evidence that the 12/23 rule is not the sole constraint operating at this level. Specific 12-RSS/23-RSS combinations appear to influence differential gene segment utilization, and this regulatory function may be particularly important for ensuring that appropriate rearrangements occur at the IgH and TCR␤ loci. The IgH locus is unique inasmuch as its D gene segments are flanked on both the 5′ and 3′ sides by a 12-RSS, while VH and JH gene segments are each flanked by a 23-RSS (Figs. 1 and 2A). This prevents direct V-to-J rearrangement, but it poses a new problem: while the 12/23 rule permits D-to-J rearrangement to occur by either inversion or deletion, deletion is the overwhelmingly favored outcome. How is this achieved? In transient transfection experiments with test substrates containing IgH gene segments, J RSSs rearranged more efficiently to the RSS 3′ of D than to the RSS 5′ of D (Gauss and Lieber, 1992; VanDyk et al., 1996), whereas under certain circumstances, a V RSS exhibited the opposite pattern, rearranging preferentially to the RSS 5′ of D (VanDyk et al., 1996). These artificial, extrachromosomal substrates thus recapitulate the preference observed for the endogenous locus, but the studies left unresolved what aspects of the gene segments, their RSSs, and their flanking genomic sequences were responsible for these effects. Recent studies of the murine TCR␤ locus in the Alt and Sleckman laboratories have provided novel insights into the constraints that operate beyond those of the 12/23 rule to determine how specific pairs of RSSs are used (Bassing et al., 2000; Sleckman et al., 2000). In the TCR␤ locus, direct V-to-J rearrangement is permitted by the 12/23 rule (Fig. 1), but it almost never occurs, despite the fact that prior D-to-J rearrangement almost invariably leaves some J gene segments available for V-to-J rearrangement. Using both transgenic recombination substrates (Sleckman et al., 2000) and targeted mutations of the germline (Bassing et al., 2000; Sleckman et al., 2000), these groups demonstrated that if D-to-J recombination is blocked (either by removal of the D element entirely, or mutation of the RSS 3′ of D), then very little direct V-to-J rearrangement occurs; but direct V-to-D rearrangement, in the absence of D-to-J rearrangement, occurs efficiently. Thus, the 23-RSSs of the V␤ genes appear to discriminate between different 12-RSSs, displaying a strong preference to recombine with the 12-RSS of the D␤ gene segment instead of those associated with the J␤ gene segments. What feature(s) of the D␤1 gene segment account for this remarkable targeting? By replacing the RSS adjacent to J␤1.2 (the second J segment in the first J cluster) with the RSS 5′ of D␤1, it was shown that the recombination targeting activity moved with the 5′ D␤1 RSS: that is, essentially all rearrangements in such a locus are V␤-to-J␤1.2 (Bassing et al., 2000). Therefore, in the context of the endogenous locus, the


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heptamer-spacer-nonamer sequences 5′ of D␤1 are sufficient for the recombination targeting phenomenon. Several types of explanations exist for these provocative results. First, it is possible that the 5′ D␤1 RSS is intrinsically a much better substrate for V(D)J recombination than the J␤ RSSs. Arguing against this interpretation is the observation that a number of the J␤ RSSs serve as efficient substrates for D-to-J␤ recombination (Livak et al., 2000). In addition, comparison of the heptamer/nonamer sequences of the 5′ D␤1 RSS (5′ -CACAATG-spacer-ACAAAAAAG-3′ ) (nonconsensus residues underlined) with those of the J␤ RSSs reveals no consistent features to explain the differences observed. Perhaps the presence of six consensus spacer residues in 5′ D␤ RSSs (which is more than is found in J␤ RSS spacers), is relevant (Bassing et al., 2000). A second possibility is that the 5′ D␤1 RSS works particularly well with V␤ RSSs (Bassing et al., 2000), while J␤ RSSs work well with the 3′ D␤1 RSS. Such preferential RSS compatibility would suggest mechanistic similarities between this phenomenon and that described above for Ig heavy chain gene segments. If either of these possibilities is correct, it should be possible to recapitulate the findings with artificial recombination substrates either in vivo or in vitro. A third possibility is raised by the existence of a functional TATA box in the 5′ D␤1 RSS spacer. This, or some other less obvious feature of the RSS, could serve as a binding site for a factor that facilitates loading of the RAG proteins onto this RSS (Bassing et al., 2000). Such a factor might alter local nucleosome binding or positioning. One could imagine that all TCR␤ locus rearrangements involve stable loading of the RAG proteins onto either the 3′ or the 5′ D␤ RSS, followed by capture of free J␤ or V␤ RSSs. Such a model would explain the paucity of direct V-to-J␤ rearrangements. Whatever the explanation for these results, it seems likely that some of the fundamental rules governing pairwise RSS usage have yet to be delineated. VII. Models

Despite extensive study during the past two decades, our knowledge of the molecular events that lead to RSS accessibility and synapsis remains rudimentary. To a large extent, this reflects our poor understanding of the structure and dynamics of the nucleus and its contents. Major unresolved questions include: What forces control the localization and movement of chromatin domains? Within what general chromatin structure does transcription (and V(D)J recombination) occur? How do cis-acting elements, and the factors that bind them, exert effects over long distances? While detailed models are not possible at this point, one can make reasonable inferences about the broad steps that might take place to render a segment of DNA accessible to binding by the RAG proteins. The presumed starting point in

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FIG. 5. Models of accessibility during V(D)J recombination. Three distinct pathways of events (labeled A, B, and C) are depicted. The pathways need not be mutually exclusive. See Models section of text for details.

Fig. 5 is an antigen receptor locus that is methylated, hypoacetylated, nuclease resistant and transcriptionally inert. It may also be associated with heterochromatin. A plausible first step is binding of a factor to a cis-acting element in the locus. For the IgH locus, one attractive possibility for this would be the binding of PU.1 and other Ets-family factors to the intronic enhancer. It seems likely that additional factors would subsequently bind, such as E2A-encoded proteins, leading to formation of a multicomponent complex on the regulatory region. This could then trigger histone modification (e.g., phosphorylation and acetylation) and chromatin remodeling, and subsequent local changes in chromatin structure (manifest as increased nuclease sensitivity in the locus). Note that E2Aencoded factors have been shown to interact with HATs and chromatin remodeling complexes. As a result, additional factors could bind at nearby promoters, leading to sterile transcription. Because RNA polymerase II has been shown to associate with HATs, this in turn could lead to further chromatin remodeling and histone modification. In some cases, transcription-dependent alterations in the


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locus might be essential for the nucleosome remodeling/repositioning needed for RAGs to bind nearby RSSs (pathway A of Fig. 5). An example of such a situation might be transcription from the PD␤1 element in the TCR␤ locus. Alternatively, in some instances sterile transcription might not be causally related to RSS accessibility (pathway C of Fig. 5). One can also imagine that sterile transcription plays a particularly central and early role, being crucial for histone modification and subsequent chromatin structural changes (pathway B of Fig. 5). Other types of changes found in complex loci are more difficult to place in the chain of events leading to V(D)J recombination. Movement of an antigen receptor locus away from heterochromatin might be required at a very early stage, or might not be required at all. Similarly, demethylation of the locus might in some instances be an important early event to establish an appropriate chromatin template on which other processes can occur. More likely, however, is the possibility that demethylation occurs as a consequence of the other changes in the locus, and plays an important role in the stable maintenance and propagation of the open, accessible configuration (Fig. 5). Not addressed in Fig. 5 are the issues of potential long-range effects of enhancer elements, and synapsis of two RSSs. Long-range effects may be mediated by a combination of looping and linking mechanisms. Synapsis and preferential targeting to certain RSSs within a group of accessible RSSs (as occurs for the 5′ D␤1 RSS) are not understood. It seems likely that these long-range and higher-order interactions will be some of the most difficult processes to understand mechanistically in the field of V(D)J recombination. Note Added in Proof

Numerous relevant studies have been published since the submission of this review for publication. These studies include: Cherry, S. R., Beard, C., Jaenisch, R., and Baltimore, D. (2000). V(D)J recombination is not activated by demethylation of the kappa locus. Proc. Natl. Acad. Sci. USA 97, 8467–8472. Kwon, J., Morshead, K. B., Guyon, J. R., Kingston, R. E., and Oettinger, M. A. (2000). Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA. Mol. Cell 6, 1037–1048. Mathieu, N., Hempel, W. M., Spicuglia, S., Verthuy, C., and Ferrier, P. (2000). Chromatin remodeling by the T cell receptor (TCR)-␤ gene enhancer during early T cell development: Implications for the control of TCR-␤ locus recombination. J. Exp. Med. 192, 625–636. Nitschke, L., Kestler, J., Tallone, T., Pelkonen, S., and Pelkonen, J. (2001). Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage. J. Immunol. 166, 2540–2552. Wang, Q. F., Lauring, J., and Schlissel, M. S. (2000). c-Myb binds to a sequence in the proximal region of the RAG-2 promoter and is essential for promoter activity in T-lineage cells. Mol. Cell. Biol. 20, 9203–9211. Whitehurst, C. E., Schlissel, M. S., and Chen, J. (2000). Deletion of germline promoter PD␤1 from the TCR␤ locus causes hypermethylation that impairs D␤1 recombination by multiple mechanisms. Immunity 13, 703–714.

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ACKNOWLEDGMENTS The authors are extremely grateful to a large number of individuals with whom we discussed aspects of this review, and whose ideas and helpful suggestions contributed significantly to its writing: Mark Schlissel, Richard Flavell, Frank Grosveld, Fred Alt, Pierre Ferrier, Gene Oltz, David Raulet, Alfred Lee, Nina Papavasiliou, Sebastian Fugmann, Isabelle Villey, Leon Ptaszek, Ferenc Livàk, Quinn Eastman, Penny Shockett, and other members of the Schatz lab. We thank Marjorie Oettinger, Fred Alt, and Barry Sleckman for sharing data with us prior to publication. We are very much indebted to those people who made the effort to read and comment upon the manuscript: Barry Sleckman, Michael Krangel, Alfred Lee, Isabelle Villey, Leon Ptaszek, Sebastian Fugmann, Ashley Eversole, and Nina Papavasiliou. We apologize to our colleagues whose work may not have been cited. Finally, we thank Debra Gilhuly for her assistance in tracking down innumerable references. D. G. T. H. was supported by Training Grant AI07019 from the National Institutes of Health. D. G. S. is an associate investigator of the Howard Hughes Medical Institute.

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Weill, J. C., and Reynaud, C. A. (1996). Rearrangement/hypermutation/gene conversion: When, where and why? Immunol. Today 17, 92–97. West, R. B., and Lieber, M. R. (1998). The RAG-HMG1 complex enforces the 12/23 rule of V(D)J recombination specifically at the double-hairpin formation step. Mol. Cell. Biol. 18, 6408–6415. Whitehurst, C. E., Chattopadhyay, S., and Chen, J. (1999). Control of V(D)J recombinational accessibility of the D␤1 gene segment at the TCR␤ locus by a germline promoter. Immunity 10, 313–322. Wilson, A., Held, W., and Macdonald, H. R. (1994). Two waves of recombinase gene expression in developing thymocytes. J. Exp. Med. 179, 1355–1360. Wilson, R., Kiledjian, M., Shen, C., Benezra, R., Zwollo, P., Dymecki, S. M., Desiderio, S. V., and Kadesch, T. (1991). Repression of immunoglobulin enhancers by the Helix-Loop- Helix protein Id: Implications for B-Lymphoid-cell development. Mol. Cell. Biol. 11, 6185–6191. Wolffe, A. (1992). “Chromatin: Structure and Function,” Academic Press, San Diego. Wolffe, A. P., and Hayes, J. J. (1999). Chromatin disruption and modification. Nucleic Acids Res. 27, 711–720. Xu, Y., Davidson, L., Alt, F. W., and Baltimore, D. (1996). Deletion of the Ig-kappa light chain intronic enhancer/matrix attachment region impairs but does not abolish V-kappa-J-kappa rearrangement. Immunity 4, 377–385. Yamagami, T., ten Boekel, E., Andersson, J., Rolink, A., and Melchers, F. (1999). Frequencies of multiple IgL chain gene rearrangements in single normal or kappa L chain-deficient B lineage cells. Immunity 11, 317–327. Yancopoulos, G. D., and Alt, F. W. (1985). Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell 40, 271–281. Yancopoulos, G. D., Blackwell, T. K., Suh, H., Hood, L., and Alt, F. W. (1986). Introduced T cell receptor variable region gene segments recombine in pre-B cells: Evidence that B and T cells use a common recombinase. Cell 44, 251–259. Ye, S. K., Maki, K., Kitamura, T., Sunaga, S., Akashi, K., Domen, J., Weissman, I. L., Honjo, T., and Ikuta, K. (1999). Induction of germline transcription in the TCR gamma locus by Stat5: Implications for accessibility control by the IL-7 receptor. Immunity 11, 213–223. Yi, M., Wu, P., Trevorrow, K. W., Claflin, L., and Garrard, W. T. (1999). Evidence that the Igkappa gene MAR regulates the probability of premature V-J joining and somatic hypermutation. J. Immunol. 162, 6029–6039. Yu, K. F., and Lieber, M. R. (1999). Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination. Mol. Cell. Biol. 19, 8094–8102. Yu, W., Misulovin, Z., Suh, H., Hardy, R. R., Jankovic, M., Yannoutsos, N., and Nussenzweig, M. C. (1999a). Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5′ of RAG2. Science 285, 1080–1084. Yu, W., Nagaoka, H., Jankovic, M., Misulovin, Z., Suh, H. Y., Rolink, A., Melchers, F., Meffre, E., and Nussenzweig, M. C. (1999b). Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization. Nature 400, 682–687. Zhong, X. P., Carabana, J., and Krangel, M. S. (1999). Flanking nuclear matrix attachment regions synergize with the T cell receptor delta enhancer to promote V(D)J recombination. Proc. Natl. Acad. Sci. USA 96, 11,970–11,975. Zhong, X. P., and Krangel, M. S. (1997). An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. Proc. Natl. Acad. Sci. USA 94, 5219–5224. Zhuang, Y., Barndt, R. J., Pan, L., Kelley, R., and Dai, M. (1998). Functional replacement of the mouse E2A gene with a human HEB cDNA. Mol. Cell. Biol. 18, 3340–3349. Zhuang, Y., Soriano, P., and Weintraub, H. (1994). The helix-loop-helix gene E2A is required for B cell formation. Cell 79, 875–884.


T Cell Effector Subsets: Extending the Th1/Th2 Paradigm TATYANA CHTANOVA AND CHARLES R. MACKAY* Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia

I. Introduction

T cells are at the heart of the adaptive immune response, and facilitate a number of immunological processes. These include help for antibody production, cytotoxicity, antiviral and antitumor responses, and defense against large extracellular parasites. Despite the fundamental role of effector T cells in all of these processes, the distinction between the different functional subsets has been unclear. The relationship between effector cells and memory cells has also remained elusive. However, two avenues of research have recently allowed the distinction of various subsets of effector T cells. The first of these has been differential gene screening, using techniques such as subtractive polymerase chain reaction (PCR) and more recently, DNA microarrays. The second avenue of research has been the enormous effort directed toward understanding chemokine biology. This effort was given a significant boost with the discovery that chemokine receptors serve as human immunodeficiency virus (HIV) coreceptors. Recently, immunologists have come to appreciate that virtually all leukocytes rely on cell movement for their immunological role, and chemokine receptors often provide the clearest definition of a cell at a particular functional stage of its life. This review will outline the developments over the past several years that have helped to define and characterize the various effector T cell responses. II. T Cell Effector Subsets

T cell maturation and selection occurs in the thymus, and thereafter na¨ıve T cells leave the thymus and begin recirculating from blood to organized lymphoid tissues (Mackay, 1993; Butcher and Picker, 1996). T cell receptor (TCR) engagement together with the appropriate costimulating signals leads to activation and expansion of antigen-specific clones. At some point, effector T cells develop with the capacity for cytotoxicity and /or cytokine secretion. The pathway for effector T cell differentiation is unclear; the simplest model has na¨ıve T cells transforming to activated T cells, then to effector T cells. Memory cells probably derive from activated or effector T cells, although other permutations have been suggested ∗ To

whom correspondence should be addressed. Phone: 61-2-92958405; fax: 61-2-92958404; E-mail: [email protected]. 233 C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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FIG. 1. Effector T cell subsets. Several distinct effector T cell subsets mediate a number of different protective and pathogenic immune responses. The precise functions of some T cell subsets still remain to be determined.

(Sallusto et al., 1999). Besides the well-characterized CD8+ cytotoxic T cells and Th1 and Th2 subsets, other subsets exist including natural killer (NK) T cells and ␥ ␦ T cells (Fig. 1). The significance of these subsets, particularly ␥ ␦ T cells, remains uncertain (Hayday, 2000) and they will be discussed only briefly in this review. A. THE TYPE 1 AND TYPE 2 PARADIGM: Th1 AND Th2 CELLS Two distinct patterns of cytokine production for CD4+ effector T cells were originally defined by Mosmann et al. (1986). Th1 cells producing interleukin 2 (IL-2), interferon ␥ (IFN-␥ ), and LT-␣ function in the defense against intracellular pathogens such as viruses, which typically involves close cooperation with phagocytic macrophages and neutrophils. Conversely, Th2 cells secreting IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 do two things. They enhance B cell-mediated humoral immunity, and they also promote defense against large extracellular pathogens (Abbas et al., 1996). The role of Th2 cells in humoral responses has

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probably been overstated, since IFN-␥ induces switching of B cells to several IgG isotypes and thus plays a role in antibody-mediated immunity (Snapper and Paul, 1987; Finkelman et al. 1988a; Snapper et al., 1988; Stevens et al., 1988). As discussed below, it is likely that B cell help can also be delivered by a non-Th2 or -Th1 effector T cell. It is important to note that T cells that simultaneously produce both Th1 and Th2 cytokines also exist in humans and mice (Sher et al., 1992; Abbas et al., 1996; O’Garra, 1998). This type of cell is usually referred to as Th0. The relevance of Th0 cells is still uncertain, although they appear to be precursors of polarized Th1 or Th2 subsets (Kamogawa et al., 1993). Finally, cytokine production by Th1 or Th2 cells can be quite heterogeneous. For instance, polarization is usually assessed at the population level, but at the single cell level there is considerable heterogeneity (Kelso et al., 1999). Not all Th2 cytokines are expressed in single Th2 cells, suggesting independent regulation of cytokine gene expression. The functional role of Th1 and Th2 cells can largely be defined based on the cytokines they produce. For instance, IFN-␥ (the hallmark Th1 cytokine) activates macrophages and stimulates production of IgG, which in turn enhances phagocytosis (Boehm et al., 1997). IL-2 and IFN-␥ promote differentiation and activation of CD8+ effector T cells. Th1 cytokines also promote recruitment and activation of inflammatory cells, and are associated with inflammatory reactions and tissue injury (Abbas et al., 1996). The cytokines produced by each type of T cell in turn promote polarization of cells of the same phenotype and inhibit the opposing subset. For instance, IFN-␥ induces macrophages to produce IL-12 which in turn promotes Th1 responses, and also directly inhibits the Th2 response (Maggi et al., 1992; Boehm et al., 1997). The cytokines produced by Th2 cells have a different role. IL-4, the signature Th2 cytokine, acts on a range of cells. It stimulates proliferation of Th2 cells while suppressing Th1 polarization (Hsieh et al., 1992; Seder et al., 1992b). Another very important effect of IL-4 is to induce isotype switching of B cells to IgE production (Coffman et al., 1986; Finkelman et al., 1988b; Gascan et al., 1992). IL-5 is a cytokine that promotes eosinophil production and mobilization from the bone marrow (Kopf et al., 1995). The function of eosinophils is to blast large extracellular parasites such helminths. Allergic reactions and the presence of eosinophils in tissues such as the airways in asthma probably represent an anomaly of this antiparasitic defense mechanism. Likewise, IL-4 and IL-13 are designed for antiparasitic defense. These cytokines have a variety of effects on numerous cell types: IL-13 stimulates mucus production by epithelial cells and contraction of smooth muscle (Grunig et al., 1998; Wills-Karp et al., 1998). Both IL-13 and IL-4 induce chemokine production (particularly eotaxin) by epithelial cells (Li et al., 1999; Stellato et al., 1999; Fujisawa et al., 2000), and adhesion molecule expression by endothelial cells (Thornhill et al., 1991; Bochner et al., 1995; Wills-Karp et al., 1998; Woltmann et al., 2000). Many of these actions are

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consistent with the role of Th2 cells in anti-infectious inflammatory responses, particularly to extracellular parasites, and probably aid in physical clearance of the parasite. In addition to their protective role, these two types of effector T cells participate in several pathological responses. Autoreactive CD4+ T cells (such as those in multiple sclerosis or type 1 diabetes) have Th1 phenotype and function (O’Garra et al., 1997), while the T cells in the airways of asthmatics have Th2 phenotype and function (Wills-Karp, 1999). In allergic asthma, allergens somehow simulate parasitic infection and promote Th2 cell development and functional effects. In autoimune disease, a breakdown of tolerance by CD4+ or CD8+ T cells gives rise to a delayed type hypersensitivity (DTH) type reaction that ultimately leads to antibody/complement or cellular destruction of the target organ. B. Tc1 AND Tc2 Cytokine production was traditionally considered the sole domain of CD4+ T helper cells. However, type 1 and type 2 cytokine producing subsets have been identified for CD8+ effector T cells, and these T cells produce cytokines while retaining their cytolytic capability (Paliard et al., 1988; Seder et al., 1992a; Sad et al., 1995b). CD8+ T cells differentiate to type 1 cytokine producing cells by default, whereas the type 2 pattern is induced through stimulation by IL-4 (Fong and Mosmann, 1990; Croft et al., 1994). Tc1 and Tc2 cells are more than a test tube curiosity, since the phenotypes are stable in vivo after adoptive transfer (Cerwenka et al., 1998) and have been isolated from patients (Maggi et al., 1994; Paganelli et al., 1995). Some studies suggest that Tc2 cells might provide help for B cells (Maggi et al., 1994; Cronin et al., 1995; Paganelli et al., 1995), although other studies showed Tc1 and Tc2 cells to be inefficient in providing cognate help; Tc2 cells could, however, provide noncognate help via expression of cytokines and cell surface molecules (Sad et al., 1995a, 1997). CD8+ T cells have been shown to play a pivotal role in diabetes in nonobesediabetic (NOD) mice (Dilts et al., 1999; Wong and Janeway, 1999). It is widely accepted that both CD4+ and CD8+ T cells are required for the development of autoimmune diabetes (Shizuru et al., 1988; Serreze et al., 1994; Sumida et al., 1994; Wicker et al., 1994; Wang et al., 1996; Wong et al., 1998). However, several studies have isolated CD8+ T cell clones, which can mediate insulin dependent diabetes mellitus (IDDM) development independently of CD4+ cells (Wong et al., 1996; Graser et al., 2000). Although it is not clear yet whether it is Tc1 or Tc2 cells that are involved in IDDM, CD8+ T cells play an important role in this disease. Tc1 and Tc2 cells also play a role in antitumor immunity and tumor regression (Dobrzanski et al.,1999, 2000). CD8+ T cells may also provide some explanation for the correlation between virus infections of the lung and asthma, observed in several studies (Busse,


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1990; Cypcar et al., 1992; Schwarze et al., 1997; Folkerts et al., 1998; Matsuse et al., 2000). CD8+ T cells are an important component of the host response to virus infection. They have also been shown to be essential for the development of respiratory syncytial virus (RSV)-induced lung eosinophilia and airway hyperreactivity (Schwarze et al., 1999). In a mouse model of virus peptide-stimulated CD8+ T cell immune responses in the lung, bystander Th2 immune responses to ovalbumin switched the virus peptide-specific CD8+ T cells in the lung to IL-5 production (Coyle et al., 1995). When these IL-5-producing CD8+ T cells were challenged via the airways with virus peptide, a significant eosinophil infiltration was induced (Coyle et al., 1996). Nevertheless, the role of viral infections and CD8+ T cells in asthma remains uncertain. Several studies suggest that some infections may exert a protective effect by inducing Th1 and inhibiting Th2 responses, and CD8+ cells may also directly inhibit Th2 responses (Hussell et al., 1997; Hansen et al., 2000). In addition, Tc1 cells were shown to provide superior protection against viral infections, which could be due to differential expression of chemokine receptors and adhesion molecules by the two subsets (Cerwenka et al., 1999a, 1999b; Wirth et al., 2000). In addition to the well-studied CD4+ and CD8+ effector subsets, other subsets with more obscure roles exist. Following is a brief description of two such effector T cell subsets, although the review will mainly focus on the conventional CD4+ and CD8+ effector subsets. This review will not discuss cytolysis by CD8+ cells or other T cells, and readers are referred elsewhere for such discussion (Harty et al., 2000). C. NK1 T CELLS NK1 T cells constitute a specialized subset of ␣␤ T cells that coexpress markers usually associated with the natural killer (NK) lineage such as NK1.1. These T cells can secrete large amounts of cytokines upon primary stimulation. Most NK1 T cells are CD4+ or CD4−CD8− cells, with an activated phenotype (Bendelac, 1995; Bendelac et al., 1997; Hammond et al., 1999; Emoto et al., 2000). These T cells express mostly an invariant TCR (consisting of an invariant ␣ chain and a restricted set of ␤ chains, Lantz and Bendelac, 1994) and recognize a major histocompatibility complex (MHC) class IB molecule, CD1 (Bendelac et al., 1995). NK1 T cells are likely to play an important immunoregulatory role because they are capable of secreting large amounts of IL-4 within minutes of primary activation (Yoshimoto and Paul, 1994; Yoshimoto et al., 1995b). Thus, NK T cells may regulate the Th1/Th2 balance, but their precise role remains unclear. For instance, normal Th2 responses can develop without the involvement of NK1 T cells (Brown et al., 1996a; Guery et al., 1996; Zhang et al., 1996; Chen et al., 1997; Smiley et al., 1997). Furthermore, upon primary stimulation, NK1.1 cells have been shown to secrete Th2 cytokines such as IL-4, IL-5, and IL-10, as well as the Th1 cytokines IFN-␥ and TNF-␤ (Chen and Paul, 1997).

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In addition to the regulation of Th1 and Th2 responses, several other functions have been attributed to NK1 T cells. They can induce slow Fas-mediated killing (Arase et al., 1994), and several studies have linked NK1 T cells to autoimmune diseases (Yoshimoto et al., 1995a; Gombert et al., 1996; Hammond et al., 1998; Lehuen et al., 1998; Wilson et al., 1998). There is also evidence to suggest that NK1 T cells can provide help to effector cells such as CD8+ T cells (Denkers et al., 1996) or B cells in an antigen nonspecific manner (Bendelac et al., 1997). The precise role of this subset in the immune responses still remains to be defined. D. ␥ ␦ T CELLS Most T cells in humans and mice express the ␣␤ TCR, and only a minor subset expresses the ␥ ␦ TCR. However, in many other species, particularly ruminants and birds, a major proportion of T cells express ␥ ␦ receptors, especially around birth (Cooper et al., 1991; Hein and Mackay, 1991). In mice (and to a lesser extent humans), ␥ ␦ T cells represent less than 5% of peripheral blood and lymphoid organ T cells, although they are more prominent in skin and mucosal epithelia, especially in mice where they are the major T cell population (Haas et al., 1993; Havran and Boismenu, 1994; Kaufmann, 1996; Salerno and Dieli, 1998; Born et al., 1999; Hayday, 2000). One fundamental difference between ␣␤ T cells and ␥ ␦ T cells lies in their different antigen specificities. ␣␤ T cells recognize antigenic peptides when they are presented in association with the appropriate MHC, whereas ␥ ␦ T cells recognize antigens directly (Haas et al., 1993; Havran and Boismenu, 1994). The precise role of these cells in the immune response is still not clear. ␥ ␦ T cells seem to be able to perform every action that has been described for ␣␤ T cells. They can secrete cytokines, and both Th1- and Th2-like patterns of cytokine production have been noted, although the Th1 pattern is more common (Ferrick et al., 1995). In addition, ␥ ␦ T cells have cytolytic capability; they can provide help to B cells; and they have been portrayed as growth factor-producing cells. Experiments with mice lacking ␥ ␦ T cells have shown that this subset is required for some infections, but not others. In mice at least, their unique positioning in the epithelium and their array of antigenic specificities suggests an important role for protection against bacterial infections (Hayday, 2000). E. T HELPER CELLS FOR ANTIBODY PRODUCTION The precise role of Th1 or Th2 cells as helper cells for antibody production has been unclear, although this function has often been ascribed to the Th2 subset (Coffman et al., 1988; Abbas et al., 1996). Recent evidence suggests that a third subset of effector T cells probably provides this function. Two research groups identified a subset of follicular homing T cells that were able to provide help to B cells for antibody production (Breitfeld et al., 2000; Schaerli et al.,


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2000). These T cells have been dubbed follicular B-helper T cells (TFH). TFH are particularly prominent in lymphoid tissue (tonsils) and express the chemokine receptor CXCR5 (see below). These CXCR5+ tonsilar T cells express the costimulatory molecule ICOS (discussed below) and are mainly CCR7− and CD69+, a phenotype consistent with recent activation and effector function. The analysis of the cytokine profile of these cells revealed an absence of Th1 or Th2 cytokines (although a proportion did produce IL-2). Whether these T cells produce a B cell helper cytokine is not yet known, although it is conceivable that they deliver help to B cells purely through cell–cell contact and activation through ICOS and other surface molecules. The involvement of CXCR5+ TFH cells in B cell responses was also suggested by their localization in the mantle and light zone of germinal centers. In contrast to tonsilar TFH cells, the phenotype of circulating blood CXCR5+ T cells was different and indicative of a more resting phenotype, possibly corresponding to memory function. These circulating CXCR5+ T cells were CCR7+ and mostly L-selectin+, but expressed little or no activation or costimulatory molecules such as ICOS, CD69, or MHCII. III. What Determines Effector T Cell Differentiation?

A number of factors influence the direction of effector T cell differentiation. These include the nature of the pathogen, the dose of antigen and route of entry, and the genetic background of the host (Bretscher et al., 1992; Constant et al., 1995; Hosken et al., 1995; Guler et al., 1996a; Constant and Bottomly, 1997; Ruedl et al., 2000). These factors combine to produce what ultimately determines the direction of T cell polarization—the cytokine environment during T cell activation. IL-12 is critical to Th1 development. It is produced mostly by phagocytic cells in response to intracellular parasites, such as viruses and some bacteria, and drives Th1 polarization (Hsieh et al., 1993; Macatonia et al., 1995; Trinchieri, 1995; Gately et al., 1998). IL-12 also induces NK cells to produce IFN-␥ (Macatonia et al., 1993; Flesch et al., 1995). Some macrophages and dendritic cells contain bioactive IL-12 that is preformed and membrane associated, ready for release within minutes after in vitro or in vivo contact with intracellular pathogens (Quinones et al., 2000). Consequently, factors that prime macrophages or dendritic cells to store IL-12, and the encounter (or not) of T cells with these IL-12-laden cells, will affect the direction of the T cell response. IL-4 is required for Th2 responses (Kopf et al., 1993). Several cell types are known to produce IL-4, depending on the nature of the infectious agents or the mode of immunization (Holland et al., 2000). As mentioned above, CD4+ T cells bearing NK surface markers have also been shown to release large quantities of IL-4 shortly after activation (Yoshimoto and Paul, 1994). Excretory-secretory proteins of the parasitic helminth Nippostronglyus brasiliensis somehow naturally induce a type 2 response from specific T helper cells, and can also drive

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Th2 responses to bystander antigens introduced at the same time of infection (Holland et al., 2000). Cholera toxin is a potent mucosal adjuvant that stimulates predominantly a Th2-type response. Cholera toxin induces phenotypic and functional maturation of blood monocyte-derived dendritic cells (DC) to a cell type that is “licensed” to prime na¨ıve T cells and polarize them toward the Th2 phenotype (Gagliardi et al., 2000). Although IL-12 and IL-4 are considered to be the critical determinants of T cell differentiation, other cytokines can contribute (O’Garra, 1998; Murphy et al., 2000). For example, cytokines such as IL-1 and IL-18 have been shown to influence the direction of polarization. IL-1␣ was found to act as a cofactor for IL-12-induced Th1 development in some strains of mice (Shibuya et al., 1998). IL-18 [initially described as interferon gamma inducing factor (IGIF)] is a proinflammatory cytokine that has been found to augment the cytotoxicity of NK cells and induce proliferation of T cells (Okamura et al., 1995). It also acts in synergy with IL-12 to stimulate Th1 cells to produce IFN-␥ (Micallef et al., 1996). This was demonstrated by crossing IL-18 deficient mice with IL-12 deficient mice, which resulted in a much more severe deficiency in IFN-␥ production than in either strain separately (Takeda et al., 1998). IL-18 knockout mice have reduced NK cell activity and Th1 responses, including IFN-␥ production in response to Propionibacterium acnes and BCG (Takeda et al., 1998). The full extent of the action of IL-18 remains unclear, as IL-18 also seems to be an inducer of several Th2 cytokines and IgE expression by B cells (Micallef et al., 1996; Hoshino et al., 1999c, 2000). Antigen dose may also influence the balance between Th1 and Th2, although the exact effect of antigen dose on T cell polarization remains unclear (Constant and Bottomly, 1997; Ruedl et al., 2000). In some studies, it was found that low antigen concentrations and low-dose infections favor Th1 responses while higher doses induce type 2 polarization (Bretscher et al., 1992, Hosken et al., 1995). However, in other studies, the opposite response was noted (Pfeiffer et al., 1995; Ruedl et al., 2000). This probably indicates that other overriding factors in addition to antigen dose affect the direction of T cell polarization. The development of both subsets of T cells is also dependent on costimulatory signals provided by interaction of receptors CD28 and CTLA-4 with their ligands B7-1 and B7-2 on antigen-presenting cells (APCs) (Corry et al., 1994). Furthermore, the differences in the resistance or susceptibility to infections between different strains of inbred mice serve as evidence that genetic background of the host can influence the direction of T cell polarization (Hsieh et al., 1995; Guler et al., 1996b, 1997). IV. Transcription Factors for T Cell Differentiation

Several transcription factors have been identified that play a role in Th1 or Th2 differentiation or function (Ho et al., 1996; Rincon and Flavell, 1997; Kuo and Leiden, 1999; Dong and Flavell, 2000). Most of these factors were identified


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by substractive PCR or similar approaches. Stat6, GATA-3, c-Maf, NFATc, AP-1, and NF-IL6 all appear to positively control Th2 responses (Zheng and Flavell, 1997). In contrast, activation of Stat4, T-bet, IRF-1, and NFATp leads to polarized Th1 responses (Glimcher and Singh, 1999; Murphy et al., 2000). Much has been gleaned from studies with mice deficient in each factor. Mice lacking Stat6 or Stat4 are unable to mount Th2 and Th1 responses, respectively (Kaplan et al., 1996a, b; Shimoda et al., 1996; Takeda et al., 1996; Thierfelder et al., 1996). Stat6 is activated in response to IL-4 and was shown to play an important role in regulating Th2 development and IL-4 mediated responses. Stat4 was shown to have an essential role in IL-12 mediated Th1 responses. There is some evidence that Stat4 plays different roles in CD4+ and CD8+ T cells, since Stat4 exerts different effects on regulation of IFN-␥ expression in the two subsets. While both lineages require Stat4 for IL-12/IL-18-dependent IFN-␥ induction, only CD4+ T cells require Stat4 for IFN-␥ induction via the TCR pathway, whereas CD8+ T cells can produce IFN-␥ in response to antigen independently of Stat4 (Carter and Murphy, 1999). GATA-3 deficient mice are embryonic lethal (Pandolfi et al., 1995); however, an alternative approach has provided insight into the gene’s function. Inhibition of GATA-3 activity in transgenic mice expressing a dominant-negative mutant of GATA-3 led to a reduction of the Th2 cytokines IL-4, IL-5, and IL-13. It also led to attenuation of Th2 responses, including airway eosinophilia, mucus production, and IgE synthesis, highlighting the therapeutic potential of targeting GATA-3 (Zhang et al., 1999). Furthermore, recent studies have shown that expression of GATA-3 in fully committed Th1 clones caused them to produce IL-4 and IL-5, and in Stat6-deficient cells, expression of GATA-3 could rescue Th2 development (Ouyang et al., 2000). Another transcription factor that is involved in Th2 polarization is c-maf. This transcription factor binds to the IL-4 promoter and is able to induce IL-4 expression in non-T cells (Ho et al., 1996). Mouse strains deficient in this gene have a severe impairment in the expression of IL-4 in Th2 and in NK1.1 T cells but normal levels of other Th2 cytokines (Kim et al., 1999). The NFAT family of transcription factors also plays an important role in T cell polarization. These transcription factors bind to DNA complexed to other factors such as AP-1 (Rao et al., 1997). NFAT binding sites in the IL-4 promoter are essential for promoter activity (Rooney et al., 1995). Both NFATc and NFATp bind to the IL-4 promoter and activate transcription in cell lines; however, studies in knockout mice showed that they play opposing roles in Th2 differentiation. NFATc knockout mice showed defective Th2 differentiation, while NFATp showed an exaggerated Th2 response (Hodge et al., 1996; Ranger et al., 1998; Yoshida et al., 1998). In contrast to Th2-specific transcription factors, less is known about factors specific to Th1 polarization. Recently, a new Th1-specific transcription factor, T-bet, was identified that seems to control IFN-␥ production in Th1 cells

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(Szabo et al., 2000). Remarkably, in addition to inducing IFN-␥ production in Th1 cells, both developing and committed Th2 cells can be converted to produce IFN-␥ by this factor. T-bet seems to repress the Th2 program and enhance the Th1 program. Other important transcription factors for Th1 differentiation are interferon regulatory factor (IRF)-1 and IRF-2 (Lohoff et al., 2000). In mice deficient in IRF-1, both macrophages and CD4+ T cells are impaired in terms of Th1 differentiation. These mice lack functional NK cells and consequently are incapable of producing IFN-␥ upon administration of IL-12 in vivo, suggesting that IRF-1 operates in multiple cell types to affect Th1 differentiation. IRF-2 was originally described as an antagonist of IRF-1 mediated transcriptional regulation. IRF-2 deficient mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation (Lohoff et al., 2000). NK cell development is compromised in both IRF-1(-/)- and IRF-2(-/)- mice, but the underlying mechanisms differ. NK (but not NK T) cell numbers are decreased in IRF-2(-/)- mice, and the NK cells that are present are immature in phenotype. Mechanistically, IRF-2 may act as a functional agonist rather than antagonist of IRF-1 for some, but not all, IFN-stimulated regulatory element (ISRE)-responsive genes. Although some of these transcription factors (e.g., GATA-3 and c-Maf) are selectively expressed only in Th1 or Th2 cells, other transcription factors are present in both cell types but mediate transcription in only one cell type. Thus, the upstream signaling pathways coupled to the activation or repression of transcription factors may be differentially regulated. Signaling pathways are important in mediation of T cell differentiation. As mentioned before, STAT4 and STAT6 play crucial roles in T helper differentiation. ERK-mitogen activated protein kinase pathway was shown to be required for Th2 cell differentiation. It enhances IL-4 induced STAT6 and IL-4 receptor phosphorylation (Yamashita et al., 1999). In addition, the p38 MAP kinase pathway is required for induced expression of IFN-␥ in effector Th1 cells but does not affect Th1 cell differentiation (Rincon et al., 1998). The JNK2 signaling pathway is also required for the early initiation of the differentiation of precursor CD4+ T cells into effector Th1 cells (Yang et al., 1998). On the other hand mice deficient in JNK1 show an enhanced Th2 response (Dong et al., 1998). V. The Link between Chemokine Receptors and T Cell Effector Function

T cells migrate continuously throughout the body, to facilitate immune protection of tissues. The migration patterns of T cells can be highly specialized and complex, reflecting a need to place the right functional T cells in the right tissue. This requires the coordinated action of numerous cell migration molecules. For instance, there are greater than 20 adhesion pairs, and 50 or more chemokines (Rossi and Zlotnik, 2000; Zlotnik and Yoshie, 2000), which together allow enormous combinatorial diversity for the control of cell migration. Most


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T cells circulate through the body by passing from blood to lymph nodes via high endothelial venules (HEV). A period of time in lymphoid tissue and lymphatics sees them returned to the blood to continue the process. A minority of lymphocytes circulate through peripheral tissues, by crossing so-called “flat” endothelium or inflamed endothelium. It is predominantly L-selectinhi na¨ıve T cells that cross HEV (Mackay, 1993), although a subset of memory T cells also crosses lymph node HEV, particularly in inflamed lymph nodes (Mackay et al., 1992). The T cells that enter via the afferent lymphatics are entirely memory phenotype (CD45RO), and the majority of these are probably effector-memory type T cells. Chemoattractants stimulate cell movement by signaling through G-protein coupled seven transmembrane spanning receptors (7TMR). Chemokines constitute the majority of chemoattractants; however, other diverse molecules also bind 7TMR and mediate chemotaxis, such as C5a, fmlp, serum amyloid A, and lipid metabolites (leukotriene B4). T and B cells tend to use chemokines over other types of chemoattractants, probably because adaptive immune responses rely on these molecules, whereas innate and early responses rely predominantly on other chemoattractants. The 50 or more chemokines identified to date can be classified according to the configuration of cysteine residues near the N-terminus into four major families: CC, CXC, C, and CX3C (Rossi and Zlotnik, 2000; Zlotnik and Yoshie, 2000). An extensive database has been compiled on most of the chemokines and receptors that outlines information such as their discovery, functional properties, and disease relevance (The Cytokine Reference, http://www.psynix.co.uk/cytweb/). Chemoattractant receptors have an important bearing on the migration pathway of T cells. As leukocytes roll along blood vessel endothelium, certain chemoattractant receptors signal a change in integrin conformation and affinity, which leads to firm arrest of the cell (Butcher, 1991; Springer, 1994). Other chemoattractants operate for subsequent steps, such as guiding leukocytes through the tissue interstitium. The importance of chemoattractant receptors versus adhesion molecules for directed migration varies, although for most leukocytes, specificity is determined combinatorially (Butcher, 1991; Springer, 1994). This may be why there are so many of them. Blocking chemokines or receptors has proved to be surprisingly effective for inhibiting cell migration, demonstrating the obligatory role these molecules play in cell movement. This may explain why a number of pathogens (as well as pharmaceutical companies) have targeted chemoattractant receptors (Lalani et al., 2000). Knowledge of the precise expression of many of the chemoattractant receptors is now emerging, with the development of specific mAbs. Some of the receptors appear to be important for particular types of immune responses, or for particular subsets of leukocytes. One subdivision that has emerged is “inflammatory” chemokines and receptors, and “lymphoid” or “constitutive” chemokines and receptors (Yoshie et al., 1997; Sallusto et al., 1998a). Most chemokines are

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the inflammatory type, which are induced in inflamed tissues by inflammatory cytokines such as IL-1 or IL-4. Indeed, our studies using Affymetrix gene microarrays show that inflammatory chemokines are the most actively transcribed genes in fibroblasts following TNF or IL-1 stimulation. Inflammatory responses involve the participation of Th1 or Th2 effector T cells and leukocytes such as neutrophils, eosinophils, and macrophages. In contrast, lymphoid responses typically involve na¨ıve T and B cells, DCs, or TFH. The chemokine receptor on T cells and B cells that facilitates lymphocyte homing to lymph nodes via HEV is CCR7, although other receptors may operate for inflamed nodes. The probable chemokines for signaling lymphocyte arrest on HEV are SLC and MIP-3␤. SLC is expressed by HEV, and induces integrin activation and firm arrest of blood T cells under flow conditions (Tangemann et al. 1998). T cells and DCs localize to the T cell zone. Antigen-specific activation leads to the development of activated T cells, and subsequently effector T cells, although the distinction between the two is ill defined. Activated T cells lose CCR7 and gain other receptors. Several groups have reported the induction of a variety of chemokine receptors on Th1, Th2, or TFH cells (Gerber et al., 1997; Sallusto et al., 1997, 1998a,b; Bonecchi et al., 1998; Breitfeld et al., 2000; Schaerli et al., 2000). A. CHEMOKINES AND THEIR RECEPTORS FOR Th1 RESPONSES CCR5 and CXCR3 are the receptors expressed most abundantly on Th1 cells (Bonecchi et al., 1998; Sallusto et al., 1998b); however, these receptors are by no means strict markers, since they are weakly expressed by some Th2 and TFH cells, and indeed some Th2 clones can even express high levels of CCR5. Interestingly, the CXCR3 ligands such as IP-10, Mig, and ITAC are IFN-␥ inducible, thus Th1 cells may promote further recruitment of Th1 cells through upregulation of these chemokines by IFN-␥ . Analysis of T cells in various inflammatory tissues confirms the CXCR3/CCR5 association with Th1 responses. In rheumatoid arthritis (considered Th1 and possibly autoimmune), virtually all of the synovial fluid T cells express CCR5 and CXCR3, in comparison to only 5–15% of T cells in blood (Qin et al., 1998). Expression of CCR5 and CXCR3 also correlates with disease activity in multiple sclerosis (MS, a disease where increased IFN-␥ precedes clinical attacks). In one study, CXCR3+ T cells were increased in the blood of relapsing-remitting MS patients, and both CCR5+ and CXCR3+ T cells increased in progressive MS compared with controls (Balashov et al., 1999). MIP-1␣ was strongly expressed by microglia/macrophages in the brains of MS patients, and IP-10 was expressed by astrocytes in MS lesions but not unaffected white matter of control or MS subjects. The same conclusions were also reached in another study, where cerebrospinal fluid T cells in MS patients were significantly enriched for cells expressing CXCR3 or CCR5, compared with blood T cells (Sorensen et al., 1999). IP-10 has also been shown to be essential for


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the development of a protective Th1 response against viral infection of the CNS (Liu et al., 2000). In type 1 diabetes, expression of CCR5 and its ligands may have an important influence on the course of the disease. In NOD mice, an elevated ratio of MIP-1␣:MIP-1␤ in the pancreas correlated with destructive insulitis and progression to diabetes. T cells infiltrating the islets were CCR5+. NOD × MIP-1␣ -/- mice have reduced destructive insulitis and are protected from diabetes, and neutralization of MIP-1␣ with antibodies delays the onset of diabetes (Cameron et al., 2000). The preferential migration of Th1 and Th2 cells to particular tissues and inflammatory sites also relies on adhesion molecules. Th1 cells but not Th2 cells are able to bind to P- and E-selectin (Austrup et al., 1997; Syrbe et al., 1999). In mouse models, Th1 cells and not Th2 cells entered the inflamed sites of Th1based diseases, such as sensitized skin or arthritic joints. The immigration of Th1 cells into inflamed skin could be blocked by antibodies against P- and E-selectin. Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is also expressed on Th2 cells; however, Th2 expressed PSGL-1 does not support binding to P-selectin (Borges et al., 1997). B. CHEMOATTRACTANTS AND RECEPTORS FOR Th2 RESPONSES Stimulation of epithelial cells, endothelial cells, and fibroblasts with inflammatory mediators such as TNF, IL-1, or IFN-␥ produces a dramatic upregulation of chemokine transcription (Luster et al., 1985). Indeed, chemokines are perhaps the most actively transcribed inflammatory genes. For instance, IP-10 expression is induced in various cells by IFN-␥ , and is expressed abundantly in Th1 lesions, such as DTH responses in skin (Kaplan et al., 1987), and in experimental allergic encephalomyelitis (Ransohoff et al., 1993). However, conspicuous by their absence from many Th1 type tissues are eotaxin and other Th2 related chemokines. Rather, these chemokines are regulated by IL-13 and IL-4 (Li et al., 1999). A likely scenario is that IL-4 and IL-13 producing T cells localize in tissues in response to allergic inflammation or parasite infection and induce epithelial and endothelial cells to produce eotaxin and related chemokines (Ponath et al., 1996; Ying et al., 1997a). This in turn results in the recruitment of eosinophils and other allergic leukocytes. In our studies, we have been unable to detect eotaxin expression in the Th1 lesions we have examined. To date, the best-characterized chemoattractant receptor for allergic or antiparasitic responses is the eotaxin receptor, CCR3. CCR3 is expressed on essentially all the leukocyte types associated with allergic inflammation, notably Th2 cells, eosinophils, basophils, and mast cells (Gerber et al., 1997; Sallusto et al., 1997; Uguccioni et al., 1997). An analysis of T cells in Th2 type inflammatory lesions has demonstrated the presence of CCR3+ Th2 cells, as well as CCR3+ eosinophils and basophils (Gerber et al., 1997). The presence of CCR3+ leukocytes in allergic inflammation also correlates with the production

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of eotaxin at these sites (Ying et al., 1997b). Nevertheless, CCR3 is not an ideal marker, since it is absent from many Th2 cells, and is expressed at relatively low levels. CCR4 is another receptor that is preferentially upregulated on Th2 cells, at least under in vitro polarizing conditions (Bonecchi et al., 1998; Sallusto 1998a; Randolph et al., 1999). However, this receptor is also expressed on other T cells, including skin homing CLA+ Th1 and Th2 cells (Campbell et al., 1999), as well as recently activated Th1 cells. Presently, CCR4 cannot be considered a Th2 marker, and there is no clear connection between its expression on skin homing cells and Th2 cells. CCR8 (a receptor for the chemokine I309) is preferentially expressed on Th2 cells, especially following TCR and CD28 engagement (D’Ambrosio et al., 1998; Sozzani et al., 1998). The ligand of CCR8, I309, is preferentially produced by type 2 T cells (unpublished). Perhaps the clearest marker for human Th2 cells is the orphan chemoattractant receptor CRTh2 (Nagata et al., 1999b), which marks about 3–6% of T cells in human blood. CRTh2 is also expressed by basophils and eosinophils (Nagata et al., 1999a). It must be stressed that many of the receptors characterized as Th2 markers are in fact expressed by other leukocytes, including in some instances Th1 or Th0 cells (i.e., CCR4). Likewise, CCR5 and CXCR3 are expressed by some Th2 cells, although usually at much lower levels than Th1 cells. C. CHEMOKINES AS MODULATORS OF T CELL POLARIZATION Chemokines themselves affect Th1 and Th2 cytokine secretion, and can influence the Th1/Th2 polarization process. This is best illustrated in MCP-1 deficient mice, which are unable to mount Th2 responses (Gu et al., 2000). MCP-1 deficient mice are unable to produce T cells that secrete appreciable levels of IL-4, IL-5, or IL-10, although IFN-␥ and IL-2 production are unaffected. The MCP-1 deficient mice also fail to accomplish Ig class switching and are resistant to Leishmamia major. Surprisingly, mice deficient in CCR2 (the receptor for MCP-1) present with defects in both Th1 and Th2 responses (Warmington et al., 1999), which suggests that MCP-1 may bind to another receptor (D6 or CCR4?). MCP-1 might mediate its effects by enhancing the secretion of IL-4 by T cells. The involvement of MCP-1 in Th2 responses might explain the ability of anti-MCP-1 antibodies to show dramatic inhibition of asthma pathogenesis in mouse models (Gonzalo et al., 1998). D. INHIBITORS OF Th1 OR Th2 CHEMOKINE RECEPTORS Because of their essential role in cell migration, and their selectivity, chemoattractant receptors are considered ideal drug targets for inflammatory diseases. Chemoattractant receptor antagonists might allow selective disruption of Th1 or Th2 responses. Considerable effort has already been devoted by the pharmaceutical industry toward the development of numerous chemoattractant receptor


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antagonists, particularly the “inflammatory” receptors CCR3, CCR2, CCR5, and CXCR3. Several potent small molecule antagonists of chemokine receptors have been published, including the Th1 receptor CCR5 (Baba et al., 1999), as well as numerous antagonistic mAbs (Heath et al., 1997; Wu et al., 1997a,b; Qin et al., 1998). A number of viruses use the chemokine system to subvert effector T cell responses (Lalani et al., 2000). A chemokine-like molecule termed vMIPII, encoded by Kaposi’s sarcoma-related herpesvirus, displays broad spectrum binding activity, binding with high affinity to a number of the CC and CXC chemokine receptors. Interestingly, vMIPII antagonizes many of the Th1-associated receptors such as CCR1, CCR2, and CCR5, but is also agonistic for Th2-associated receptors such as CCR3 and CCR8 (Boshoff et al., 1997; Kledal et al., 1997; Sozzani et al., 1998). Likewise, vMIPI is antagonistic for many of the Th1 chemokine receptors, but is agonistic on CCR8 (Endres et al., 1999). vMIP-III, another Kaposi’s sarcoma-related herpesvirus chemokine, is an agonist for CCR4 (another Th2 expressed receptor; Stine et al., 2000). Hence, these viral chemokines may act to selectively inhibit the recruitment of Th1 cells and macrophages (the cells that protect against viral spread), and recruit Th2 cells (which produce cytokines that inhibit Th1 responses). E. CXCR5 AND FOLLICULAR T HELPER CELL MIGRATION B cell response to antigen requires interactions between antigen-specific B cells and T helper cells within secondary lymphoid tissues (MacLennan et al., 1997; Garside et al., 1998). These T helper cells are activated by antigen in the T cell area of lymphoid tissue, and then migrate specifically to the outer edge of B cell follicles, where they meet antigen-specific B cells that have also migrated to this location (Garside et al., 1998). The B cell presents antigen to the T cell and receives stimulatory signals from antigen-specific T cells via cell surface molecules or soluble factors. The signals delivered to B cells enable clonal expansion and differentiation, as well as antibody production and isotype switching. The role of Th1 or Th2 cells as helper cells for antibody production has been unclear, although B cell help has often been ascribed to the Th2 subset. However, Th2 cells may not be essential for B cell proliferation and differentiation, since IL-4 knockout mice (which are deficient in Th2 responses) produce antibodies and develop germinal centers upon antigenic stimulation. One study that sought to address the role of Th1 and Th2 cells in B cell help found that both Th1 and Th2 cells were able to migrate into B cell follicles and support B cell clonal expansion and antibody production in a CD154-dependent manner (Smith et al., 2000). In another study, transferred Th1 and Th2 cells were assessed for their ability to provide help for antibody responses, and the cells were tracked for their localization within lymphoid tissue, and this was correlated with their expression of chemokine receptors. Th2 cells showed a characteristic localization pattern,

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forming rings around the outer PALS near the B cell zones. In contrast, Th1 cells clustered within the central PALS. Expression of CCR7 was found to be responsible for the differences in localization patterns between Th1 and Th2 cells, in that Th1 cells preferentially expressed CCR7, and forced expression of CCR7 in Th2 cells changed their localization pattern, driving them away from B cell areas. The chemokine BLC (also called BCA-1) and its receptor CXCR5 are particularly important for B cell responses (Forster et al., 1996; Ansel et al., 2000). CXCR5 is expressed on B cells, and also on a small subset of effector/memory CD4+ T cells (Forster et al., 1994). In lymphoid tissue, these CD4+ T cells (termed TFH see above) provide help for B cell stimulation by migrating to the B cell area through the actions of BLC and CXCR5 (Breitfeld et al., 2000; Schaerli et al., 2000). BLC is expressed selectively within primary and secondary follicles of lymphoid tissue. The importance of this receptor–ligand pair is underscored by the fact that CXCR5-deficient mice have malformed follicles and germinal centers (Forster et al., 1996; Ansel et al., 2000).

VI. Cell Surface and Costimulatory Molecules That Distinguish T Cell Effector Functions

A. ICOS T cell activation requires a signal from the TCR and costimulatory signals delivered by APCs (Bretscher, 1999). CD28 is the most important costimulatory signal for the activation of resting T cells, and blockade of CD28 attenuates a variety of responses including both Th1-type diseases (Cross et al., 1995) and Th2/allergic-type diseases (Keane-Myers et al., 1997). Na¨ıve T cells in particular depend on CD28-mediated signaling for their activation (Schweitzer and Sharpe, 1998), whereas effector T cells rely on additional molecules, since optimal activation of certain T helper subsets occurs independently of CD28 and its ligands (Schweitzer and Sharpe, 1998). Moreover, in CD28 gene-targeted mice, normal Th2 effector immune responses can be generated, suggesting the participation of other costimulatory signals (Brown et al., 1996b). Recently, a CD28 homologue termed ICOS was identified that appears to be the signaling molecule critical for effector T cell activation (Hutloff et al., 1999; Yoshinaga et al., 1999). In one study, ICOS was identified as part of a strategy to isolate effector T cell costimulatory molecules, using subtractive libraries from activated murine Th1 versus Th2 clones (Coyle et al., 2000). ICOS was induced upon T cell activation; however, repetitive antigenic stimulation in the presence of IL-12 lead to down-regulation of ICOS mRNA in Th1 cells and over-expression in Th2 cells. Studies in vitro and in vivo demonstrated a role for ICOS in Th2 activation. ICOS-Ig was able in inhibit secretion of Th2 cytokines


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by Th2 cells, but had no effect on Th1 cells. In a model of allergic airway inflammation mediated by adoptive transfer of antigen-specific Th1 or Th2 effector cells, ICOS blockade inhibited Th2- but not Th1-mediated lung mucosal inflammation (Coyle et al., 2000). In another study, ICOS was found to be important for both CD28-dependent and CD28-independent CD4+ Th1 and Th2 responses, but not CTL responses (Kopf et al., 2000). Thus, ICOS is important for Th2 responses, but also plays a role in TFH functions. Its role in Th1 responses is less clear. B. IL-1 RECEPTOR-LIKE MOLECULES T1/ST2 is an IL-1 receptor-like orphan receptor with unknown function. It is widely expressed, particularly by mast cells (Moritz et al., 1998). In the mouse, its notable feature is that it is expressed on the surface of Th2 but not Th1 effector cells (Lohning et al., 1998; Coyle et al., 1999). In vitro blockade of T1/ST2 signaling with an Ig fusion protein suppressed both differentiation to, and activation of, Th2 but not Th1 effector populations (Coyle et al., 1999). In a Th2-mediated allergic airway model, anti-T1/ST2 mAb inhibited eosinophil infiltration, IL-5 secretion, and IgE production (Coyle et al., 1999). The immune response of T1/ST2-deficient mice is also dramatically affected. For instance, in a pulmonary granuloma model induced with Schistosoma mansoni eggs, granuloma formation (characterized by eosinophil infiltration) is abrogated in T1/ST2-deficient mice, and levels of Th2 cytokine production are severely impaired. Nevertheless, two separate studies have shown that an absence of T1/ST2 does not affect the development and function of Th2 cells (Hoshino et al., 1999a; Senn et al., 2000). The expression and function of T1/ST2 in humans may not be as clear as it is in the mouse. While T1/ST2 is associated with inflammation (Kumar et al., 1997), there appears to be no preferential association with Th2 responses. C. IL-18 AND IL-18 RECEPTOR As discussed above, IL-18 synergizes with IL-12 to induce IFN-␥ production by Th1 but not Th2 cells. The receptor for IL-18 (IL-18R, which is probably composed of two components, IL-1Rrp or IL-1R-related protein and AcPL or IL-1R accessory protein-like) was shown to be preferentially expressed on Th1 cells compared with Th2 cells (Xu et al., 1998a). Similar to IL-18 deficient mice, IL-18 receptor deficient mice have impaired Th1 responses (Hoshino et al., 1999b). The expression of IL-18 receptor was found to be up-regulated in response to IL-12 (Yoshimoto et al., 1998). These findings suggest that IL-18 receptor can be used as a Th1 specific marker to differentiate between Th1 and Th2 cells. As a Th1 specific marker, IL-18 receptor may have some utility

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in treating Th1 mediated immunopathologies. It was found that an anti-IL-18 receptor antibody caused a shift from Th1 to Th2 response with a decrease in Th1 cytokine production and increase in IL-4 and IL-5. It also caused a reduction in local inflammation and lipopolysaccharide (LPS)-induced mortality when administered to mice (Xu et al., 1998b). D. TETRASPANS The tetraspan (TM4) superfamily comprises at least 20 members, all of which span the membrane four times. The precise function of these molecules is unknown but they are involved in diverse processes such as cell activation, proliferation, adhesion, and cell differentiation. Many of the tetraspans associate with other molecules, such as lineage-specific proteins and integrins. It has been suggested that tetraspans act as “molecular facilitators,” that group certain cell-surface proteins and allow the formation and stability of functional signaling complexes (Maecker et al., 1997). The tetraspan Chandra was identified as a Th1 expressed gene using a PCR-based subtraction method to identify novel Th1 genes (Venkataraman et al., 2000). Expression of Chandra is not regulated by IL-12 or IFN-␥ , but is dependent on the absence of IL-4 signaling in activated T cells. The function of Chandra is not yet resolved. CD81 (TAPA-1) is a widely expressed tetraspan molecule. Mice lacking CD81 have impaired Th2 dependent antibody responses, and antigen-specific IL-4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice (Maecker et al., 1998). CD81 may interact with a ligand on T cells to signal IL-4 production.

VII. Microarrays for the Identification of T Cell Subset Expressed Genes

Despite the evidence discussed above that different effector T cell subsets can be defined by the expression of different sets of chemokine receptors and other surface molecules, the picture is incomplete, and markers for Th1 and Th2 and other subsets are generally imperfect. The expression of some molecules remains stable following polarization, but the expression of others varies depending on cytokine stimulation and other factors. Lack of definitive markers for different T cell subsets significantly compromises efforts to fully characterize different T cell responses. Clearly, our understanding of T lymphocyte biology would greatly benefit from identification of novel genes differentially expressed in different T cell subsets. Perhaps one of the most powerful techniques for differential gene screening that is currently available is gene microarrays, and comparison of gene expression in different populations of effector T cells is an obvious application. Two studies utilizing DNA microarrays to look at T cell subset expressed genes in human and mouse are reviewed below.

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TABLE I DIFFERENTIALLY EXPRESSED GENES IN CD4+ AND CD8+ TYPE 1 AND TYPE 2 T CELLS A. Gene IL-4 IL-5 IL-10 c-maf I-309 HLF CCR1 SDF-3 c-fos Ctla-2 ␣ IL-3 PPAP-␥ 2 Ctla-2 ␤ Eta-1 GM-CSF GATA-3 Zyxin TRAF5 CXCR4 CD27 Integrin ␤7 ZAP-70 IL-6 IL-7 R Amhiregulin IFN-␥ TRAF4 STAT4 MOK2 EBF IL-2 IL-18R Lumican

Fold Change 25.3 20.9 19.7 10.5 7.7 6.6 5.5 5.4 5.1 4.7 4.3 4.2 3.7 3.4 3.1 2.9 2.6 2.3 2.3 2.3 2.1 2.1 2.1 2 2 −2.3 −2.3 −2.9 −4.1 −4.8 −10.9 −12.4 −12.6

B. Gene

Fold Change

GATA-3 c-fos c-maf GM-CSF C/EBP␤ PPAP-␥ 2 IL-4 Amphiregulin IL-10 HLF IL-5 CCR1 IL-1R2 IL-6 IL-3 IL-7R B-ATF Eta-1 Ctla-2 ␤ ZAP-70 CXCR4 SDF-3 CD27 Ctla-2 ␣ I-309 TRAF5 TRAF4 IFN-␥ Lumican MOK2 CD36 IL-18R LT-␣

27.3 23.7 15.9 13.5 8.6 8.6 8.4 8.4 8.2 8.1 7.8 7.1 6.7 6.2 5.2 5 3.8 3.1 3.1 3.1 2.7 2.4 2.4 2.3 2.3 2.1 −2.8 −3.7 −4.6 −6.5 −7.5 −10.9 −16.3

Note. The fold change in gene expression between (A) Th1 and Th2 and (B) Tc1 and Tc2 cells as determined from microarray experiments is shown. Negative-fold change indicates higher expression in type 1 cells; positive-fold change indicates higher expression in type 2 cells (unpublished data).

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A. HUMAN STUDIES Rogge et al. used oligonucleotide microarrays to analyze gene expression in human T helper cells (Rogge et al., 2000). Their analysis (using a chip containing 6000 full-length human genes) identified 215 differentially expressed genes. As expected, the analysis of differentially expressed genes identified some genes that are well established as markers of T helper cell polarization, such as IFN-␥ and the transcription factor GATA-3. In addition, genes whose role in T cell polarization had not yet been defined were also differentially expressed. These were genes involved in transcriptional regulation such as ETS-1, NF-IL-6, ROR␣2, STAT1, IRF-1, and IRF-7A; and genes involved in apoptosis and proteolysis, such as perforin, granzyme B, TRAIL, caspase-8, and BAK-2. The preferential expression of genes involved in apoptosis and proteolysis in Th1 cells correlated well with the increased susceptibility of Th1 cells to activationinduced cell death. Importantly, a number of genes involved in cell adhesion and migration were differentially expressed between the two T helper subsets, confirming previous studies (see above). Several genes involved in mediating adhesion and effector functions of Th1 cells were further regulated by IL-12 in the absence of antigenic stimulation (Rogge et al., 2000). In vitro and in vivo experiments should determine if the differential expression determined by Genechip is consistent and stable, and establish the utility of these molecules as markers for functional subsets of CD4+ effector T cells. B. MOUSE STUDIES In a complementary study, we have analyzed gene expression in murine polarized CD4+ and CD8+ T cells. One striking but perhaps not unexpected result was the observation that over half of the 11,000 known full-length genes and ESTs analyzed were expressed by at least one of the cell types. This high number of genes expressed in effector T cells seems quite remarkable, although caution should be exercised, as the subset of known genes and ESTs may not provide an unbiased sample of the whole genome. The other broad conclusion of the study was that the vast majority of genes expressed in T cells are in fact expressed at similar levels in both type 1 and type 2 T cells. While the overall gene expression is largely similar in both types of effector T cells, we identified a large number of genes that are differentially expressed in type 1 and type 2 effector T cells. Among these were genes that have been identified previously as type 1 or type 2 specific, such as IL-4, IL-5, IL-10, IL-13, and IFN-␥ , and transcription factors such as c-maf, GATA-3, and Stat4. In addition, we identified a number of genes that have not been associated with T cell polarization. We also noted a significant similarity between gene expression programs for CD4+ and CD8+ type 1 and type 2 T cells. Listed in Table I are some of the genes differentially expressed in our experiment. Although our


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study confirmed the overall conclusions of the human study, it also highlighted some important differences. Protocols used for polarization, as well as species differences, yielded some notable discrepancies in gene expression. In summary, the two studies have illustrated the utility of using microarrays to identify the differences in gene expression between different subsets of effector T cells. Further studies using microarrays should lead to identification of novel markers that should reliably differentiate between various effector T cell subsets. VIII. Conclusions

The T cell immune system of mammals has attained a degree of specialization, so as to effectively deal with various pathogens. The identity of different patterns of cytokine secretion by T cells led to the development of the Th1/Th2 paradigm, which has dominated modern immunology. However, it is becoming increasingly clear that effector T cell responses are more complicated. CD8+ subsets also secrete type 1 and type 2 cytokines, and other subsets exist such as TFH, whose relationship to Th1 or Th2 cells is uncertain. A complete understanding of all of these subsets should flow from the identity of the cytokines, transcription factors, chemokine receptors, and cell surface molecules that function in different T cell responses. Toward this end, DNA microarrays will undoubtedly make a significant contribution. As T effector subsets are dissected and become better understood, prospects will improve for new therapeutics for inflammatory diseases. ACKNOWLEDGMENTS Tatyana Chtanova and Charles Mackay are supported by the Glazebrook Trust and the CRC for Asthma.

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Yoshida, H., Nishina, H., Takimoto, H., Marengere, L. E., Wakeham, A. C., Bouchard, D., Kong, Y. Y., Ohteki, T., Shahinian, A., Bachmann, M., Ohashi, P. S., Penninger, J. M., Crabtree, G. R., and Mak, T. W. (1998). The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production. Immunity 8, 115. Yoshie, O., Imai, T., and Nomiyama, H. (1997). Novel lymphocyte-specific CC chemokines and their receptors. J. Leukocyte Biol. 62, 634. Yoshimoto, T., Bendelac, A., Hu-Li, J., and Paul, W. E. (1995a). Defective IgE production by SJL mice is linked to the absence of CD4+, NK1.1+ T cells that promptly produce interleukin 4. Proc. Natl. Acad. Sci. USA 92, 11931. Yoshimoto, T., Bendelac, A., Watson, C., Hu-Li, J., and Paul, W. E. (1995b). Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production. Science 270, 1845. Yoshimoto, T., and Paul, W. E. (1994). CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179, 1285. Yoshimoto, T., Takeda, K., Tanaka, T., Ohkusu, K., Kashiwamura, S., Okamura, H., Akira, S., and Nakanishi, K. (1998). IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: Synergism with IL-18 for IFN-gamma production. J. Immunol. 161, 3400. Yoshinaga, S., Whoriskey, J., Khare, S., Sarmiento, U., Guo, J., Horan, T., Shih, G., Zhang, M., Coccia, M., Kohno, T., Tafuri-Bladt, A., Brankow, D., Campbell, P., Chang, D., Chiu, L., Dai, T., Duncan, G., Elliott, G., Hui, A., McCabe, S., Scully, S., Shahinian, A., Shaklee, C., Van, G., and Mak, T. et al. (1999). T-cell co-stimulation through B7RP-1 and ICOS. Nature 402, 827. Zhang, D. H., Yang, L., Cohn, L., Parkyn, L., Homer, R., Ray, P., and Ray, A. (1999). Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3. Immunity 11, 473. Zhang, Y., Rogers, K. H., and Lewis, D. B. (1996). Beta 2-microglobulin-dependent T cells are dispensable for allergen-induced T helper 2 responses. J. Exp. Med. 184, 1507. Zheng, W., and Flavell, R. A. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587. Zlotnik, A., and Yoshie, O. (2000). Chemokines: A new classification system and their role in immunity. Immunity 12, 121.


MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens INGELISE BJERRING KASTRUP,* MADS HALD ANDERSEN,* TIM ELLIOTT,† AND JOHN S. HAURUM*,‡ *Institute of Cancer Biology, Danish Cancer Society, 2100 Copenhagen OE, Denmark, and †Cancer Sciences

Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom

I. Introduction

It is well established that antigen recognition by T cells involves the engagement of the T cell receptor (TcR) with a complex between major histocompatibility complex (MHC) molecules and antigenic peptides. Antigens presented by class I MHC molecules for recognition by cytotoxic T lymphocytes (CTL) normally consist of 8 to 10 amino acid long peptides of endogenous origin, whereas antigens presented by class II MHC for recognition by T helper cells (Th) consist of peptides with an average length of 16 amino acids derived from exogenous proteins. The repertoire of peptides presented by MHC molecules has been characterized extensively and a vast majority of peptides presented naturally by class I or class II MHC molecules consist of unmodified peptides. However, a large proportion of proteins carry posttranslational modifications, the exact structure of which may be difficult to predict from the genetic sequence, and these modifications may persist on peptide fragments after proteolytic degradation of the modified protein. This opens the possibility that peptides carrying posttranslational modifications may be presented by MHC molecules for T cell recognition. In agreement with this, peptides derived from posttranslationally modified proteins have indeed in recent years been identified among peptide ligands presented by class I and class II MHC molecules in vivo (Chicz et al., 1993; Dustin et al., 1996; Haurum et al., 1999; Kastrup et al., 2000; Skipper et al., 1996). However, very little is yet known about the overall impact of naturally occurring posttranslational modifications on MHC-restricted antigen presentation in vivo. Theoretically, such modifications may affect all levels of antigen presentation (antigen processing, MHC binding, and interaction with T cells). Recently, several groups, including our own, have provided data concerning the effect of glycosylation on all these aspects of antigen presentation. Also, naturally occurring T cell responses toward posttranslationally modified peptides ‡ Address correspondence to John Haurum, Gustav Adolfsgade 6, DK-2100 Copenhagen, Denmark. Email: [email protected].

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have been reported for both class I and class II MHC-restricted specificities. Interestingly, it seems that for several of these examples the recognition of a posttranslational modification might be directly relevant to disease pathology, underlining the biological relevance of such modified peptide antigens. Here, we will review the current knowledge of the influence of posttranslational protein modifications on antigen presentation and T cell recognition of class I and class II MHC-restricted antigens. Examples of naturally occurring MHC-restricted T cell responses directed toward a posttranslational modification will be discussed with an emphasis on examples where recognition of a posttranslational modification might be directly relevant to disease pathology. II. Posttranslational Modifications of Proteins

Over 200 different posttranslational modifications of amino acid side chains have been characterized and the list of modifications is constantly growing (Creighton, 1993). The most frequently found include glycosylation, phosphorylation, sulfation, hydroxylation, carboxylation, ubiquitination, acetylation, methylation, and deamidation. Many posttranslational modifications are carried out by enzymes that display specificity for particular amino acid residues. These enzymes are located in specific compartments within the cell, and hence the nature of modification of any particular protein depends primarily on the cellular distribution of the particular protein. The enzymatically regulated posttranslational modification of proteins may also result in molecular heterogeneity due to partial modification or, e.g., the modification with different glycan isoforms at the same glycosylation site. Glycosylation is one of the most ubiquitous eukaryotic posttranslational modifications, yet the exact structure, regulation, and function of most protein glycans remains largely unknown (Ploegh and Neefjes, 1990). Furthermore, information is still scarce as to what extent such glycoproteins give rise to glycopeptides for MHC-restricted antigen presentation. However, four examples of glycosylated antigens presented naturally by MHC class II have been reported to date, including helper T cells recognizing bee venom phospholipase A2 in human individuals allergic to bee stings (Dudler et al., 1995), as well as an immunodominant epitope involved in the initiation of collagen-induced arthritis in mice (Corthay et al., 1998; Michaelsson et al., 1994). There is also one report of a tumor-specific glycopeptide presented by MHC class I (Zhao and Cheung, 1995). In all cases, where investigated glycopeptide-specific T cells were found to depend on the glycan group for peptide recognition. There are two major types of glycosylation, denoted as N-linked or O-linked, depending on the carbohydrate linkage to the protein. Thus, N-linked glycosylation occurs on asparagine (Asn) amino acid side chains, and is the major glycosylation of membrane bound or secretory proteins. It is acquired cotranslationally

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by the transfer of a preassembled core structure, (GlcNAc)2(Man)9(Glc)3, from a dolichol phosphate precursor immediately after the Asn residue of the protein emerges into the endoplasmic reticulum (ER) lumen. Subsequently, the glycan will be extensively modified by compartmental enzymes during passage through the ER and Golgi apparatus, finally giving rise to three distinct N-glycans: the high-mannose type, the hybrid type, or the complex type, each with distinct structural features (Creighton, 1993). The so-called O-linked glycosylation mainly occurs on the oxygen atoms of serine (Ser) or threonine (Thr) residues, but also on hydroxylated Pro (hyPro) and hydroxylated Lys (hyLys) side chains. At least seven different monosaccharides are utilized: galactose (Gal), glucose (Glc), fucose (Fuc), mannose (Man), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and xylose (Xyl) linked in a variety of glycoside linkages. The O-linked types of glycosylation can be subdivided in at least three groups based on structure and topology: the collagen type, the mucin type, and the cytosolic type. In the collagen type certain hyLys (and to a lesser extent hyPro) collagen side chains are glycosylated with Gal, and in some instances the Gal structure is further modified with a Glc moiety. The mucin type glycosylation (so-called because it is particularly abundant on mucin glycoproteins) is initiated in the ER or cis-Golgi where GalNAc moieties are transferred enzymatically to the hydroxyl amino acids of Ser or Thr. Then, the glycoprotein may be further modified with Gal, GalNAc, sialic acid, Xyl, or Fuc in the Golgi compartment in a stepwise process specific for the type of cell. If elongation does not occur, the so-called Tn antigen (O-␣-GalNAc) is generated, which is frequently expressed by tumor cells (Van den Steen et al., 1998). For many years glycosylation was thought to be confined to proteins of the ER and Golgi compartments, and none of the accepted models of glycoprotein biosynthesis or transport predicted the existence of glycoproteins in the nucleus or cytoplasmic compartments (Kornfeld and Kornfeld, 1985). However, a plentitude of studies have now firmly documented the ubiquitous presence of glycoproteins in the nucleus and cytosol carrying single monosaccharide GlcNAc residues in O-␤-linkages to the hydroxyl groups of Ser or Thr residues. Proteins so modified have been identified in all eukaryotic tissue or prokaryotic organisms examined to date, and appear to be exclusively nuclear and cytosolic (Kearse and Hart, 1991). Peptides carrying posttranslational modifications other than glycosylation have also been found to be presented naturally by classical class I and class II molecules. Deamidation of Asn or Gln residues is the most common nonenzymatic modification of proteins which can also be mediated by the action of enzymes such as tissue transglutaminase (tTGase) on Gln residues of gliadin (Molberg et al., 1998). Disulfide bond formation between Cys residues is another common posttranslational modification of proteins in the ER. A large proportion

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of eukaryotic proteins synthesized in the cytoplasm are isolated with acetylated N-termini. A variety of N-␣-acetyltransferases are thought to catalyze this reaction, using acetyl-CoA as the acetyl donor. Finally, an increasing number of proteins are known to be phosphorylated. The phosphoryl groups are added by specific protein kinases, and removed by specific phosphatases in a tightly controlled manner, regulating the activities of the phosphorylated proteins. The phosphorylation sites usually include the hydroxyl groups of specific Ser, Thr, or tyrosine (Tyr) residues, and it primarily occurs in the cytoplasm (Creighton, 1993). III. Posttranslational Modifications and Antigen Processing

Two distinct pathways exist for presentation of antigen in association with class I and class II MHC molecules. Thus, peptide antigens derived from the proteolytic degradation of cytosolic and nuclear proteins by the proteasome are subsequently translocated across the ER membrane by the so-called TAP transporter associated with antigen processing (Elliott, 1997; Heemels and Ploegh, 1995; Howard and Seelig, 1993; Michalek et al., 1993). Once in the lumen of the ER, peptides bind to newly synthesized class I MHC heavy chain and ␤2m molecules assisted by chaperones. After stable assembly of the peptide–MHC complex, it is transported through the Golgi apparatus and secretory vesicles to the cell surface for presentation to T cells. Class II MHC molecules on the other hand acquire their peptides in the endosomal or lysosomal compartment. Immediately after synthesis, class II MHC ␣␤ chain heterodimers associate with membrane bound invariant chain (Ii) (Riberdy et al., 1992). A part of the Ii occupies the peptide binding cleft and thus prevents the class II MHC molecule from binding peptides present in the lumen of the ER and Golgi compartment. The invariant chain also facilitates emigration of the class II MHC molecules from the ER through the Golgi apparatus to the acidified endosomes, at which point the class II MHC molecules encounter peptides derived from bacteria or engulfed extracellular proteins. Then invariant chain is cleaved, leaving the so-called CLIP-peptide bound to the class II MHC molecule (Matsumura et al., 1992). Release of the CLIP peptide and subsequent binding of peptides is catalyzed by interaction of the complex with the class II-like molecule HLA-DM (Kelly et al., 1991; Sanderson et al., 1996). Finally, the class II MHC-peptide complex is transported to the cell surface for presentation to T cells. Posttranslational modifications may affect the presentation and T cell recognition of peptide antigens at all levels of antigen presentation (antigen processing, MHC binding, and interaction with T cells). First, the presence of posttranslational modifications on proteins may prevent the access of proteolytic enzymes of the class I or class II MHC antigen processing pathway and thereby inhibit the generation of the antigenic peptides. Thus, T cell responses specific for a


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class II MHC-restricted determinant of influenza hemagglutinin was abrogated when N-glycans were attached to an Asn residue just outside the T cell determinant, indicating that the presence of a glycan can convert an immunodominant epitope into a cryptic determinant. However, it is unclear whether the blocking of the T cell response occurred at the level of antigen presentation or T cell recognition (Drummer et al., 1993). Wood and Elliott found that N-glycosylation of ER-targeted influenza A nucleoprotein (NP) inhibited the generation of epitopes distal to the site of glycosylation (Wood and Elliott, 1998). The authors suggest that N-glycosylation of proteins targeted to the secretory pathway may, in some instances, prevent the generation of class I MHC binding peptides within the ER by enzymes which normally function to trim peptides delivered by TAP. It is not known to what extent the interaction between these ER-targeted antigens and ER-resident chaperones contribute to the processing events, but it might be significant that the glycosylated antigens, which were not processed, could be found in association with the ER resident chaperone calreticulin (T. Elliott, unpublished data). The presence on a peptide antigen of a posttranslational modification may also potentially influence TAP transport from the cytosol into the ER. Recently, our group demonstrated that the TAP molecule was able to transport either peptides glycosylated with the natural cytosolic type of O-linked N-acetylglucosamine (O-GlcNAc) (Haurum et al., 1999) or phosphorylated peptides (Andersen et al., 1999). The transport of the phosphorylated peptide was just as efficient as the transport of the nonphosphorylated analogue. On the other hand, the translocation efficiency of the glycopeptide was slightly lower than for the nonglycosylated control peptide, although it was well within the range of other known peptides. For class II MHC molecules, the uptake of antigen by antigen-presenting cells may be influenced by the presence of posttranslational modifications on the antigen. The 45/47-kDa antigen complex (Apa) of Mycobacterium tuberculosis has been found to be glycosylated with up to nine mannose residues per mole of protein, and with only a small number of molecules not being glycosylated (Dobos et al., 1996). Furthermore, in a recent study by Romain et al. (1999), Apa was found to have a significantly lower capacity to elicit delayed-type hypersensitivity reactions in vivo upon deglycosylation, and a reduced T cell stimulatory activity in vitro. This indicates that the presence of the mannose residues on the Apa protein is essential for the antigenicity of the molecules in T cell dependent immune responses in vivo and in vitro. It is, however, not known at what level of antigen presentation deglycosylation reduced the T cell stimulatory potency. Thus, it might be caused by altered processing of deglycosylated Apa molecules, reduced binding of deglycosylated Apa-derived peptides to the MHC molecule or reduced T cell recognition of the deglycosylated Apa peptides in complex with MHC.

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IV. Posttranslational Modifications and MHC Binding

A. BINDING TO MHC MOLECULES OF SYNTHETIC PEPTIDES CARRYING POSTTRANSLATIONAL MODIFICATIONS Using synthetic peptides carrying different types of posttranslational modifications, we and other groups have investigated the effect of such modifications on the binding to MHC molecules. The results of these experiments naturally fall in three categories, depending on whether the peptide modification results in (1) reduced binding to the MHC molecule, (2) unaltered binding, or in a few cases (3) increased binding to the MHC molecule. The first two studies examined glycopeptide binding to class II MHC molecules. Ishioka et al. (1992) investigated the binding to the class II MHC molecule I-Ad of a model T cell epitope, OVA323–339, carrying a single N-linked GlcNAc residue at various positions. When the glycan was located on residues outside the core MHC-binding region, no effect on MHC binding was observed. When the glycan was located within the core MHC-binding region, either the glycopeptide maintained binding to the MHC molecule or binding was abolished. These experiments demonstrated for the first time that a GlcNAc monosaccharide modification is tolerated even within the core of the class II MHC peptide binding groove. Harding et al. (1993) modified the amino terminus of a T cell peptide epitope from hen egg lysozyme (HEL) with the disaccharide galabiose (Gal␣(1-4)Gal␤). It was then found that the glycopeptide bound as well to the MHC molecule I-Ak as the nonglycosylated analogue, confirming that peptides carrying posttranslational modifications are able to bind to MHC molecules. We have analyzed what affect the natural cytosolic type of O-linked monosaccharide GlcNAc glycosylation of Ser or Thr residues had on class I MHC peptide binding using the synthetic glycopeptide [FAPS(-O-␤-GlcNAc)NYPAL] (Haurum et al., 1994). This glycopeptide is an analogue of the immunodominant H-2Kb-restricted Sendai virus NP epitope [FAPGNYPAL], where the glycine at position 4 was substituted with Ser or Ser-O-␤-GlcNAc residues. This position was chosen because the crystal structure of the wild-type peptide in complex with H-2Kb indicated that the GlcNAc would be likely to point out of the MHC binding groove and interact with the TcR (Fremont et al., 1992). We found that both the glycosylated and the nonglycosylated variant of this and other related peptides bound with high affinity to both H-2Kb and H-2Db. However, introducing an asparagine-linked (N-linked) GlcNAc residue at peptide position 5, which is an anchor residue required for efficient peptide binding to H-2Db, blocked binding of the glycopeptide to H-2Db, but as expected, not to H-2Kb. In another study, the mouse hemoglobin-derived decapeptide Hb67–76, which binds well to I-Ek and is nonimmunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn. The peptide was glycosylated with


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the Tn antigen on Ser or Thr residues at all 10 different peptide positions, and the different glycopeptides tested for binding to I-Ek. As expected, the glycopeptides carrying the Tn glycosylation antigen on an MHC-contact residue completely lost the ability to bind to I-Ek, whereas glycopeptides with the Tn antigen on a nonMHC contact residue in most cases preserved their capacity to bind (Jensen et al., 1996). Recently, we also reported the binding to class I MHC molecules of a number of synthetic phosphopeptide sequences derived from natural viral phosphoproteins and phosphorylated oncogene products (Andersen et al., 1999). In two cases the natural phosphopeptides bound with a similar high affinity as the nonphosphorylated version to class I MHC. Thus, the BCR174–182 peptide [KPFY(PO32−)VNVEF] binds with high affinity to both HLA-B∗ 0702 and HLAB∗ 3501, and similarly the Sendai virus peptide analogue [FAPS(PO32−)NYPAL] binds with high affinity to both H-2Kb and H-2Db. Surprisingly, we also found that a phosphorylated version of the CRKL-derived peptide [Y(PO32−) AQPQTTTPL] bound with higher affinity to HLA-A∗ 0201 than the nonphosphorylated counterpart. A similar example was described by Mouritsen et al. (1994) where a 16-mer peptide derived from HEL bound better to I-Ek when N-glycosylated with a branched pentaglucose at the N-terminus. Likewise, we had reported that substituting the position 5 anchor residue Asn of the Sendai virus NP peptide [FAPGNYPAL] with Ser abrogated binding to H-2Db. Surprisingly, however, glycosylation of the Ser residue with O-GlcNAc partially restored peptide binding to Db (Haurum et al., 1995, see Table I). Together, these studies demonstrate that, with certain exceptions, a posttranslational modification on a peptide epitope will have a neutral or positive effect on MHC binding if positioned on a non-MHC contact residue or a residue outside the peptide binding cleft, whereas modifications of MHC contact residues in most cases reduced or abrogated binding of the modified peptide, although examples of enhanced binding upon posttranslational modification also exist. B. POSTTRANSLATIONALLY MODIFIED PEPTIDES PRESENTED in vivo Several reports have now documented that peptides carrying posttranslational modifications are presented by MHC molecules in vivo. Thus, in a study by Chicz et al. (1993), naturally processed peptides that had been acid extracted from immunoaffinity purified class II MHC molecules were sequenced. In addition to plain peptide, sequence analysis by mass spectrometry confirmed the existence of an HLA-DR8 binding peptide derived from the protein LAM Blast-1, which carried a GlcNAc carbohydrate residue on the Asn104 sidechain. The authors suggest that this is the remnant of a degraded N-linked glycan, and that the remainder of the N-linked carbohydrate structure had been hydrolyzed either during antigen processing or during the peptide isolation procedures.

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TABLE I STUDIES OF ANTIGEN PRESENTATION AND T CELL RECOGNITION OF POSTTRANSLATIONALLY MODIFIED PEPTIDES in Vitro Binding of posttranslationally modified peptides to MHC molecules in vitro Binding of OVA323–339-derived synthetic glycopeptides to I-Ad Binding of galabiose modified HEL52–61-derived synthetic glycopeptides to I-Ak Binding of O-GlcNAc modified SEV NP324–332-derived synthetic glycopeptides to H-2Kb and H-2Db Binding of HEL81–96-derived synthetic glycopeptides to I-Ek Binding of Hb67–76-derived synthetic glycopeptides to I-Ek Binding of human oncogene and viral phosphoproteinderived synthetic phosphopeptides to class I MHC molecules Crystal structure of class I MHC-glycopeptide complexes Crystal structure of class I MHC-glycopeptide complexes

References (Ishioka et al., 1992) (Harding et al., 1993) (Haurum et al., 1994, 1995) (Mouritsen et al., 1994) (Galli Stampino et al., 1997; Jensen et al., 1996) (Andersen et al., 1999)

(Glithero et al., 1999) (Speir et al., 1999)

T cell recognition of posttranslationally modified peptides in vitro I-Ad-restricted Th cell recognition of OVA323–339derived synthetic glycopeptides I-Ak-restricted T cell recognition of HEL52–61derived synthetic glycopeptides H-2Kb-and H-2Dh-restricted T cell recognition of O-␤-GlcNAc modified SEV NP324–332 glycopeptide analogues Fine specificity of I-Ak-restricted T cells recognizing HEL52–61 derived synthetic glycopeptides I-Ek-restricted T cell recognition of glycosylated peptide analogues of Hb67–76 I-As-restricted T cell recognition of an ␣-B-crystallin-derived phosphopeptide HLA-A2- and H-2Kb-restricted T cell recognition of oncogene and virus derived phosphopeptides

(Ishioka et al., 1992) (Harding et al.,1993) (Haurum et al., 1994, 1995) (Deck et al., 1995, 1999) (Galli Stampino et al., 1997; Jensen et al., 1996) (van Stipdonk et al., 1998) (Andersen et al., 1999)

Dustin et al. (1996) studied antigens presented naturally from a human B lymphoblastoid cell line (B-LCL) and identified a mannose-6-phosphate (Man-6-P) glycopeptide derived from lysosomal acid lipase. The persistence of the Man-6-P on the glycopeptides suggests that the glycans do not spend an extended period of time in the lysosomal compartment where phosphatases would be expected to cleave the phosphate from Man-6-P. Together these studies demonstrated for the first time that posttranslational modifications are tolerated on peptides presented by class II MHC molecules in vivo, even a modification as large as mannose-6-phosphate.


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Posttranslationally modified peptides have also been isolated from class I MHC-peptide complexes presented in vivo (Table II). The first identification of a modified peptide was by Skipper et al. (1996), who described an HLAA∗ 0201-restricted tyrosinase peptide antigen, which resulted from deamidation of an Asn to an aspartic acid (Asp). Meadows et al. (1997) isolated a HLA-A∗ 0201restricted peptide originating from the protein SMCY, where the peptide had been modified at a Cys residue to form a disulfide bond with a second Cys residue. Similarly, Pierce et al. (1999) identified a HLA-A∗ 0101-restricted HY minor histocompatibility antigen originating from the protein product of DFFRY (a Y chromosome gene) containing a cysteinylated Cys residue as identified by a novel mass spectrometry technique. It is still unclear whether the cysteinylation occurs after peptide binding to MHC or whether the cysteinylated peptide is generated by processing of a disulfide bonded protein. Hudrisier et al. (1999) studied the processing of a H-2Db-restricted lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein-derived peptide (GP92–101) which contains a glycosylation motif for N-linked glycosylation that is utilized in the mature viral glycoprotein. Multiple isoforms of GP92–101 were generated form the viral glycoprotein and copresented on the surface of LCMVinfected cells. Thus, the peptides eluted from H-2Db corresponded to both nonglycosylated and posttranslationally modified sequences such as the deglycosylated sequence resulting in an Asn to Asp conversion and peptides containing oxidized forms of Cys, whereas the N-glycosylated peptide remained undetectable. Very recently, a natural ligand of class I MHC with a blocked NH2-terminus was identified (Yague et al., 2000). By mass spectrometry sequencing analysis a peptide matching the NH2-terminal sequence of two human helicases eluted from HLA-B39 was shown to be N-␣-acetylated. This finding contradicts the general idea of a universal binding mode for peptide NH2 termini in the A pocket of the class I molecule. This finding is especially surprising since N-␣-acetylation of peptides has been reported to markedly decrease binding of peptides to class I MHC molecules (Elliott et al., 1992; Matsumura et al., 1992). The discovery of the cytosolic O-␤-GlcNAc protein glycosylation led us to speculate whether such modifications might also be present on peptides presented by class I MHC molecules in vivo. Since the O-␤-GlcNAc modification takes place on nuclear and cytosolic proteins, and since cytosolic protein is the preferred source of peptides for presentation by class I MHC molecules, it seemed possible that peptides carrying O-␤-GlcNAc modifications might enter the class I MHC presentation pathway. Indeed, we have recently provided evidence to support that peptides presented by human spleen class I MHC molecules in vivo encompass a small, but significant, amount of glycopeptides, as verified by their ability to serve as substrates for the terminal GlcNAc-specific enzyme galactosyltransferase (Haurum et al., 1999).

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TABLE II STUDIES OF ANTIGEN PRESENTATION AND T CELL RECOGNITION OF POSTTRANSLATIONALLY MODIFIED PEPTIDES in Vivo Presentation of posttranslationally modified peptides in vivo HLA-DR8-restricted presentation of N-GlcNAc modified peptides derived from the protein LAM Blast-1 H-2q-restricted presentation of an immunodominant type II collagen glycopeptide modified with O-␤-Gal Evidence for hydroxylation of a H-2Ld-restricted octapeptide derived from ␣-ketoglutarate HLA class II-restricted presentation of a monosaccharide glycosylated peptide from bee venom phospholipase A2 H-2b-restricted presentation of a ganglioside GD2 modified glycopeptide HLA-DR-restricted presentation of a mannose-6-phosphatecontaining N-linked glycopeptide Deamidated HLA-A2-restricted tyrosinase peptide presented on melanoma cells Class I HLA-restricted presentation of cysteinylated peptide antigens HLA-DQ2-restricted presentation of a deamidated gliadin peptide Class I MHC-restricted presentation of O-␤-GlcNAc modified glycopeptides on human spleen tissue H-2Kd-restricted presentation of cysteinylated influenza virus peptides Presentation of deamidated and oxidized variants of the LCMV peptide GP92–101 Presentation of N-acetylated peptides by HLA-B39

References (Chicz et al., 1993) (Corthay et al., 1998; Kjellen et al., 1998; Michaelsson et al., 1994, 1996) (Wu et al., 1995) (Dudler et al., 1995) (Zhao and Cheung, 1995) (Dustin et al., 1996) (Skipper et al., 1996) (Meadows et al., 1997; Pierce et al., 1999) (Arentz-Hansen et al., 2000; Molberg et al., 1998) (Haurum et al., 1999) (Chen et al., 1999) (Hudrisier et al., 1999) (Yague et al., 2000)

T cell recognition of posttranslationally modified peptides in vivo H-2q-restricted T cell recognition of an immunodominant type II collagen-derived glycopeptide glycosylated with O-␤-Gal HLA class II-restricted carbohydrate-dependent T cell recognition of the bee venom allergen phospholipase A2 H-2b-restricted T cell recognition of peptide linked GD2 HLA-A2-restricted T cell recognition of a deamidated tyrosinase antigen Class I HLA-restricted T cell recognition of cysteinylated peptides HLA-DQ2-restricted T cell recognition of gliadin peptide epitopes containing a deamidated Gln residue

(Corthay et al., 1998; Michaelsson et al., 1994, 1996) (Dudler et al., 1995) (Zhao and Cheung, 1995) (Skipper et al., 1996) (Meadows et al., 1997; Pierce et al., 1999) (Arentz-Hansen et al., 2000; Molberg et al., 1998)


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Furthermore, as sequence analysis by Edman degradation demonstrated that the glycopeptides isolated by lectin affinity chromatography did indeed originate from the class I MHC antigen presentation pathway, this provides further evidence for the natural presentation by human class I MHC of glycopeptides carrying terminal O-␤-GlcNAc residues in vivo (Kastrup et al., 2000).

V. Posttranslational Modifications and T Cell Recognition

A. T CELL RECOGNITION OF SYNTHETIC MODIFIED PEPTIDES The ability to induce T cell responses against small organic molecules such as the trinitrophenyl (TNP) derivatives trinitrochlorobenzene or trinitrobenzene sulfonic acid was first reported in the 1970s (Finberg et al., 1979; Schmitt Verhulst et al., 1978; Shearer, 1974). These hapten compounds are thought to form covalent complexes with protein before or during class I or class II MHCrestricted presentation of the peptide fragments. CTL raised against haptenated peptide were hapten-specific, i.e., they generally did not cross-react with the nonhaptenated peptide control (Martin et al., 1992; Ortmann et al., 1992). The fact that the TcR repertoire encompasses receptors which are able to recognize antigens which differ from conventional peptide side-chains raised the question of whether posttranslationally modified peptides can also be presented by MHC molecules for recognition by T cells. Since then, a number of studies have demonstrated that it is possible to raise T cells against posttranslationally modified peptides bound to MHC molecules utilizing glycosylated, phosphorylated, or cysteinylated peptide analogues of known T cell epitopes. In general, three patterns of T cell responses result from the posttranslational modification of peptides, including loss of recognition, cross-reactivity, or specific recognition of the posttranslational modification. Ishioka et al. (1992) observed different effects regarding T cell recognition of the OVA323–339 peptide when glycosylated at different positions. When the glycan was located on residues outside the core MHC-binding region, no effect on T cell recognition was observed. However, when the glycan was located within the core MHC-binding region, a T cell response to the glycopeptide could be generated in mice if the glycosylation did not block MHC-binding. Furthermore, the data indicated that the glycan was an important part of the antigenic determinant recognized by the T cells, since T cells from animals immunized with GlcNAc-substituted peptides failed to recognize nonglycosylated peptide or peptides displaying a structurally related cyclohexyl-Asn glycan analogue instead of GlcNAc-Asn. In the study by Harding et al. (1993) T cell hybridomas were produced after immunization of CBA/J mice with the peptide HEL52–61 carrying galabiose (Gal␤(1-4)Gal␤) at the amino terminus. Many of the T cell hybridomas

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were glycopeptide specific and responded only to the galabiose-modified HEL peptide but not to the nonglycosylated HEL peptide or to the HEL peptide modified with a single galactosyl residue or galabiose bound to a different I-Ak binding peptide. However, the T cells directed to the galabiose modified HEL peptide also recognized glycopeptides having significant variation in the disaccharide structure such as HEL glycopeptides carrying lactose (Gal␤(1-4)Glc␤), cellobiose (Glc␤(1-4)Glc␤, or acetylated galabiose. These results indicate that the T cells recognize a peptide conformation specific to glycopeptide-I-Ak complexes, and that the recognition does not involve a direct interaction between the carbohydrate moiety and the TcR (Harding et al., 1993). Our own studies on the immunization of C57/BL6 mice with the O-␤-GlcNAc modified CTL epitope from Sendai virus NP gave rise to both Kb- and Db-restricted CTL responses with a high degree of specificity for the glycosylated peptide. The T cells showed only marginal cross-reactivity toward the nonglycosylated peptide or a glycopeptide containing the same core peptide but an O-␣-linked GalNAc structure instead of the natural O-␤-linked GlcNAc structure (Haurum et al., 1994). These results clearly demonstrated the ability to generate specific class I MHC-restricted CTL responses to glycopeptides, and suggested that the glycan is involved in a specific contact with the TcR. This contrasts with the findings of Harding et al. (1993), although the difference may be explained by the position of the posttranslational modification in the peptide being critical for T cell recognition. Thus, modification of central peptide residues may be essential for glycan specificity of the T cells. In support of this, a recent study showed that galabiose attached in the middle of the HEL52–61 peptide resulted in T cells recognizing both the carbohydrate and the peptide in an MHC-restricted way (Deck et al., 1995). The same group also investigated the antigenic specificity of two T cell hybridomas generated against the disaccharide galabiose attached to the fifth residue of the I-Ak binding peptide HEL 52–61. The results indicated that the distal Gal residue of the galabiose, together with exposed side chains of the peptide, were directly recognized by the T cells, again confirming the fine specificity of T cells raised against carbohydrate molecules on MHC binding peptides (Deck et al., 1999). In a study by Jensen et al. (1996), Tn-modified analogues of the mouse hemoglobin-derived Hb67–76 were tested for immunogenicity in CBA/J mice. They found that some of the MHC-binding peptides became more immunogenic when glycosylated. In particular, glycopeptides with Tn attached to the Ser or Thr at peptide position 72, which is known to be the dominant T cell receptor contact residue of Hb67–76, was more immunogenic when glycosylated. The fine specificity of T cell hybridomas raised against the Hb67–76 modified


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with the Tn antigen at position 72 was analyzed against a panel of Hb67–76derived glycopeptides modified at position 72 with different glycans (Jensen et al., 1997). The hybridomas showed a high degree of specificity for the ␣-GalNAc moiety and only few and weak responses were found to glycopeptides containing other glycans, although some of these structurally were very similar to ␣-GalNAc. The fine specificity toward the peptide moiety was investigated by testing the responses of the hybridomas to a panel of Hb67–76-␣-GalNAc glycopeptides substituted with alanine (Ala) at all positions except for the two MHC anchor positions and position 72 to which the ␣-GalNAc was attached. These results showed that substituting amino acids which pointed toward the TcR often resulted in a decreased stimulation of the hybridomas, while substituting amino acids pointing down into the MHC binding cleft did not affect hybridoma activation. This indicates that both the glycan as well as solvent-accessible parts of the peptide are recognized with a high degree of specificity by the T cells (Jensen et al., 1996). Recently, Glithero et al. (1999) determined the crystal structure of glycopeptides in complex with H-2Db (Fig. 1). It was found that the O-␤-GlcNAc of two slightly different H-2Db-binding glycopeptides (K2G and K3G) were solvent exposed and directly available for recognition by the TcR. Furthermore, H-2Db-restricted CTL were raised against both of these glycopeptides, and it was found that the H-2Db-restricted K2G-specific clones exhibited a strong degree of cross-reactivity toward K3G, whereas K3G-specific clones displayed very limited cross-reactivity toward K2G. Modeling of the glycopeptide-specific TcR from CTL clones raised against K2G or K3G suggested a molecular basis for this CTL cross-reactivity. The O-␤-GlcNAc of K3G adopts two conformations ◦ which together occupy a relatively restricted volume of space around 260 A3, whereas the same sugar in K2G adopts at least three conformations that may occupy a volume of space up to double that in K3G. The highly cross-reactive K2G CTL clones seemed to recognize both glycopeptides by virtue of a large cavity required to accommodate the K2G glycan, and this space would be anticipated to also accommodate the K3G sugar conformations. The specificity of the K3G clones seemed to be determined in part by a longer ␤CDR3 loop that may limit the size of the “glycan binding pocket” in such a way as to restrict the access of O-␤-GlcNAc of K2G, which occupies several conformations within a larger volume of space and in slightly different positions (Glithero et al., 1999). In another crystallographic study, the structure of H-2Kb in association with the glycopeptide, RGY8-6H-Gal2 (where H refers to a homocysteine-ethylene linker) was solved (Speir et al., 1999). Immunization with this glycopeptide generates a population of CTL that expresses both ␣/␤ TcR, specific for glycopeptide,

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FIG. 1. Crystal structure of FAPS(O-GlcNAc)NYPAL (K3G, left hand panel) and FAPGS (O-GlcNAc)YPAL (K2G, right hand panel) bound to H2-Db. Only the peptide-binding groove of the H2-Db molecule is shown. A model of the putative peptide-contacting region of T cell receptors raised against K3G and K2G are also shown superimposed on their respective ligands. Both receptors share an alpha chain (green) and the beta chains (red or blue) differ only slightly in the CDR3 loop. These differences explain the fact that the K3G-specific receptor (green and blue) does not recognise K3G, wheras the K2G-specific receptor (green and red) cross-reacts strongly with K3G (Reproduced with permission from Glithero et al. 1999.) (See color insert.)

and ␥ /␦ TcR, specific for the disaccharide structure, even on glycolipids. The structure presented in this study reveals that the peptide and H-2Kb structures are not affected by the peptide glycosylation, but that the central region of the putative TcR binding region is dominated by the exposure of the disaccharide structure. The crystallographic data provide further evidence that small carbohydrates attached to short, centrally placed linkers allow ␣/␤ TcR binding, and that carbohydrate of the same size attached to longer linkers also may satisfy ␥ /␦ TcR antigen binding. Phosphorylation is another common posttranslational modification with potential importance for T cell immunity. Larson et al. (1992) described the effect of phosphorylating a synthetic I-Ak-binding peptide constituting the immunodominant epitope from rabies virus phosphoprotein. This significantly reduced the antigenic potency of the peptide. However, it was not examined whether the reduction in antigenic potency reflected decreased binding to I-Ak or impaired T cell recognition. We recently investigated the immunogenicity of synthetic phosphopeptides derived from phosphorylated viral proteins or oncogene products and found that phosphopeptides presented by class I MHC molecules can elicit phosphopeptide-specific CTL (Andersen et al., 1999). Furthermore, CTL raised against a non-phosphorylated peptide recognize target cells pulsed with the nonphosphorylated peptide, most generally without recognizing target cells pulsed


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with the phosphorylated peptide. Thus, T lymphocytes are able to discriminate between phosphorylated and nonphosphorylated peptides. In another study, Van Stipdonk et al. (1998) described how murine class II MHC-restricted T cells raised against differentially phosphorylated forms of ␣-crystallin discriminate between the phosphorylated and the nonphosphorylated peptides. Finally, in the study by Meadows et al., (1997), T cells were identified which required cysteinylated Cys residues for recognition. Also, Chen et al.(1999) investigated two H-2Kd-restricted cysteine-containing influenza virus NP determinants (NP39–47 and NP218–226). It was found that the antigenicity of synthetic peptides was enhanced between 10 and 100 fold by treatment with reducing agents, although the affinity for Kd was not enhanced. Similar effects were obtained by substituting Cys residues with Ala or Ser in the synthetic peptides, indicating that sulfhydryl modification of Cys residues may result in reduced antigenicity of the determinants NP39–47 and NP218–226. In addition, it was shown that T cells specific for cysteinylated NP218–226 are also induced during influenza virus infection in mice, strongly indicating that this modification occurs in vivo. These findings suggest that posttranslational modification of peptides must be considered when studying peptide immunodominance hierarchies. In conclusion, it has clearly been demonstrated that T cells are able to recognize posttranslationally modified peptides carrying small nonprotein structures such as carbohydrate or phosphate groups as well as modified protein structures such as cysteinylated Cys. It appears very likely that for many of these examples, both the posttranslational modification and the peptide side-chains make contact with the TcR binding site. However, it might be expected that if peptides carrying larger modifications (e.g., N-linked glycans) bind to MHC molecules, then these will block access of the TcR to the MHC molecule, resulting perhaps in modification-specific but peptide-independent and/or MHC-unrestricted T cell responses. Importantly, T cells which recognize the carbohydrate in a peptide and MHC independent manner have indeed been described. Abdel-Motal et al. (1996) found that immunization of mice with galabiose bound to homocysteine in position 6 in a Kb-binding vesicular stomatitis virus NP-derived peptide generated both glycopeptide-specific Kb-restricted CTL and unrestricted glycan-specific CTL. Preliminary data indicated that the CTL belong to two different T cell populations with regard to the TcR expression (␣/␤ and ␥ /␦, respectively). B. T CELL RESPONSE AGAINST MODIFIED PEPTIDES in vivo A number of reports have described naturally occurring T cell responses against posttranslationally modified peptide presented by MHC, and in several cases the T cell response seems directly involved in disease pathology.

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Thus, in a model for the autoimmune disease rheumatoid arthritis, immunization of mice with rat type II collagen induces a collagen-specific, I-Aq-restricted immune response resulting in arthritis due to a cross-reactive autoimmune response to type II collagen in cartilage, the so-called collagen-induced arthritis (CIA). The peptide CII256–270 has been identified as the immunodominant class II MHC-restricted T cell epitope in CIA (Michaelsson et al., 1992). Interestingly, it was found that several T cell hybridomas, which responded to the CNBrcleaved type II collagen fragment CB11 (containing the immunodominant determinant), did not respond to the synthetic peptide CII256–270 (Michaelsson et al., 1992). The peptide CII256–270 contains two lysine residues which are susceptible to hydroxylation and glycosylation with ␤-Gal or Glc(1-2)Gal␤ residues. Biochemical elimination of the carbohydrate on type II collagen blocked recognition by those T cell hybridomas, which did not respond to the synthetic nonmodified peptide (Michaelsson et al., 1994). Furthermore, it was found that the incidence, time of onset, and severity of arthritis strongly correlated with the presence of carbohydrate on the collagen antigen, suggesting that carbohydrate on posttranslationally modified tissue-specific proteins may in some circumstances be a critical factor for development of autoimmune disease. Another study indicated that T cell recognition of the naturally glycosylated immunodominant determinant CII256–270 specifically involved the carbohydrates (Michaelsson et al., 1996), and was not merely due to the T cells recognizing a distorted peptide conformation induced by the carbohydrates, as proposed by Harding et al. (1993). Recently, it was established that the majority of hybridomas obtained by immunization of mice with type II collagen are specific for glycosylated CII256–270 containing the monosaccharide ␤-Gal attached to hyLys at peptide position 264 (Broddefalk et al., 1998; Corthay et al., 1998). Another exciting example of a naturally occurring T cell response against a posttranslationally modified peptide antigen has been described in the HLADQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation observed in individuals suffering from CD is caused by exposure to wheat gliadin and is believed to be driven by a CD4+ T cell response to this dietary antigen after it has been specifically modified by tissue transglutaminase (tTGase). This enzyme has been shown to specifically deamidate particular Gln residues within gliadin, resulting in the generation of modified peptides displaying increased binding to HLA-DQ2 and -DQ8, thereby creating neoepitopes for recognition by gut-derived T cells (Molberg et al., 1998). Recently, the same group demonstrated that the intestinal T cell response to gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides. A panel of gliadin-recombinant antigens was tested to characterize the T cell response to gliadin using gluten-specific T cell lines and clones cultured from intestinal biopsies of DQ2-positive CD patients. Two distinct but overlapping epitopes were identified, and in the seven-residue overlapping fragment


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common to the two epitopes, tTGase converts a Gln residue into a glutamic acid (Glu). This enzymatic posttranslational modification of Gln to Glu is critical for T cell recognition and probably also for disease pathology. Interestingly, the binding studies revealed that the deamidated peptides displayed increased affinity for DQ2, and that the modified Gln of the two overlapping peptide epitopes was accommodated in different pockets of DQ2. Thus, the modifications of anchor residues lead to an improved affinity for MHC, and it was suggested that altered conformation of the peptide–MHC complex may be a critical factor leading to T cell responses to gliadin (Arentz-Hansen et al., 2000). Importantly, enzymatic modifications such as deamidation may also take place on self-antigens and be relevant for the breaking of tolerance and initiation of autoimmune disease. Another important example of a naturally occurring T cell response against posttranslationally modified peptide antigens in vivo was reported from a study of the fine specificity of the human T cell response against the major bee venom allergen phospholipase A2. Phospholipase A2 is a 16- to 20-kDa protein which is glycosylated on a single Asn residue (Dudler et al., 1995). Several class II MHC-restricted T cell clones were identified which proliferated in response to the glycoprotein but not to nonglycosylated variants. Antibody directed against the carbohydrate structure inhibited T cell proliferation, and peripheral blood mononuclear cells isolated from individuals immune to bee venom phospholipase A2 showed higher proliferation in response to glycosylated compared to nonglycosylated antigen. Together these findings suggested that a glycan was required for T cell recognition of phospholipase A2 following natural exposure to the glycoprotein. Zhao and Cheung (1995) identified GD2 oligosaccharide-specific CTL. The disialoganglioside GD2 is a glycolipid expressed at high levels in certain human tumors and a small group of murine lymphomas such as EL4. Immunization of C57/B16 mice with EL4 cells stimulated a H-2b-restricted, GD2-specific CTL response. The CTL response could be completely inhibited by GD2oligosaccharide-specific antibodies. These findings again supported the notion that natural carbohydrates can function as antigenic epitopes for T cells in vivo. In a study of H-2Ld-restricted CTL recognition of peptides derived from the enzyme ␣-ketoglutarate dehydrogenase (␣-KGDH), a naturally processed peptide was identified which differed by one oxygen atom from a previously described natural peptide, consistent with a phenylalanine (Phe) to Tyr substitution at peptide position 4 (Wu et al., 1995). This was suggested to be the result of posttranslational hydroxylation by the liver microsomal Phe hydroxylase, and a possible relationship between the unusual abundance of ␣-KGDH peptides in the liver and the human autoimmune disease primary biliary cirrhosis was proposed, which may be caused by CTL responses specific against posttranslationally hydroxylated peptides derived from ␣-KGDH.

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Although not the focus of this review, it should be noted that human ␣␤ T cells have also been described which recognize nonpeptide antigens presented by the CD1 molecule. The molecule CD1 is not encoded in the MHC, but shares structural features with class I MHC molecules (Bendelac, 1995). The human CD1b isotype has been shown to present lipoglycans, and the T cell recognition appears to be dependent on both the glycan and the lipid component (Sieling et al., 1995). T cells expressing the ␣␤ TcR have also been isolated that recognize nonpeptidic antigen, most likely carbohydrate, in an MHC-independent, CD1independent, but antigen-presenting cell-dependent way (Corinti et al., 1997). These findings are important, as they extend the potential repertoire of antigens recognized by ␣␤ T cells. VI. Posttranslationally Modified Peptide Antigens: Are They Immunologically Relevant?

There are several potential outcomes of posttranslationally modifying a MHCrestricted peptide antigen, with respect to the ensuing immune response: First, a neoepitope could be created by providing a novel structure for the TcR to engage. Second, if all peptides in a given pool of epitopes are posttranslationally modified, an existing immune response to the unmodified peptide could be blocked at the level of T cell recognition. Third, an existing response could be blocked at the level of processing and binding of the peptide to the MHC molecule (especially if an anchor residue is modified). Fourth, a neoepitope could be generated by providing a novel MHC anchor structure. Fifth, the T cell response may be unchanged, for example, due to T cell specificity for a peptide sequence distal to the site of the modification. Presentation of posttranslationally modified peptides may be of great relevance in the immune defense against infectious diseases, since many posttranslationally modified proteins have been identified from viruses and parasites. Thus, the repertoire of T cell responses against microbial targets very likely includes posttranslationally modified epitopes in vivo. In addition, the posttranslational modification of viral or parasitic proteins might constitute a new immune escape mechanism, preventing the immune system from eliminating the pathogen through interference at the level of antigen processing or T cell recognition. For example a viral point mutation could introduce a novel glycosylation site, potentially resulting in T cell epitope conversion from a recognized epitope to an escape determinant. Many studies have demonstrated that the pattern of posttranslational modifications of proteins reflects the intracellular milieu in which they are expressed. Thus, numerous studies have associated alterations in the pattern of glycosylation and phosphorylation with autoimmune disease (Malhotra et al., 1995) or malignant transformation (Dennis et al., 1987; Itzkowitz, 1992; Nilsson, 1992; Orntoft,


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1992; Springer, 1984). The mechanisms behind disease-associated changes in the pattern of posttranslational modifications are not well understood. Possible mechanisms are deregulation of transferase function, or increased/decreased transferase expression (Smets and Van Beek, 1984). Changes in the pattern of posttranslational modifications in various disease states opens the possibility that altered posttranslational modification of intracellular proteins could result in the conversion of a silent (cryptic) nonmodified peptide sequence into a modified neoepitope or vice versa. This could give rise to a tumor neoepitope (if resulting from malignant transformation), or to an autoantigen (if in autoimmune disease), particularly if negative selection of T cells specific for the novel peptide structure has not occurred. Although the number of examples of naturally occurring posttranslationally modified MHC-restricted peptide antigens is still limited, evidence that posttranslational modifications can be recognized by T cells is indeed accumulating. Thus, it is not known just how common are posttranslational modifications of MHC-restricted peptides. Most T cell epitope mapping efforts are still based on the use of synthetic peptides, which would miss posttranslationally modified epitopes. Also, current preparative or analytical methods may simply not be sufficiently sensitive toward the detection of modified peptides, e.g., glycosylated peptides. The techniques currently applied to characterize posttranslational modifications of protein include high-sensitivity mass spectroscopy but are generally carried out on protein of known sequence. This is in stark contrast with the task of identifying individual MHC-derived posttranslationally modified peptides, where the starting material consists of complex peptide mixtures of unknown sequence, and where only the major individual peptide species reach picomole amounts. Therefore, the techniques used to examine the repertoire of peptides presented naturally by MHC molecules need to be especially adapted to the detection of natural posttranslational modifications, before a detailed understanding of the importance for antigen presentation of peptide modifications can be reached. Hence, the potential importance of posttranslationally modified peptides for MHC-restricted antigen presentation may have been underestimated previously due to an underestimation of their natural occurrence. However, the methodology for determining sequence information by mass spectrometry is continuously improving, and still more complex mixtures of peptides can be sequenced. Thus, using tandem quadropole mass spectrometry Skipper et al. (1996) identified the first posttranslationally modified class I MHC-restricted antigen, and recently Pierce et al. (1999) have identified yet another posttranslationally modified class I MHC-restricted antigen using novel mass spectrometric instrumentation. By a combination of nanoflow liquid chromatography with electrospray ionization on a Fourier transform mass spectrometer, detection of peptides at levels as low as 2–10 amol with mass measurement accuracy in the millimass range is

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possible. Thus, this continuous improvement of instrument sensitivity and sample handling techniques in the methodology of mass spectrometry will undoubtedly lead to identification of new posttranslationally modified peptides and firmly establish such peptides as an immunologically important category of natural MHC-restricted antigens. REFERENCES Abdel Motel, U. M., Berg, L., Rosen, A., Bengtsson, M., Thorpe, C. J., Kihlberg, J., Dahmen, J., Magnusson, G., Karlsson, K. A., and Jondal, M. (1996). Immunization with glycosylated Kb-binding peptides generates carbohydrate-specific, unrestricted cytotoxic T cells. Eur. J. Immunol. 26, 544–551. Andersen, M. H., Bonfill, J. E., Neisig, A., Arsequell, G., Sondergaard, I., Valencia, G., Neefjes, J., Zeuthen, J., Elliott, T., and Haurum, J. S. (1999). Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL. J. Immunol. 163, 3812–3818. Arentz-Hansen, H., Korner, R., Molberg, O., Quarsten, H., Vader, W., Kooy, Y. M., Lundin, K. E., Koning, F., Roepstorff, P., Sollid, L. M., and Mc-Adam, S. N. (2000). The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J. Exp. Med. 191, 603–612. Bendelac, A. (1995). CD1: Presenting unusual antigens to unusual T lymphocytes. Science 269, 185–186. Broddefalk, J., Bäcklund, J., Almqvist, F., Johansson, M., Holmdahl, R., and Kihlberg, J. (1998). T cells recognize a glycopeptide derived from type II collagen in a model for rheumatoid arthritis. J. Amer. Chem. Soc. 120, 7676. Chen, W., Yewdell, J. W., Levine, R. L., and Bennink, J. R. (1999). Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants. J. Exp. Med. 189, 1757–1764. Chicz, R. M., Urban, R. G., Gorga, J. C., Vignali, D. A., Lane, W. S., and Strominger, J. L. (1993). Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. J. Exp. Med. 178, 27–47. Corinti, S., Palma, R. D., Fontana, A., Gagliardi, C., Pini, C., and Sallusto, F. (1997). Major histocompatibility complex-independent recognition of a distinctive pollen antigen, most likely a carbohydrate, by human CD8+ alpha/beta T cells. J. Exp. Med. 186, 899–908. Corthay, A., Bäcklund, J., Broddefalk, J., Michaelsson, E., Goldschmidt, T. J., Kihlberg, J., and Holmdahl, R. (1998). Epitope glycosylation plays a critical role for the T cell recognition of type II collagen in collagen-induced arthritis. Eur. J. Immunol. 28, 2580–2590. Creighton, T. E. Ed. (1993). Biosynthesis of proteins. In Proteins. Structures and Molecular Properties. W. H. Freeman and Company: New York, pp. 49–100. Deck, B., Elofsson, M., Kihlberg, J., and Unanue, E. R. (1995). Specificity of glycopeptide-specific T cells. J. Immunol. 155, 1074–1078. Deck, M. B., Sjolin, P., Unanue, E. R., and Kihlberg, J. (1999). MHC-restricted glycopeptide specific T cells show specificity for both carbohydrate and peptide residues. J. Immunol. 162, 4740. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L., and Kerbel, R. S. (1987). Beta 1–6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236, 582– 585. Dobos, K. M., Khoo, K. H., Swiderek, K. M., Brennan, P. J., and Belisle, J. T. (1996). Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. J. Bacteriol. 178, 2498–2506.


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Gastrointestinal Eosinophils in Health and Disease MARC E. ROTHENBERG,* ANIL MISHRA, ERIC B. BRANDT, AND SIMON P. HOGAN Division of Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039

I. Introduction

Eosinophils are multifunctional proinflammatory leukocytes implicated in the pathogenesis of numerous inflammatory processes, especially allergic disorders (Gleich and Adolphson, 1986; Weller, 1991); in addition, it has been recently recognized that eosinophils may have a physiological role in organ morphogenesis (e.g., postgestational mammary gland development) (Gouon-Evans et al., 2000). Eosinophils express numerous receptors (for cytokines, immunoglobulin, and complement proteins) that when engaged lead to eosinophil activation, resulting in several processes, including the release of toxic secondary granule proteins (Fig. 1) (Rothenberg, 1998). The secondary granule contains a crystalloid core composed of major basic protein (MBP) and a granule matrix which is mainly composed of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO). These proteins elicit potent cytotoxic effects on a variety of host tissues at concentrations similar to those found in biological fluid from patients with eosinophilia. The cytotoxic effects of eosinophils may be elicited through multiple mechanisms, including degrading cellular ribonucleic acid, since ECP and EDN have substantial functional and structural homology to a large family of ribonuclease genes (Rosenberg et al., 1995; Slifman et al., 1986). ECP also inserts ion-nonselective pores into the membranes of target cells, which may allow the entry of the cytotoxic proteins (Young et al., 1986). Further proinflammatory damage is caused by the generation of unstable oxygen radicals formed by the respiratory burst oxidase apparatus and EPO. Furthermore, direct degranulation of mast cells and basophils is triggered by MBP. In addition to being cytotoxic, MBP directly increases smooth muscle reactivity by causing dysfunction of vagal muscarinic M2 receptors (Jacoby et al., 1993). Activation of eosinophils also leads to the generation of large amounts of leukotriene (LT)C4, which induces increased vascular permeability, mucous secretion, and smooth muscle constriction (Lewis et al., 1990). Additionally, activated eosinophils generate a wide range of cytokines, including interleukin (IL)-1, IL-3, IL-4, IL-5, IL-13, GM-CSF, TGF-␣/␤, TNF-␣, RANTES, macrophage inflammatory protein (MIP)-1␣, vasoactive intestinal ∗ To whom correspondence should be addressed. Telephone: (513) 636-7210; Fax: (513) 636-3310;

E-mail: [email protected]. 291 C 2001 by Academic Press Copyright All rights of reproduction in any form reserved. 0065-2776/01 $35.00

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FIG. 1. Schematic diagram of an eosinophil and its diverse properties. Eosinophils are bilobed granulocytes that respond to diverse stimuli including allergens, helminths, viral infections, allografts, and nonspecific tissue injury. The secondary granules contain four primary cationic proteins designated eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). All four proteins are cytotoxic molecules; in addition, ECP and EDN are ribonucleases. In addition to releasing their preformed cationic proteins, eosinophils can also release a variety of cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, TGF␣/␤, GM-CSF, TNF␣, and IFN␥ ), chemokines (e.g., eotaxin, RANTES, and MIP-1␣), neuromediators (vasoactive intestinal peptide [VIP] and substance P), and can generate large amounts of leukotriene (LT)C4. Lastly, eosinophils can be induced to express MHC class II and costimulatory (e.g., B7.2) molecules, and may be involved in propagating immune responses by presenting antigen to T cells.

peptide, substance P, and eotaxin, indicating that they have the potential to sustain or augment multiple aspects of the immune response, inflammatory reaction, and tissue repair process (Kita, 1996). Finally, eosinophils have the capacity to initiate antigen-specific immune responses by acting as antigen-presenting cells. Consistent with this, eosinophils express relevant costimulatory molecules (CD40, CD28, CD86, B7) (Ohkawara et al., 1996; Woerly et al., 1999), secrete cytokines capable of inducing T cell proliferation and maturation (IL-2, IL-4, IL-6, IL-10, IL-12) (Kita, 1996; Lacy et al., 1998; Lucey et al., 1989), and can be induced to express major histocompatibility complex (MHC) class II molecules (Lucey et al., Weller, 1989). Interestingly, experimental adoptive transfer of antigen-pulsed eosinophils induces antigen-specific T cell responses in vivo (Shi et al., 2000). Increased levels of eosinophils are seen in the gastrointestinal tissue obtained from patients with a variety of disorders (Table I). In some of these diseases (e.g., eosinophilic gastroenteritis), eosinophils are believed to be the principal

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TABLE I EOSINOPHIL-ASSOCIATED GASTROINTESTINAL DISEASESa Primary Eosinophil Disorders Eosinophilic colitis Eosinophilic esophagitis Eosinophilic gastroenteritis Gastrointestinal Disorders Allergic colitis Inflammatory bowel disease Food allergy Gastroesophageal reflux Protein-sensitive enteropathy Systemic Disorders Idiopathic hypereosinophilic syndrome Parasitic infections (helminthic) a

These exclude primary diseases of the biliary tract.

effector cell, whereas in other diseases (e.g., inflammatory bowel disease and gastroesophageal reflux), the finding of eosinophils in tissues is an enigma, since eosinophils do not always appear to be degranulating, implying that they may have a regulatory function (Furuta et al., 1995; Gleich and Adolphson, 1986; Rothenberg, 1998; Weller, 1991). Understanding the processes that regulate eosinophil trafficking in the gastrointestinal tract is not only important in clinical diseases but may also have important implications in further understanding the role of eosinophils in innate immune responses and in immune surveillance of healthy tissues. Whereas numerous reviews have recently been written concerning eosinophils (Weller, 1991), their regulation (Hirai et al., 1997; Walsh, 1997), and role in allergic respiratory diseases (Capron and Desreumaux, 1997; Desreumaux and Capron, 1996; Gleich, 2000; Seminario and Gleich, 1994), this article is a comprehensive review focused on the properties and role of gastrointestinal eosinophils in health and disease. II. Gastrointestinal Eosinophils in Healthy States

Eosinophils have been noted to be present at low levels in numerous tissues. When a large series of biopsy and autopsy specimens were analyzed, the only organs that demonstrated tissue eosinophils (at substantial levels) were the gastrointestinal tract, spleen, lymph nodes, and thymus (Kato et al., 1998). Interestingly, eosinophil infiltrations were only associated with eosinophil degranulation in the gastrointestinal tract. For the purpose of systematically characterizing eosinophils, recent analyses have been primarily conducted in mice, since this species allows unique experimental manipulation. Identification of eosinophils

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has been facilitated by development of antiserum against murine MBP, since this reagent allows for eosinophil-specific recognition (Larson et al., 1995; Mishra et al., 1999). Interestingly, similar to human tissues, eosinophils predominantly reside in the hematopoietic organs, gastrointestinal tract, and thymus of mice (Matthews et al., 1998). A. LAMINA PROPRIA EOSINOPHILS Eosinophils throughout the gastrointestinal tract of conventional healthy mice (untreated mice maintained under pathogen-free conditions) are present in the lamina propria of the stomach, small intestine, cecum, and colon. No eosinophils are detected in the esophagus or tongue, which is a pattern of distribution that has been observed in humans (Furuta et al., 1995; Kato et al., 1998). Eosinophils are at similar levels in all mouse strains analyzed (BALB/c, C57BL/6, 129 SvEv, and Black Swiss Webster) and are predominantly localized in the submucosa and not in the mucosal or serosal layers of the gastrointestinal tract (Mishra et al., 1999). The distribution of eosinophils varies in different regions of the small intestine. For example, in the duodenum, eosinophils are primarily detected through the entire length of villi. Whereas in the jejunum and ileum, most eosinophils are at the base of villi in the region of the crypt of Lieberkuhn ¨ (Mishra et al., 1999). B. PEYER’S PATCH AND INTEREPITHELIAL EOSINOPHILS The identification of eosinophils as resident cells of the gastrointestinal lamina propria during normal healthy states (Matthews et al., 1998; Mishra et al., 1999) prompted the question whether eosinophils also resided in intestinal Peyer’s patches. B and T lymphocytes recirculate between three predominant compartments in the gastrointestinal tract (lamina propria, Peyer’s patches, and interepithelial regions). Using immunohistochemical techniques with the anti-MBP antibody, eosinophils were barely detected in any region of the Peyer’s patches or in the interepithelial compartment; whereas, the same mice had detectable eosinophils within the lamina propria (Matthews et al., 1998; Mishra et al., 1999). Thus, in contrast to lymphocytes, gastrointestinal eosinophils reside only in the lamina propria during healthy conditions. C. EOSINOPHILS IN PERINATAL MICE AND THE ROLE OF ENDOGENOUS FLORA Early studies have demonstrated a significant reduction in the number of lymphoid cells in the gastrointestinal tract of perinatal mice (Ferguson and Parrott, 1972a, 1972b). Lymphocytes and plasma cells do not appear in the lamina propria of the gastrointestinal tract until 3 weeks after birth (i.e., around the time of weaning) and only reach adult levels by 6 weeks of age in mice. Similarly, mast cells home into the gastrointestinal tract primarily after the first month of life in


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rats (Watkins et al., 1976; Woodbury and Neurath, 1978). An exception to this homing pattern is seen with ␥ /␦-T cells; these cells home to their interepithelial location in the absence of intestinal flora (Bandeira et al., 1990). Thus, it was of interest to determine whether eosinophils migrate into the gastrointestinal tract as a normal developmental process during gestation or if they migrate during the postnatal period in response to an extrinsic stimulus. Migration during the postnatal period would suggest that factors such as gastrointestinal colonization with bacteria are involved in initiating eosinophil homing. The absence of resident neutrophils in the gastrointestinal tract at baseline, suggested that granulocyte homing was unlikely to be a mere consequence of intestinal flora, since neutrophils would be expected to respond to this stimulus. We therefore examined the number of intestinal eosinophils in the mice 1 day prior to birth (embryonic Day 19) and during the first 2 weeks postpartum. Prenatal mice had readily detectable eosinophils located in similar regions as observed in adults (Mishra et al., 1999). In order to make comparisons between the experimental groups (e.g., to control for the growing size of the gastrointestinal tract during this time period), eosinophil numbers were normalized per unit area of the lamina propria and their concentrations were found to be similar at all ages (Fig. 2). Staining for CD45, a pan-leukocyte marker, assessed the presence

FIG. 2. Gastrointestinal eosinophil numbers in embryonic and perinatal mice. The number of eosinophils (A) and total leukocytes measured by CD45+ cells (B) residing in the small intestine of wild-type mice was determined in Day 19 embryos and postnatal mice during the first 2 weeks of life and in adult mice. Cell levels were normalized to the unit area (mm2) of the lamina propria. The mean ±SEM for littermate 129 SvEv mice (n = 5–12 mice in each group) is shown. In (A), there was no significant difference between any of the groups (p > 0.05). Reprinted in part with permission (Mishra et al., 1999).

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of total leukocytes in the gastrointestinal tract. CD45+ cells were substantially reduced in the perinatal mice compared with adult mice (Fig. 2), indicating that eosinophil homing was unique compared to most other gastrointestinal leukocytes. In an attempt to identify the stimulus for eosinophil homing, the role of endogenous intestinal bacterial flora on eosinophil homing into the gastrointestinal tract was examined by analyzing eosinophil levels in germ-free mice. Germ-free mice have never come in contact with viable bacteria and have been shown to have decreased levels of lymphocytes in the lamina propria (Ferguson, 1976). However, germ-free mice were found to have eosinophil levels that were similar to control colonized mice (Mishra et al., 2000b). Taken together, these results suggest that eosinophil homing into the gastrointestinal tract occurs prenatally, is independent of the presence of viable bacterial flora, and appears to be regulated by mechanisms distinct from those regulating other gastrointestinal leukocytes (e.g., mast cells and lymphocytes). D. ROLE OF CONSTITUTIVE EOTAXIN Numerous inflammatory mediators have been implicated in regulating eosinophil accumulation including IL-1, IL-3, IL-4, IL-5, IL-13, and granulocyte/ macrophage-colony stimulating factor (GM-CSF) and the chemokines RANTES, monocyte chemoattractant protein (MCP)-3, MIP-1␣, and eotaxin (Silberstein, 1995; Teixeira et al., 1995). Interleukin-3 and GM-CSF, in association with IL-5, have been shown to enhance eosinophil development, migration, and effector function, while IL-1, IL-4, IL-13, and tumor necrosis factor (TNF)-␣ regulate eosinophil trafficking by promoting adhesive interactions with the endothelium (Bochner et al., 1995; Bochner and Schleimer, 1994). In collaboration with IL-5, chemokines and lipid mediators [platelet-activating factor (PAF) and leukotriene B4] induce eosinophil trafficking by promoting chemoattraction. Of the mediators implicated in modulating eosinophil accumulation, only the recently described subfamily of chemokines, termed eotaxin, is specific for eosinophils (Rothenberg, 1999). Eotaxin was originally described as the chief eosinophil chemoattractant generated in a guinea pig model of allergic lung disease (Jose et al., 1994). Subsequently, several other chemokines (of the CC family) have been shown to be eosinophil-selective and have been termed eotaxin-2 (identified in mice and humans) and eotaxin-3 (identified only in humans) (Forssmann et al., 1997; Kitaura et al., 1999; Shinkai et al., 1999; Zimmermann et al., 2000). It is important to note that although several chemokines have activity on eosinophils, eotaxin is the only chemokine that was discovered based on a biological assay designed to identify eosinophil-specific chemoattraction, attaching further biological significance to this molecule. When the eotaxin cDNA from guinea


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pigs, mice, and humans was identified, the mRNA for this chemokine was noted to be constitutively expressed in a variety of tissues (Garcia-Zepeda et al., 1996; Rothenberg et al., 1995a,b). A finding in all species was that the intestine expressed relatively high levels of eotaxin mRNA. Examination of multiple segments of the gastrointestinal tract of mice for expression of eotaxin mRNA indicated that this chemokine was ubiquitously expressed, at variable levels, in all segments from the tongue to the colon. Notably, the constitutive expression of eotaxin was distinct from the expression of related chemokines (MCP-1, MCP-2, MCP-3, MIP-1␣), which were not readily detectable. Only RANTES and eotaxin-2 were detectable, but they were not ubiquitously expressed (Mishra et al., 1999; Zimmermann et al., 2000). The expression patterns of eosinophil-active chemokines at baseline indicated that eotaxin may be involved in the selective regulation of eosinophil homing in the gastrointestinal tract. Furthermore, eotaxin mRNA expression in the small intestine is localized to mononuclear cells that reside in the lamina propria, the region where most gastrointestinal eosinophils reside (Matthews et al., 1998). In order to test the role of eotaxin in regulating eosinophil homing to the gastrointestinal tract, the number of eosinophils in the small intestine of mice deficient in eotaxin (through gene targeting) (Rothenberg et al., 1997) was shown to be significantly lower when compared with wild-type mice (Matthews et al., 1998). For example, the duodenum, jejunum, and ileum of wildtype mice had 6.2 ± 1.0, 2 ± 0.4, and 1.7 ± 0.5 eosinophils/villus in comparison to eotaxin-deficient mice which had 0.52 ± 0.14, 0.09 ± 0.02, and 0.05 ± 0.03 eosinophils/villus, respectively. Other gastrointestinal segments were also examined in order to determine if eotaxin had a similar role in other regions of the gastrointestinal tract. Eosinophils were only rarely encountered in any segment of the gastrointestinal tract of eotaxin-deficient mice. The eosinophil numbers (mean ± SEM) in the stomach, cecum, and colon of wild-type mice were 36.7 ± 7.5, 69.3 ± 14, and 39 ± 10 cells/mm2, respectively, whereas eotaxin gene-targeted mice had 2.6 ± 0.6, 8.9 ± 2.9, and 7.4 ± 2.8 cells/mm2, respectively. No significant differences were observed in the level of bone marrow and peripheral blood eosinophils in these two groups of mice, indicating that eotaxin was having a tissue specific effect rather than a primary effect on eosinophil hematopoiesis (Mishra et al., 1999). To further support the hypothesis that eotaxin had a critical role in regulating eosinophil recruitment to the gastrointestinal tract, the level of gastrointestinal eosinophils in mice that were deficient in another eosinophil-active CC chemokine gene, MIP-1␣, was also examined. These gene-targeted mice did not have an alteration in the level of gastrointestinal eosinophils compared to wild-type mice (Mishra et al., 1999). Collectively, these data suggest that eotaxin has a key role in the modulation of eosinophil accumulation in the gastrointestinal tract and that the effect is primarily tissue specific.

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E. EOSINOPHIL HEMATOPOIETINS Interleukin-3, IL-5, and GM-CSF have been shown to be critically involved in the proliferation and accumulation of eosinophils in response to allergic stimuli (Lopez et al., 1986; Owen et al., 1987; Rothenberg et al., 1988, 1989). These three cytokines also regulate the postmitotic differentiation of eosinophils, including their survival, activation, and responsiveness to other signals (Silberstein et al., 1989). Additionally, activated eosinophils and eosinophils exposed to tissue components such as fibronectin have been shown to produce these cytokines, in particular GM-CSF (Anwar et al., 1993). It has been hypothesized that an autocrine process may be responsible, at least in part, for eosinophil tissue survival. The physiological role of IL-5 and the related cytokine, GM-CSF, was therefore investigated for their involvement in the regulation of baseline levels of gastrointestinal eosinophils. The IL-5 deficient mice but not the GM-CSF deficient mice had ∼50% reduction in gastrointestinal eosinophils compared with age, sex, and background controlled mice (Mishra et al., 1999). Mice that were deficient in the functional receptor for IL-5 and GM-CSF (␤c-gene-targeted mice) were also examined (Mishra et al., 1999). The ␤c-deficient mice (C57BL/6) had ∼80% reduction in gastrointestinal eosinophils compared with control mice. The effect of IL-5 and ␤c was not restricted to the gastrointestinal tract because mice deficient in these molecules had a ∼75% reduction in circulating levels of eosinophils. These data suggest that GM-CSF and IL-5 have a combined effect in regulating eosinophil levels in the gastrointestinal tract; however, this effect appears to be secondary to their effect on regulating the pool of circulating eosinophils. F. REGULATION BY LYMPHOCYTES T cells and their products (e.g., IL-5) are known to regulate the development of peripheral blood and pulmonary eosinophilia following allergen challenge (Gavett et al., 1994; Hogan et al., 1998b). It was therefore of interest to analyze the role of T cells in the maintenance of gastrointestinal eosinophil levels at baseline. Athymic nude mice are deficient in the majority of T cells and have impaired eosinophil responses to allergens and parasites (Hamelmann et al., 1997b). However, these mice have no difference in the level of eosinophils in the jejunum (1.7 ± 0.3 vs. 1.07 ± 0.15 [mean ± SEM, n = 8]) for control and athymic mice, respectively (Mishra et al., 1999). To investigate the role of residual T cells (e.g., intraepithelial ␥ /␦-T cells) and B cells in regulating eosinophil accumulation in the gastrointestinal tract, mice deficient in the recombinase gene-1 (RAG-1) were examined (Mombaerts et al., 1992). These mice also had a ∼50% reduction in jejunum eosinophils compared with strain matched controls. Analysis of blood and bone marrow levels of eosinophils in the RAG-1 deficient and athymic nude mice revealed significant increases in eosinophils


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(Mishra et al., 1999). These data indicate that in contrast to their critical role in regulating eosinophils during inflammatory conditions, lymphocytes do not have a major role in maintaining homeostatic levels of eosinophils in the gastrointestinal tract. III. Gastrointestinal Eosinophils in Disease States

The accumulation of eosinophils in the gastrointestinal tract is a common feature of numerous disorders such as drug reactions (Rothenberg, 1998), helminth infections (Behm and Ovington, 2000), idiopathic hypereosinophilic syndrome (Assa’ad et al., 2000; Bauer et al., 1996; Weller, 1994), eosinophilic esophagitis (Kelly, 2000), eosinophilic gastroenteritis (Katz et al., 1984; Keshavarzian et al., 1985; Torpier et al., 1988), allergic colitis (Hill and Milla, 1990; Odze et al., 1995; Sherman and Cox, 1982), inflammatory bowel disease (Dvorak, 1980; Sarin et al., 1978; Walsh and Gaginella, 1991), and gastroesophageal reflux (Brown et al., 1984; Liacouras et al., 1998; Winter et al., 1982) (Table I). A subset of these disorders may comprise a distinct set of hypersensitivity disorders that lies in the middle of a spectrum ranging from anaphylaxis to celiac disease (Fig. 3) (Moon and Kleinman, 1995; Saavedra-Delgado and Metcalfe, 1985; Sampson, 1999). Although the underlying causes of eosinophilic gastrointestinal disorders are not yet understood, several investigations have demonstrated an association between atopy and eosinophil-associated gastrointestinal disorders (Furuta et al., 1995; Sampson, 1999). For example, nearly half of the patients with eosinophilic gastrointestinal disorders are atopic as defined by elevated levels of

FIG. 3. The spectrum of inflammatory disorders of the gastrointestinal tract associated with eosinophil accumulation. Increased levels of eosinophils in the gastrointestinal tract occur in a wide variety of primary gastrointestinal disorders. These diseases vary in spectrum from strong dependence (e.g., food allergy) to low dependence (e.g., celiac disease) on IgE. Diseases in the intermediate spectrum are characterized by specific organ inflammation primarily associated with eosinophil accumulation (e.g., eosinophilic esophagitis, eosinophilic gastroenteritis, and eosinophilic colitis). In the latter set of diseases, increased levels of IgE have been associated with the disorders in a subset of patients, but the etiological role of IgE is not clear.

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total IgE or food-specific IgE (Caldwell et al., 1975; Cello, 1979; Furuta et al., 1995; Iacono et al., 1996; Sampson, 1997; Scudamore et al., 1982). In addition, IgE-mediated mast cell degranulation in eosinophilic gastroenteritis has been demonstrated (Oyaizu et al., 1985). Nevertheless, food-induced IgE-mediated reactions have been shown to be critical in only a minority of patients (Sampson, 1999; Talley et al., 1990). The accumulation of gastrointestinal eosinophils can occur in all regions of the gastrointestinal tract and can involve all depths of the tissue, including the mucosa, muscularis, and/or serosal layers (Kelly, 2000). In vitro studies have shown that eosinophil granule constituents are toxic to a variety of tissues including heart (Tai et al., 1984), brain (Venge et al., 1980), bronchial epithelium (Frigas et al., 1980), and intestinal epithelium (Gleich et al., 1979). Clinical investigations have demonstrated extracellular deposition of MBP and ECP in the small bowel of patients with eosinophilic gastroenteritis (Dvorak, 1980; Keshavarzian et al., 1985; Tajima and Katagiri, 1996; Talley et al., 1990; Torpier et al., 1988) and have shown a correlation between the level of eosinophils and disease severity (Desreumaux et al., 1996; Talley et al., 1990). Electron microscopy studies have revealed ultrastructural changes in the secondary granules (indicative of eosinophil degranulation and mediator release) in duodenal samples from patients with eosinophilic gastroenteritis (Torpier et al., 1988). Furthermore, Charcot–Leyden crystals, remnants of eosinophil degranulation, are commonly found on microscopic examination of stools obtained from patients with eosinophilic gastroenteritis (Cello, 1979; Klein et al., 1970). Despite these clinical findings, there is currently only a limited understanding of the biological and pathological significance of eosinophils in the gastrointestinal tract. Most studies on eosinophils in vivo have concentrated on trafficking and activation of these cells in the lung. It remains to be determined if the mechanisms involved in the regulation of eosinophil recruitment in the lung are conserved in the gastrointestinal tract. Therefore, elucidating the properties of gastrointestinal eosinophils and the molecular mechanisms of their tissue localization has important implications in further understanding this cell type and the pathogenesis of various gastrointestinal disorders. A. EOSINOPHIL-ASSOCIATED GASTROINTESTINAL DISEASES In classic food allergy, IgE-mediated hypersensitivity initially leads to mast cell activation leading to immediate clinical responses. In other forms of food allergy, an adverse immunologic response to food leads to a delayed clinical response, and eosinophils have been shown to be elevated in various inflamed segments of the gastrointestinal tract (Moon and Kleinman, 1995). Another set of patients have eosinophilic gastroenteritis, an idiopathic disease characterized by selective infiltration of eosinophils into the stomach, with variable involvement of the esophagus and/or large intestine (Katz et al., 1984; Keshavarzian et al., 1985;


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Torpier et al., 1988). Eosinophilic gastroenteritis encompasses multiple disease entities and can occur independent of peripheral blood eosinophilia ∼50% of the time, indicating the potential significance of gastrointestinal-specific mechanisms for regulating eosinophil levels. The disease has been divided, based on the degree of histological involvement. Most patients have mucosal involvement, whereas others have eosinophil infiltration in the muscularis and/or serosal layers. Atopy and food hypersensitivity are common in patients who have the mucosal pattern of the disease. In the atopic subset, IgE levels are elevated, and food-specific IgE has been detected. In another disorder, eosinophilic colitis, eosinophil accumulation occurs in the first few months of life and is a frequent cause of bloody diarrhea (Hill and Milla, 1990; Odze et al., 1995; Sherman and Cox, 1982). In these patients, eosinophils predominantly accumulate in the colon in the presence or absence of peripheral blood eosinophilia. The eosinophil infiltration appears to be triggered by cow’s milk protein hypersensitivity and improves upon withdrawal of the allergic triggers from the diet (Hill and Milla, 1990; Maluenda et al., 1984; Saavedra-Delgado and Metcalfe, 1985). In patients with gastroesophageal reflux, a common disorder than affects nearly 50% of the population, eosinophils are often found in the esophagus and are part of the diagnostic criteria for the disease (Brown et al., 1984). The magnitude of esophageal eosinophilia has been proposed to be a negative prognostic indicator (Liacouras et al., 1998; Winter et al., 1982) and adversely predicts response to conventional antigastroesophageal reflux medication (Ruchelli et al., 1999). It remains unclear if the trigger responsible for initiating the eosinophil accumulation in this disorder is secondarily related to the reflux of acidic gastric contents into the esophagus or if eosinophils themselves are pathogenically involved. In at least a subset of refractory patients, the severity of the gastroesophageal inflammation is reversed by institution of an allergen-free diet (Kelly et al., 1995). Lastly, patients with inflammatory bowel disease also have an accumulation of eosinophils in the gastrointestinal tract. Both forms of inflammatory bowel disease, Crohn’s disease and ulcerative colitis, are characterized by gastrointestinal eosinophilia; however, eosinophils usually represent only a small percentage of the infiltrating leukocytes (Desreumaux et al., 1999; Walsh and Gaginella, 1991). Interestingly, both diseases are associated with overproduction of eotaxin-1; however, there is controversy concerning whether IL-5 is overproduced in both disorders (Fuss et al., 1996; Hankard et al., 1997). Crohn’s disease is thought to be a predominantly T helper1 (Th1) and TNF-␣ associated response, whereas ulcerative colitis is predominantly a Th2-associated process accompanied by IL-5 overproduction. The level of eosinophils in inflammatory bowel disease lesions has been proposed to be a negative prognostic indicator (Desreumaux et al., 1999; Nishitani et al., 1998).

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IV. Experimental Dissection of Eosinophilic Gastrointestinal Inflammation

It is currently thought that eosinophils may augment and sustain gastrointestinal inflammatory responses through the release of proinflammatory mediators and/or granule cationic proteins that are toxic to the mucosa (Dvorak et al., 1993; Furuta et al., 1995; Kato et al., 1998; Sampson, 1999). However, it should be noted that these conclusions are primarily based on the recent exploitation of murine models of asthma and on evaluation of clinical tissue from patients with a variety of eosinophil-associated gastrointestinal disorders. Although substantial progress has been made in elucidating the inflammatory mechanisms involved in allergic responses in the lung, there has been limited progress in understanding the pathogenesis of allergic disorders of the gastrointestinal tract. The development of experimental models of allergy has provided important insights into the immunological mechanisms regulating systemic (e.g., anaphylaxis) and pulmonary (e.g., asthma) allergic diseases. Collectively, these studies have identified a central role for cytokines (e.g., IL-4, IL-5, and IL-13), CD4+ T cells, mast cells, and in particular, eosinophils, in the induction and sustainment of allergic inflammatory responses (Drazen et al., 1996; Wills-Karp, 1999). While there have been recent advances in modeling some allergic gastrointestinal disease processes (e.g., IgE-mediated anaphylaxis responses), there have been only limited models of eosinophil-associated gastrointestinal allergy, and the precise mechanisms regulating gastrointestinal eosinophilia and the immunopathological role of this leukocyte in gastrointestinal disorders remain an enigma (Furuta et al., 1995; Kelly, 2000; Sampson, 1999). In order to elucidate these processes, murine models of eosinophil-associated gastrointestinal allergy have recently been developed. A. EXPERIMENTAL EOSINOPHILIC GASTROENTERITIS One of the complexities of inducing allergic inflammation of the gastrointestinal tract is the ineffectiveness of orally administered soluble protein antigens in promoting hypersensitivity responses; rather, oral antigens generally promote immunological tolerance (Miller et al., 1992). The poor immune response and induction of oral tolerance is thought to be associated, at least in part, with gastric digestion of soluble protein antigens, which leads to the formation of nonimmunogenic peptides (Mestecky et al., 1978; Michael, 1989). In order to overcome immunological tolerance associated with oral administration of soluble antigens (Mayer et al., 1996; Weiner and Mayer, 1996), our group has used an encapsulated soluble protein antigen in order to protect against gastric digestion (Litwin et al., 1998). The encapsulated biodegradable antigen particles are resistant to degradation at gastric pH (pH 2.5); however, they are susceptible to degradation at pH 5.5 which facilitates the delivery and release of the allergen in a preserved native conformational state to the small intestine (Litwin


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et al., 1998). Extensive previous investigations have demonstrated that the particles may overcome immunological tolerance associated with oral administration of antigens and possess adjuvant and immunostimulatory activity promoting antigen-specific antibody production (Challacombe et al., 1997). Mice were intraperitoneally injected with the egg antigen ovalbumin (OVA) in the presence of adjuvant (alum) and subsequently challenged with oral OVAparticles after 12 and 15 days (Hogan et al., 2000). Administration of oral allergen to sensitized mice induced peripheral blood eosinophilia and allergen-specific IgE and IgG1 as well as expansion of a Th2-biased immune response. Histological examination of the jejunum revealed vascular congestion, edema, and a prominent cellular infiltrate in the oral allergen-challenged mice as compared to placebo-challenged mice (Hogan et al., 2000). The cellular infiltrate was predominantly localized to the mucosa and lamina propria throughout the small intestine and was primarily composed of eosinophils. The infiltrating eosinophils were observed interspersed throughout the reticular connective tissue of the lamina propria and mucosa and throughout the length of the lamina propria of the villi. Electron microscopic analysis revealed that the eosinophils were physically associated with damaged axons (Hogan et al., 2001). Placebo-challenged mice had low levels of eosinophils predominantly localized to the base of the villus in the region of the crypt of Lieberkuhn ¨ and occasional cells within the lamina propria of the villus. This distribution is similar to the location of eosinophils at baseline (na¨ıve mice), indicating that placebo challenge alone did not significantly affect eosinophil trafficking (Mishra et al., 1999). Morphometric analysis revealed that the level of eosinophils in oral allergen-challenged mice was significantly higher than that observed in placebo-challenged mice. Since mast cells have also been implicated in the pathogenesis of various gastrointestinal hypersensitivity responses, their participation in oral-antigen induced eosinophil-associated gastrointestinal allergy was preliminarily examined by determining their levels. Histological analysis of the jejunum revealed no significant difference in the level of mast cells and their degranulation status within the lamina propria and villus between placebo-challenged and oral allergen-challenged mice, suggesting that mast cells were not involved in propagating eosinophilic gastrointestinal inflammation. B. ORAL ALLERGEN-INDUCED LAMINA PROPRIA EOSINOPHILS ARE CRITICALLY REGULATED BY EOTAXIN Elevated levels of eotaxin and eosinophils have been associated with various human inflammatory disorders of the respiratory tract, and increased levels correlate with disease severity (Garcia-Zepeda et al., 1996; Lamkhioued et al., 1997; Ying et al., 1999). In addition, elevated levels of eotaxin mRNA are seen in the lesions of patients with inflammatory bowel disease (Garcia-Zepeda et al., 1996). Thus, it was critical to determine the role of eotaxin in gastrointestinal allergic

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inflammation. In particular, it was of interest to determine if the reduced level of lamina propria eosinophils, normally seen in non-allergen-exposed eotaxindeficient mice, was increased following oral allergen exposure. Oral allergen challenge of eotaxin-deficient mice induced a marked increase in peripheral blood eosinophils compared with placebo-challenged eotaxin-deficient mice. Interestingly, the peripheral blood eosinophilia was significantly higher than that of oral allergen-challenged wild-type mice. We hypothesized that the elevated level of eosinophils in the peripheral blood of oral allergen-challenged eotaxindeficient mice was due to the failure to recruit eosinophils to the gastrointestinal tract, thus preventing the transmigration of circulating eosinophils into the site of inflammation. To test this hypothesis, the level of eosinophils in the lamina propria of the small intestine of oral allergen-challenged eotaxin-deficient mice was examined. Histological analysis of the intestinal tissue from oral allergenchallenged mice revealed no significant morphological changes to the small intestine structural integrity in the absence of eotaxin. Morphometric analysis of anti-MBP stained tissue revealed that in contrast to wild-type mice, oral allergen challenge of eotaxin-deficient animals induced no significant increase in the level of eosinophils as compared to placebo-challenged eotaxin-deficient mice. The level of eosinophils in oral allergen-challenged eotaxin-deficient mice was markedly reduced compared to wild-type mice (P < 0.001). This indicates that the reduced baseline level of gastrointestinal lamina propria eosinophils in eotaxin-deficient mice is not increased by allergen challenge (Matthews et al., 1998). The reduction of intestinal inflammation in the absence of eotaxin was not due to the failure to develop allergen-specific lymphocyte responses, since eotaxin-deficient mice produced marked levels of allergen-specific IgE, IgG1, and Th2 cytokines. C. THE ROLE OF IL-5 It was of interest to define the role of IL-5 in regulating eosinophil-associated gastrointestinal allergy in the small intestine since this cytokine is a pivotal modulator of eosinophil trafficking during allergic airways inflammation (Foster et al., 1996; Hamelmann et al., 1997a; Hogan et al., 1998a; Nakajima et al., 1992). Interleukin-5 has been shown to mobilize eosinophils from the bone marrow into the circulation and to promote eosinophil tissue trafficking during allergic airway inflammation (Foster et al., 1996; Mould et al., 1997; Palframan et al., 1998). We therefore compared oral allergen-induced gastrointestinal allergy in IL-5 deficient and wild-type mice (Hogan et al., 1999). In marked contrast to wild-type mice, allergen challenge of IL-5 deficient mice did not promote a peripheral blood eosinophilia. Interestingly, after the second allergen challenge (Day 19), the level of eosinophils in the blood of these mice was significantly lower than placebo-challenged IL-5 deficient mice (p < 0.05). Levels of allergen-specific IgE and IgG1 resembled those present in wild-type mice.


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These data suggest that the limited number of circulating eosinophils present in IL-5 deficient animals were being recruited into the intestine following allergen challenge, thereby depleting the level of peripheral blood eosinophils. Morphometric analysis revealed a three-fold increase in the level of eosinophils in the intestine of allergen-challenged mice. The level of eosinophil recruitment was still lower than in allergen-challenged wild-type mice. These studies also indicated that the baseline production of eosinophils, which occurs independently of known eosinophil hematopoietins (IL-3, IL-5, and GM-CSF) (Mishra et al., 1999), provides a sufficient number of eosinophils for the development of tissue eosinophilia. In addition, combining the results of the studies conducted with eotaxin and IL-5 deficient mice allows us to propose a model to explain the dichotomy often seen between peripheral blood and tissue eosinophilia (Fig. 4). We propose that when eotaxin is overproduced relative to IL-5 (e.g., IL-5 genetargeted mice), then eosinophils predominantly accumulate in tissue locations (e.g., eosinophilic esophagitis and/or gastroesophageal reflux); whereas when IL-5 is overproduced relative to eotaxin (e.g., eotaxin gene-targeted mice), then eosinophils predominantly accumulate in the blood compartment (e.g., drug reaction). As a corollary, when IL-5 and eotaxin are both increased, then eosinophils accumulate in both the tissue and blood compartments (e.g., idiopathic hypereosinophilic syndrome). D. EFFECT OF T CELL OVEREXPRESSION ON LAMINA PROPRIA EOSINOPHILS Transgenic mice that overexpress IL-5 under the control of the T cell promoter CD2 were demonstrated to have a ∼10- to 20-fold increase in the number of eosinophils in the hematopoietic organs (Dent et al., 1990). Because T cells reside in the gastrointestinal tract, and local overexpression of IL-5 can promote eosinophil accumulation in the lung (Lee et al., 1997), we hypothesized that T cell-driven IL-5 transgenic mice would have increased levels of gastrointestinal eosinophils. Eosinophils in the lamina propria of the small intestine of IL-5 transgenic mice were 4-fold higher compared with wild-type mice (Mishra et al., 1999) (Fig. 5). Interestingly, IL-5 transgenic mice also had increased eosinophils in the esophagus (wild-type and IL-5 transgenic mice had 0.44 ± 0.23 and 21.6 ± 6.9 eosinophils/mm2, respectively [mean ± SEM, n = 3–4]), as will be discussed later. In light of known synergistic effects between eotaxin and IL-5 (Collins et al., 1995; Mould et al., 1997; Rothenberg et al., 1996), it was important to examine the relationship between eotaxin and IL-5 in regulating gastrointestinal eosinophil levels. In order to test the dependency of gastrointestinal eosinophil levels on eotaxin in the presence of elevated levels of IL-5, mice that were transgenic for IL-5 and deficient in eotaxin were generated (Mishra et al., 1999). These IL-5 transgenic/eotaxin-deficient mice had a significantly reduced level (∼10%)

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FIG. 4. Proposed model for the dichotomy between peripheral blood and tissue eosinophilia. Eosinophil accumulation can occur in the peripheral blood and/or tissue depending upon the relative expression of the eosinophil chemoattractant (eotaxin) in the tissue and the systemic level of IL-5. In the top panel, a model for peripheral blood and tissue eosinophilia (e.g., idiopathic hypereosinophilic syndrome) involving overexpression of both eotaxin and IL-5 is presented. In the middle panel, a model for peripheral blood eosinophilia in the absence of tissue eosinophilia is presented. In these disorders (drug induced peripheral blood eosinophilia), there is a relative overexpression of IL-5 compared with eotaxin (as exemplified in eotaxin deficient mice). Finally, in the bottom panel, a model for tissue eosinophilia in the absence of circulating eosinophilia is presented. In these disorders (e.g., eosinophilic gastroenteritis, eosinophilic esophagitis), a relatively higher level of eotaxin is expressed in the tissue compared with IL-5 (as seen in IL-5 deficient mice).

of eosinophils within the jejunum as compared with IL-5 transgenic wild-type eotaxin mice (Fig. 5). Furthermore, the IL-5 transgenic mice that were deficient in eotaxin had > 2-fold higher level of circulating eosinophils compared to IL-5 transgenic mice that had wild-type eotaxin. Collectively, these data suggest that the elevation in circulating eosinophils was a consequence of an inhibition of


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FIG. 5. Gastrointestinal eosinophils in mice carrying a combination of the CD2 IL-5 transgene and the wild-type or eotaxin gene-targeted allele. The level of eosinophils in the jejunum of littermate mice transgenic (Tg) or wild-type (WT) for IL-5, and carrying the wild-type (+/+) or homozygous deletion (−/−) of eotaxin is shown. Each data point represents an individual mouse. The horizontal line is the mean value. Reprinted in part with permission (Mishra et al., 1999).

eosinophil recruitment into the gastrointestinal tract in the absence of eotaxin. Furthermore, these data indicate that eotaxin, even in the presence of high levels of IL-5 and circulating eosinophils, is critical for maintaining gastrointestinal eosinophil levels. E. PEYER’S PATCH EOSINOPHILS Since IL-5 overexpression was associated with eosinophil accumulation in the lamina propria of the small intestine (Mishra et al., 1999), it was of interest to determine if eosinophils also accumulated in Peyer’s patches under conditions in which IL-5 is overproduced. Eosinophil levels were determined in the Peyer’s patches of mice transgenic for IL-5 under the control of the T cell promoter CD2 (Mishra et al., 2000). These transgenic mice were found to have a marked increase in the number of eosinophils in the Peyer’s patches as compared to wild-type mice. For example, the eosinophil levels in the Peyer’s patches of wild-type and IL-5 transgenic mice were 0.043 ± 0.025 (n = 8) and 9.71 ± 1.56 (mean ± SEM, n = 12) MBP/area (%), respectively. Interestingly, the number of eosinophils in the lamina propria of the small intestine of the same IL-5 transgenic and wild-type control mice, measured in parallel, exhibited a smaller increase in eosinophils (only 5-fold higher) (Mishra et al., 1999). Characterization of the distribution of eosinophils in IL-5 transgenic mice revealed that these cells were predominantly distributed in the outer cortex and interfollicular regions of Peyer’s patches, the T cell-rich regions.

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FIG. 6. Histological analysis of eosinophils in Peyer’s patches of wild-type mice following oral allergen treatment. OVA sensitized wild-type mice were subjected to two treatments with (A) oral saline or (B) OVA (in the form of enteric beads) (Hogan et al., 2000), and the presence of eosinophils in the Peyer’s patches was determined 3 days after the last challenge. Eosinophils in the Peyer’s patches were determined by anti-MBP immunostaining, which results in black staining of eosinophils (depicted with arrows). Representative Peyer’s patches (designated PP) are shown with eosinophils present in the outer cortex and interfollicular regions only following allergen challenge (B). Eosinophils are also readily detectable in the lamina propria following allergen challenge. Following control saline treatment, eosinophils in Peyer’s patches remain at low baseline levels (data not shown) (Mishra et al., 2000b). (See color insert.)

To determine the relationship between IL-5 and eotaxin in regulating eosinophil trafficking to Peyer’s patches, IL-5 transgenic mice that were either genetically wild-type or deficient in eotaxin were evaluated for the presence of eosinophils in Peyer’s patches (Mishra et al., 2000). The level of eosinophils in Peyer’s patches was markedly increased in IL-5 transgenic mice and reduced in eotaxin-deficient IL-5 transgenic mice. In the absence of the eotaxin, there was a ∼3-fold reduction in the number of eosinophils in Peyer’s


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patches compared to IL-5 transgenic mice. Eosinophil levels were 9.7 ± 1.6 (mean ± SEM, n = 11) and 3.7 ± 1.9% MBP/area (mean ± SEM, n = 8) in IL-5 transgenic mice and eotaxin-deficient IL-5 transgenic mice, respectively. However, the level of eosinophils in eotaxin-deficient IL-5 transgenic mice was significantly higher than that observed in wild-type mice. Collectively, these data indicate the occurrence of IL-5 mediated eotaxin-dependent and -independent pathways for eosinophil trafficking to Peyer’s patches. It was also of interest to determine if eosinophils migrated into Peyer’s patches during Th2-associated conditions induced by experimental challenge with mucosal allergens. Mice were subjected to two distinct models of mucosal allergeninduced eosinophilic inflammation (Mishra et al., 2000). In the first approach, a well-accepted model of allergic airway disease using repeated doses of intranasal Aspergillus fumigatus antigen was employed (Kurup et al., 1992; 1994). Mice exposed to nine doses of intranasal antigen developed marked increases (>50 fold) in eosinophils in the peripheral blood and lung (Mishra et al., 1999). When the Peyer’s patches from these mice were examined, they were found to have increased levels of eosinophils following the allergen challenge. Eosinophils in the allergen-challenged mice were predominantly located in the outer cortex and interfollicular regions similar to their location in IL-5 transgenic mice. Morphometric analysis of eosinophil levels confirmed a marked increase in eosinophil levels in the Peyer’s patches for Aspergillus fumigatus antigen-challenged mice compared to placebo-challenged mice. Aspergillus antigen challenge did not increase eosinophil levels in the lamina propria following allergen challenge (Mishra et al., 1999), indicating that discrete mechanisms regulate Peyer’s patch and the lamina propria compartments. Eosinophil trafficking to Peyer’s patches was also examined in the experimental model of oral antigen-induced Th2associated allergic hypersensitivity responses of the gastrointestinal tract (as described earlier) (Hogan et al., 2000). Exposure of OVA-sensitized mice to enteric-coated OVA beads induces Th2-associated accumulation of eosinophils in the peripheral blood and gastrointestinal lamina propria of the small intestine as compared to mice challenged with placebo beads (Hogan et al., 2000). Similar to the lamina propria, oral allergen-challenged animals had elevated levels of eosinophils in the Peyer’s patches compared to placebo bead treated mice (Fig. 6). These results demonstrated that eosinophils traffic to Peyer’s patches during Th2 responses following mucosal allergen exposure. It was of interest to determine next the role of eotaxin in allergen-induced recruitment of eosinophils into Peyer’s patches. Eotaxin-deficient and wild-type mice were subjected to experimental allergic airway inflammation (Mishra et al., 2000). As expected, allergen challenge of wild-type mice promoted eosinophil accumulation in the blood, lung, and Peyer’s patches (Mishra et al., 2000). In the absence of eotaxin, there was reduced accumulation of eosinophils in Peyer’s patches and the lung compared with wild-type mice. However, in the absence

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of eotaxin, allergen challenge still induced eosinophil infiltration into Peyer’s patches compared with mice that were challenged with placebo alone. In particular, wild-type mice subjected to placebo or allergen challenge had 0.014 ± 0.01 and 1.53 ± 0.46 MBP/area (%) (mean ± SEM, n = 7), respectively. Eotaxindeficient mice subjected to placebo or allergen challenge had 0.008 ± 0.004 and 0.89 ± 0.16 MBP/area (%) (mean ± SEM, n = 5), respectively. This indicates that eosinophil trafficking to Peyer’s patches during mucosal allergen exposure occurs by eotaxin-dependent and -independent pathways. The localization of eosinophils in T cell-rich regions of the Peyer’s patches, and the recent demonstration that eosinophils can present antigen to T cells, provides evidence that eosinophils may have a critical role in the induction of antigen-specific T cell responses. F. COLONIC EOSINOPHILS Eosinophils accumulate in the colon of patients with a variety of disorders, including eosinophilic colitis, allergic colitis, and inflammatory bowel disease (Furuta et al., 1995). Some of these disorders are very common; for example, allergic colitis, which is often due to milk protein hypersensitivity, is the most common cause of bloody diarrhea in the first year of life (Chong et al., 1986; Hill and Milla, 1990; Machida et al., 1994). In an attempt to dissect the immunological mechanisms involved in eosinophil-associated gastrointestinal disorders of the large intestine, a murine model of antigen-induced colitis was developed (Kweon et al., 2000). Mice were systemically sensitized to OVA in the presence of Complete Freund’s Adjuvant and subsequently challenged with repeated doses of intragastric OVA. After nine doses of antigen over 3 weeks, the mice developed diarrhea accompanied by a dramatic infiltration of eosinophils, mast cells, and CD4+ Th2 cells into the large but not small intestine. Similar experimental analysis in mice with the targeted deletion of signal transducers and activators of transcription (STAT)6 completely eliminated the colonic eosinophils and the diarrhea. Adoptive transfer experiments showed that systemically primed splenic CD4+ T cells were preferentially recruited to the large but not small intestine upon oral allergen challenge. These results indicate that eosinophilassociated inflammation of the large intestine appears to be critically regulated by Th2 cells that specifically home to the colon. While eotaxin has been shown to be essential for the baseline homing of eosinophils to the colon (Mishra et al., 1999), its importance in inflammatory disorders of the colon has yet to be evaluated. G. ESOPHAGEAL EOSINOPHILS Eosinophil infiltration into the esophagus is a commonly observed medical problem in patients with diverse diseases including gastroesophageal reflux, eosinophilic esophagitis, and allergic gastroenteritis (Furuta et al., 1995; Kelly,


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2000; Sampson, 1999). As mentioned earlier, the murine and human esophagus, even though they express eotaxin, are devoid of resident eosinophils at baseline (Kato et al., 1998; Mishra et al., 1999). This indicates that eotaxin expression is not sufficient for eosinophil homing into these segments of the gastrointestinal tract. In an effort to understand the mechanisms and significance of eosinophil accumulation in the esophagus in diseased states, we have developed a murine model for antigen-induced esophagitis (Mishra et al., 2001). Mice were repeatedly challenged with intransal Aspergillus fumigatus allergen (under conditions which promote allergic airway inflammation) and were found to develop ∼100to 125-fold increase in their levels of esophageal eosinophils and epithelial hyperplasia. In contrast, exposure to repeated doses of oral or intragastric soluble allergen did not promote esophageal inflammation. Since allergen challenge is associated with Th2 immune responses, the role of IL-5, a Th2 cell-derived cytokine, was examined for its role in regulating eosinophil accumulation in the esophagus. Intranasal allergen challenge of IL-5 gene-targeted mice resulted in the complete loss of eosinophil recruitment to the esophagus and the onset of epithelial hyperplasia. In contrast, in the absence of eotaxin, allergen-induced esophageal eosinophils were only partially reduced (∼2-fold). Consistent with the important role of Th2 cells and their cytokines, a clinical study of patients with eosinophilic esophagitis has demonstrated elevated levels of IL-4 secreting T cells in esophageal lesions (Nicholson et al., 1997). These findings have several implications: (1) they implicate aeroallergens and eosinophils in the etiology of eosinophilic esophagitis; (2) they suggest that esophageal eosinophilic inflammation is mechanistically associated with pulmonary inflammation; and (3) they suggest that targeting IL-5 (e.g., with the humanized anti-IL-5 reagent that is currently being evaluated for asthma) may be a useful strategy for patients with eosinophilic esophagitis and/or refractory gastroesophageal reflux. In addition, they suggest that distinct mechanisms regulate eosinophil accumulation in the esophagus compared with other components of the gastrointestinal tract such as the small intestine. H. GASTROINTESTINAL EOSINOPHILS IN ENTERIC HELMINTH INFECTIONS Early work indicated that the hematopoietic expansion of eosinophils but not neutrophils during helminth infections (e.g., Schistosoma mansoni, Nippostrongyloides brasiliensis, Onchocerca volvulus, and Trichinella spiralis) was reduced in athymic mice (Hsu et al., 1976; Ruitenberg et al., 1977). Subsequently, mice treated with anti-IL-5 monoclonal antiserum (or mice containing a disruption of the IL-5 gene) were shown to have dramatically reduced systemic and local eosinophilia following infection with Nippostrongyloides brasiliensis (Behm and Ovington, 2000; Coffman et al., 1989); taken together these studies identified IL-5 as the chief T cell factor responsible for mediating

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helminth-induced eosinophilia (Maizels et al., 1993; Sanderson, 1992). Experimental infection of animals with helminths has provided an opportunity to examine the mechanisms of eosinophil trafficking to the gastrointestinal tract. For example, following Trichinella spiralis infection, eosinophils accumulate in the lamina propria of the jejunum, in the mesenteric lymph nodes, and in the spleen (Friend et al., 2000). In the jejunum, eosinophils are located in the lamina propria and not in the interepithelial location (in contrast to mast cells) (Friend et al., 2000). During the recovery phase, the fate of lamina propria eosinophils was assessed by the TUNEL assay, a method for identifying apoptotic cells. Apoptotic eosinophils were rarely found in the lamina propria, but were readily detected in mesenteric lymph nodes. Additionally, many macrophages in the lymph nodes were demonstrated to be actively phagocytosing apoptotic eosinophils. Thus, although only examined in the context of Trichinella spiralis infection, gastrointestinal eosinophils may be destined for trafficking to local lymph nodes, where they undergo clearance by apoptosis. The role of adhesion molecules in the regulation of eosinophil trafficking to the gastrointestinal tract has also been evaluated following Trichinella spiralis infection. Eosinophils express several families of adhesion molecules, including members of the selectin and integrin families (summarized in Fig. 7). In brief, reversible interactions between eosinophils and endothelial cells are primarily mediated by selectins. Eosinophils express the ligands (e.g., sialylated LewisX antigen) for E- and P-selectins. In addition, eosinophils express L-selectin, but its exact ligand on endothelial cells is not known. Eosinophils have recently been demonstrated to selectively express a sialic acid binding immunoglobulinlike lectin designated Siglec-8 (Floyd et al., 2000; Kikly et al., 2000). The ligand for Siglec-8 has not yet been identified, but other members of the Siglec family of adhesion molecules have been shown to be important signaling receptors, employing immunomodulatory inhibitory motifs to interact with tyrosine phosphatases (e.g., SHP-1). The integrins expressed by eosinophils include members of the ␤1 (e.g., very late antigen (VLA)-4), ␤2 (e.g., CD18 family of molecules), and ␤7 families (e.g., ␣4␤7 molecule) (Bochner and Schleimer, 1994) (Fig. 7). The CD18 family of molecules includes lymphocyte function antigen (LFA)-1 and Mac-1 that both interact with endothelial cells via intercellular adhesion molecule (ICAM); the VLA-4 integrin (which is not expressed by neutrophils) binds to vascular cell adhesion molecule (VCAM)-1. These adhesion interactions have been shown to be important for eosinophil recruitment into the lung and skin, but their role in eosinophil recruitment to the gastrointestinal tract has not yet been evaluated. The ␣4␤7 molecule, which is coexpressed on lymphocytes and eosinophils, is perhaps the most important integrin for gastrointestinal eosinophils. This integrin binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a major adhesion molecule expressed on high endothelial venules in the intestinal lamina propria, lymph nodes, and Peyer’s patches. Mice with targeted disruption of the ␤7 gene have been examined for


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FIG. 7. Schematic diagram of eosinophil adhesion molecules and their ligands. Eosinophils express several classes of adhesion molecules, including members of the selectin and integrin families [more extensively reviewed in other publications (Bochner and Schleimer, 1994)]. Depicted are eosinophil ligands (e.g., sialylated Lewis-X antigen) for E- and P-selectins, which mediate reversible interactions between eosinophils and endothelial cells. In addition, eosinophils express L-selectin, but its exact ligand on endothelial cells is not known. Eosinophils have recently been demonstrated to selectively express a sialic acid binding immunoglobulin-like lectin designated Siglec-8. The ligand for Siglec-8 has not yet been identified, but other members of the Siglec family of adhesion molecules have been shown to be important signaling receptors, employing immunomodulatory inhibitory motifs to interact with tyrosine phosphatases (e.g., SHP-1). The integrins expressed by eosinophils include members of the ␤1 (e.g., very late antigen (VLA)-4), ␤2 (e.g., CD18 family of molecules), and ␤7 (e.g., ␣4␤7) families of molecules. The CD18 group of receptors includes lymphocyte function antigen (LFA)-1 and Mac-1 that both interact with endothelial cells via intercellular adhesion molecule (ICAM); the VLA-4 integrin (which is not expressed by neutrophils) binds to vascular cell adhesion molecule (VCAM)-1. The ␣4␤7 molecule, which is coexpressed on lymphocytes and eosinophils, binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a major adhesion molecule expressed on high endothelial venules in the intestinal lamina propria, lymph nodes, and Peyer’s patches.

inflammatory responses to helminth infection. These studies have demonstrated a delayed influx and reduced magnitude of intestinal eosinophilia in response to Trichinella spiralis (Artis et al., 2000). Consistent with this, we have found reduced eosinophil recruitment into the jejunum of ␤7-deficient mice following oral allergen challenge compared with wild-type mice (unpublished results). In

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contrast, during infection with Trichuris muris, a helminth that infects the large intestine, no difference in the level of eosinophils in the colon is observed (Artis et al., 2000). Thus, similar to the important role of ␣4␤7 in lymphocyte homing to the gastrointestinal tract (Berlin et al., 1993), eosinophil recruitment to the small intestine also appears to be regulated by this integrin. Although eosinophils have been shown to elicit powerful antihelminthic cytotoxicity in vitro (Butterworth, 1977, 1984), studies in vivo have not consistently demonstrated an important role for eosinophils in this process, with the exception of immunity against select organisms such as Strongyloides venezuelensis (Behm and Ovington, 2000; Korenaga et al., 1991), as will be discussed later. V. Function of Eosinophils

A. PROINFLAMMATORY ROLE The function of eosinophils in inflammatory states in the gastrointestinal tract has been largely derived from studies investigating the properties of these cells in vitro and by analysis of their involvement in allergic respiratory disorders. A variety of studies (primarily utilizing antibody neutralization of IL-5 and IL-5 gene deficient mice) have demonstrated a strong association of eosinophilic airway inflammation and the development of lung damage (e.g., airway hyperresponsiveness) (Hogan et al., 1998c). However, recent studies have shown that the mere presence of eosinophils in the lung is not sufficient for airway hyperresponsiveness (Mould et al., 2000), indicating that multiple signals are necessary for eosinophil activation and/or airway hyperresponsiveness. As stated earlier, eosinophils generate a variety of mediators that augment inflammatory responses. Most prominent among these molecules are the secondary granule constituents (MBP, ECP, EPO, and EDN) that are specific to eosinophils (Fig. 1). These mediators induce a variety of proinflammatory effects, including toxicity to multiple tissues (including host cells), induction of mast cell degranulation, and dysfunction of vagal muscarinic M2 receptors (Gleich and Adolphson, 1986). Further proinflammatory damage can be caused by the generation of unstable oxygen radicals by the respiratory burst oxidase apparatus and EPO. The eosinophil also generates large amounts of LTC4 (Lewis et al., 1990) which is metabolized to LTD4 and LTE4, potent smooth muscle constrictors. Additionally, activated eosinophils generate a wide range of inflammatory cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, TGF-␣/␤, TNF-␣, and eotaxin) which can be preformed or generated de novo following cellular activation. Eosinophils also produce neuroactive mediators (e.g., substance P and vasoactive intestinal peptide [VIP]) (Metwali et al., 1994). Interestingly, specimens from patients with eosinophilic gastroenteritis often display eosinophils undergoing marked degranulation near nerves, suggesting that they may indeed be involved in promoting inflammatory changes to neurons (Stead, 1992; Dvorak et al., 1993).


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Interestingly, the gastric dysmotility during experimental oral antigen-induced gastrointestinal inflammation is associated with eosinophils in the proximity of damaged nerves, suggesting a causal role for eosinophils in nerve dysfunction (Hogan et al., 2001). Additionally, experimental eosinophil accumulation in the gastrointestinal tract is associated with the development of weight loss, which is attenuated in eotaxin-deficient mice (Hogan et al., 2001). Taken together with the clinical data in patients with gastroesophageal reflux, where the level of eosinophils directly correlates with the severity of the disease, and lack of responsiveness to medical management (Ruchelli et al., 1999), these results suggest a proinflammatory detrimental role for eosinophils in the pathogenesis of gastrointestinal disorders. B. ANTIHELMINTH IMMUNITY The beneficial function of eosinophils has been primarily attributed to their ability to defend the host against parasitic helminths. This is based on several lines of evidence, including (1) the ability of eosinophils to mediate antibody (or complement) dependent cellular toxicity against helminths in vitro (Butterworth, 1977, 1984); (2) the observation that eosinophil levels increase during helminth infections and that eosinophils aggregate and degranulate in the local vicinity of damaged parasites in vivo; and (3) the results in experimental parasite-infected mice that have been depleted of eosinophils by IL-5 neutralization and/or gene targeting (Behm and Ovington, 2000). However, it should be noted that murine studies are particularly problematic since mice are not the natural hosts of many of the experimental parasites. Nevertheless, in some primary infection models, a role for IL-5 (and hence eosinophils) in protective immunity has been suggested following infection with Strongyloides venezuelensis, Strongyloides ratti, Nippostrongyloides brasiliensis, and Heligmosomoides polygyrus (Behm and Ovington, 2000; Korenaga et al., 1991). Most recently, a role for eosinophils in the encystment of larvae in Trichinella spiralis infection has been demonstrated (D. S. Friend, Harvard Medical School, personal communication). In this study, a markedly reduced level of gastrointestinal eosinophils was found in Trichinella spiralis-infected CCR3 gene-targeted mice compared with control infected mice that contained abundant degranulating eosinophils. The reduced level of eosinophils correlated with a greater number of intact encysted larvae. Thus, although the debate continues, it seems likely that eosinophils participate in the protective immunity against selected helminths. C. INTERACTIONS WITH T LYMPHOCYTES The localization of gastrointestinal eosinophils in juxtaposition with lymphocytes (e.g., lamina propria and Peyer’s patches) suggests a functional interaction between these two leukocytes. While most studies have focused on the role of T cells in the regulation of eosinophils (e.g., through IL-5), it is likely

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that eosinophils may also regulate lymphocytes. Consistent with this finding, eosinophils are known to express the necessary cellular machinery for antigen presentation such as H-2 class II and costimulatory molecules (e.g., B7-1) (Lucey et al., 1989; Tamura et al., 1996; Woerly et al., 1999). Eosinophils are also known to express a variety of cytokines that can induce the proliferation and/or maturation of T cells (e.g., IL-2, IL-4, IL-12). Furthermore, preliminary investigations with human eosinophils in vitro have shown that eosinophils have the capacity to present antigen to T cells (Lucey et al., 1989; Tamura et al., 1996). Recent studies have shown that eosinophils isolated from the mouse lung can present antigen to T cells when adoptively transferred to na¨ıve animals (Shi et al., 2000). In addition, it has been proposed that lymph node eosinophils in patients with Hodgkin’s disease may provide cellular ligands for TNF superfamily receptors and CD30, thereby transducing proliferation and antiapoptotic signals (Pinto et al., 1996, 1997). Additional support for an interaction between eosinophils and T cells has recently been derived from analysis of thymic eosinophils. As mentioned earlier, the thymus is a primary site for eosinophils under healthy conditions. In young mice, thymic eosinophils are primarily located in the corticomedullary region, express IL-4 and IL-13, and are CD11b and CD11c positive (similar to dendritic cells) (Throsby et al., 2000). In adult mice, eosinophils traffic to the medulla under the regulation of eotaxin (Matthews et al., 1998). During experimental induction of tolerance, the level of thymic eosinophils increases, and their location correlates with areas of active T cell apoptosis. Taken together, these studies indicate that regulation of T cell responses is one of the physiological functions of eosinophils. D. DEVELOPMENTAL BIOLOGY The finding that eosinophils home into the gastrointestinal tract during gestational development (Mishra et al., 1999) suggests that eosinophils may have a role in tissue or organ development. A role for eosinophils in developmental processes in the gastrointestinal tract has not yet been identified. However, a physiological function for eosinophils in postnatal mammary gland development has been recently proposed (Gouon-Evans et al., 2000). In this investigation, F4/80-positive leukocytes were identified to be present in the developing mammary gland, primarily in the region of the terminal end buds. Surprisingly, on close histological examination, the F4/80-positive cells were identified as macrophages and eosinophils. The important role for leukocytes in mammary gland development was demonstrated by depleting hematopoietic precursors by whole-body ␥ -irradiation. Following ␥ -irradiation, ductal outgrowth was impaired, and this abnormality was reversed by bone marrow transplantation. Interestingly, the level of eotaxin and eosinophils in the mammary gland was shown to increase with the development of the mammary gland during puberty. Furthermore, eotaxin-deficient mice had a near complete loss of mammary gland


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eosinophils, and this was associated with a decreased number of ductal branches and a defect in terminal end bud formation. Taken together, these data establish that eosinophils are critically involved in the branching morphogenesis of the mammary gland. The presence of constitutive eotaxin and eosinophils in other endocrine organs (e.g., uterus) (Hornung et al., 2000; Salamonsen and Lathbury, 2000; Zhang et al., 2000), as well as in the gastrointestinal tract, suggests that the involvement of tissue eosinophils in developmental processes is not likely to be restricted to the mammary gland. VI. Summary and Concluding Remarks

Eosinophils are well known as proinflammatory leukocytes that account for a small subset of circulating blood cells. The recent studies outlined in this review have identified eosinophils as constituents of the mucosal immune system in the gastrointestinal tract residing in the lamina propria and Peyer’s patches, and have identified complex regulatory pathways and characteristics of these cells (summarized in Fig. 8). Under baseline (healthy) conditions, gastrointestinal eosinophils predominantly reside in the lamina propria in the stomach and intestine, and their numbers in these organs are substantially higher than in hematopoietic tissues. Interestingly, eosinophils migrate to the gastrointestinal tract during embryonic development, and their concentrations in perinatal mice are comparable to those in adults, indicating that eosinophil homing occurs independent of intestinal flora. The chemokine eotaxin, an eosinophil-selective chemoattractant that is constitutively expressed throughout all segments of the gastrointestinal tract, is required for eosinophil homing to the lamina propria. During Th2-associated inflammatory conditions (e.g., IL-5 overexpression or allergen challenge), marked increases of eosinophils occur in the lamina propria in an eotaxin-dependent manner. In addition, allergen challenge promotes eosinophil migration to the outer cortex and interfollicular regions of Peyer’s patches, and this process is critically regulated by IL-5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Furthermore, following mucosal allergen challenge, eosinophils under the regulation of IL-5 accumulate in the esophagus, an organ normally devoid of eosinophils at baseline. Preliminary investigations have shown that gastrointestinal eosinophils express the ␣4␤7 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system, their levels are increased by antigen exposure under Th2-associated conditions, and eotaxin and IL-5 differentially regulate their trafficking in a tissue-specific manner. We propose that eosinophils are integral members of the gastrointestinal immune system and are likely to be important in innate, regulatory, and inflammatory immune responses.

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FIG. 8. Schematic representation of eosinophil trafficking to the intestine. Eosinophils develop in the bone marrow where they differentiate from hematopoietic progenitor cells into mature eosinophils. Factors that control this process have not been fully defined; however, IL-3, IL-5, and GM-CSF are important in eosinophil expansion during conditions of hypereosinophilia. Eosinophil migration out of the bone marrow into the circulation is primarily regulated by IL-5. Circulating eosinophils subsequently interact with the endothelium by processes involving rolling, adhesion, and diapedesis. In the gastrointestinal tract, the adhesion ␣4␤7 ligand on eosinophils interacts with the endothelial receptor mucosal vascular addressin MAdCAM-1. Eosinophils are mobilized into the lamina propria in response to a chemotactic gradient primarily established by the chemokine eotaxin liberated from mononuclear cells in the crypts. Additionally, eosinophils are mobilized into the interfollicular and paracortical regions of Peyer’s patches. The chemotactic response is enhanced by IL-5, an important eosinophil cytokine for eosinophil priming and survival. Depending upon the chemokine concentration gradient, gastrointestinal eosinophils can also migrate into the villi, residing in proximity to lymphocytes, and have the potential to degranulate, resulting in tissue damage. (See color insert.)

In view of the ability of eosinophils to participate in antigen presentation and to secrete cytokines, which induce T cell proliferation and maturation, the finding of eosinophils as part of gut-associated lymphoid tissue (GALT) seems logical since most lymphocytes also reside in the gastrointestinal tract. However, both cell types reside in distinct locations and are regulated by unique processes (Table II). At baseline, eosinophils only reside in the lamina propria; however, during inflammatory conditions these cells can be located in several other compartments shared with T cells (lamina propria, Peyer’s patches, and interepithelial regions). Eosinophil localization to the gastrointestinal tract is not

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TABLE II CHARACTERISTICS OF GASTROINTESTINAL T CELLS AND EOSINOPHILS

Location—baseline Peyer’s patch Lamina propria Interepithelial Location—diseased state Peyer’s patch Lamina propria Interepithelial Role for ␣4␤7 Eotaxin IL-5 Gut flora a

T Cells

Eosinophils

+ + +

– + –

+ + +

+ + +

+ – – +a

+ + + –

Excluding ␥ /␦-T cells.

simply a response to the “proinflammatory” environment of the gastrointestinal tract, since prenatal mice (free of exogenous flora), as well as germ-free mice (free of viable intestinal flora) have normal levels of eosinophils. While some of the mechanisms involved in leukocyte homing to the gastrointestinal tract appear to be conserved between lymphocytes and eosinophils (e.g., utilization of ␣4␤7 integrin), gastrointestinal eosinophils are critically regulated by local eotaxin, and less significantly by IL-5. Recent studies that have begun to uncover functional properties of eosinophils indicate that these cells are more than just pro inflammatory leukocytes. Rather, they are likely to have diverse physiological functions involved in critical arms of the immune system, including innate responses, tolerance, and antigen presentation; in addition, eosinophils may have a role in tissue morphogenesis. The accumulation of eosinophils in the gastrointestinal tract in diverse medical diseases is often associated with serious medical consequences (e.g., weight loss, malabsorption, architectural changes of the intestine such as blunting of the villi), but the role of eosinophils in the pathogenesis of these diseases has been debated. Recent progress in experimental modeling of eosinophil-associated gastrointestinal diseases has been instrumental in determining that gastrointestinal eosinophils can be directly increased by mucosal allergen challenge. These models have been useful in identifying the critical role for eotaxin and in suggesting a causal association of eosinophilic inflammation with clinical and pathological changes to the gastrointestinal tract. These findings parallel the large number of mechanistic studies concerning eosinophilic allergic inflammation in the lung (e.g., asthma). Interestingly, patients with inflammatory gastrointestinal disorders

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have been found to have an altered proallergic mucosa in the lung, including occult airway hyperresponsiveness (Louis et al., 1999; Mansi et al., 2000). Likewise, patients with asthma have been demonstrated to contain increased levels of eosinophils in the gastrointestinal tract (Wallaert et al., 1995). Taken together, it is likely that common mucosal immune responses are operational in patients with allergic disorders, perhaps controlled by trafficking T cells. It is hoped that examination of gastrointestinal eosinophils with the same scrutiny that has been applied to investigate pulmonary eosinophils and gastrointestinal lymphocytes may soon uncover the role of eosinophils in health and disease. ACKNOWLEDGMENTS This work was supported in part by the National Health Medical Research Council (Australia) C. J. Martin Post-doctoral Fellowship (S. P. H.), the Jaffe Family Fund of the American Academy of Allergy, Asthma, and Immunology (S. P. H.), NIH Grant R01 AI45898 (M. E. R.), and the Human Frontier Science Program (M. E. R.). The authors thank Drs. K. Frank Austen, Mitchell Cohen, Susan Wert, Paul Foster, Glenn Furuta, and Nives Zimmermann for helpful discussions, numerous other instrumental colleagues, and Andrea Lippelman for expert editorial assistance.

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Walsh, R. E., and Gaginella, T. S. (1991). The eosiniophil in inflammatory bowel disease. Scand. J. Gastronenterol. 26, 1217–1224. Watkins, S. G., Dearin, J. L., Young, L. C., and Wilhelm, D. I. (1976). Association of mastopoiesis with haemopoietic tissues in the neonatal rat. Experientia 32, 1339–1340. Weiner, H. L., and Mayer, L. F. (1996). Oral tolerance: Mechanisms and applications. Introduction. Ann. N. Y. Acad. Sci. 778, xiii–xviii. Weller, P. F. (1991). The immunobiology of eosinophils. N. Engl. J. Med. 324, 1110–1118. Weller, P. F. (1994). The idiopathic hypereosinophilic syndrome. Blood 83, 2759–2779. Wills-Karp, M. (1999). Immunologic basis of antigen-induced airway hyperresponsiveness. Ann. Rev. Immunol. 17, 255–281. Winter, H. S., Madara, J. L., Stafford, R. J., Grand, R. J., Quinlan, J. E., and Goldman, H. (1982). Intraepithelial eosinophils: A new diagnostic criterion for reflux esophagitis. Gastroenterology 83, 818–823. Woerly, G., Roger, N., Loiseau, S., Dombrowicz, D., Capron, A., and Capron, M. (1999). Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (Interleukin 2 and interferon gamma). Inhibition by immunoglobulin a complexes. J. Exp. Med. 190, 487–496. Woodbury, R. G., and Neurath, H. (1978). Purification of an atypical mast cell protease and its levels in developing rats. Biochemistry 17, 4298–4304. Ying, S., Meng, Q., Zeibecoglou, K., Robinson, D. S., Macfarlane, A., Humbert, M., and Kay, A. B. (1999). Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (intrinsic) asthmatics. J. Immunol. 163, 6321–6329. Young, J. D., Peterson, C. G., Venge, P., and Cohn, Z. A. (1986). Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature 321, 613–616. Zhang, J., Lathbury, L. J., and Salamonsen, L. A. (2000). Expression of the chemokine eotaxin and its receptor, CCR3, in human endometrium. Biol. Reprod. 62, 404–411. Zimmermann, N., Hogan, S. P., Mishra, A., Brandt, E. B., Bodette, T. R., Finkelman, F. D., and Rothenberg, M. E. (2000). Murine eotaxin-2: A constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression. J. Immunol. 165, 5839–5846.

INDEX

A Allelic exclusion, V(D)J recombination, 199–201 AP-1, lipopolysaccharide signaling in macrophages, 19–20 Asthma ABCD chemokine role, 148–149 gastrointestinal eosinophils, 319–320 Atopic dermatitis, ABCD chemokine role, 149–150

B B cell activation and follicle formation, 126 compartmental homing within secondary lymphoid organs B cell-rich areas, 130 microarchitecture formation role lymphotoxins and receptors, 131–133 tumor necrosis factor and receptor, 131–133 memory/effector cell migration features of cells, 143–144 plasma cells and chemokines, 144–145 T cell encounters and chemokines, 141–143 V(D)J rearrangements, see V(D)J recombination Biliary cirrhosis, T cell responses against modified peptides, 283 Bone marrow chemokine and receptor expression, 74–75 hematopoietic stem cell migration during embryogenesis integrins, 119

overview, 117–118 stem cell rolling, 118–119 BSAP, V(D)J recombination regulation, 206–207

C CD11b/CD18, lipopolysaccharide binding, 11–12 CD14 lipopolysaccharide binding, 10 signaling, see Lipopolysaccharide CD18, Taxol binding, 26 C/EBP␤, see NF-IL6 Celiac disease, T cell responses against modified peptides, 282–283 Ceramide, lipopolysaccharide signaling in macrophages, 16–17 Chemokines ABCD chemokines and disease asthma, 148–149 atopic dermatitis, 149–150 endotoxemia, 147–148 human immunodeficiency virus, 151 knockout mouse studies, 153 lymphoma, 150 prospects for study, 151–153 sepsis, 148 B cell/T cell interactions, 141–143 CCR6 interaction with ␤-defensin, 138–139 chromosomal localization chemokines, 68–70, 72 receptors, 68, 71 dendritic cell antigen uptake and migration MIP-3␣/CCR6 interaction, 137–138 overview, 136–137 329

330

INDEX

Chemokines (continued) receptor expression, 137 discovery, 58, 63 dose–response of cell migration, 115, 117 ELR motifs, 63 expression and function in immune system bone marrow, 74–75 dendritic cells CCR6, 88–89 CCR7, 88–89 chemokine production, 89–90 receptor expression in maturation, 88–89 lymph nodes, 77–80 mucosal immune system CCR6 expression by dendritic cells, 86 fractalkine, 87 LARC, 86 MEC/CCL28, 87 overview, 85 TECK/CCL25, 86 Peyer’s patches, 77–80 skin CCR4, 88 CCR6, 87–88 dendritic cells, 87 ILC/CTACK/CCL27, 88 MEC/CCL28, 88 spleen, 77–80 T helper cells CCR3, 82, 245–246 CCR4, 82–83, 246 CCR5, 81–82, 244–245 CCR7, 83–84, 244 CCR8, 83 CXCR3, 81–82, 244 CXCR5, 84–85, 239, 247–248 follicular T helper cell migration, 247–248 inhibitors of receptors, 246–247 intervention therapy, 92 overview, 242–244 receptor expression by cell type, 80–81 Th1 responses, 244–245 Th1/Th2 polarization role, 90, 140–141, 246 Th2 responses, 245–246 thymus, 75–77 functional classification, 64 gene clusters, 58

gene duplications, 68 immune chemokines and receptors, 66–67 knockout mouse studies of immunity, 90–91 letter codes, 116–117 lymphocyte compartmental homing within secondary lymphoid organs BLC, 135–136 CXCR5, 134–136 ELC, 133–134 SLC, 133–134 lymphoid cell movements into secondary lymphoid organs during T cell-dependent, antigen-specific immune response B cell activation and follicle formation, 126 CCR7 role, 129–130 circulation, 123–124, 128 dendritic cell activation and maturation, 125, 139 ELC role, 128–129 SLC role, 128–130 T cell activation, 127 phylogenetic relationships, 59–60, 63, 68, 72–73, 112 plasma cells migration, 144–145 receptors desensitization, 115 ligand specificity, 113–115 phylogenetic relationships, 65–66, 113 signal transduction, 67–68, 115, 151–152 structural homology, 65–66 structural homology, 64, 114 subfamilies, 58, 62–64 T cell memory/effector cell migration CCR4 role, 146–147 CCR6 role, 146–147 CCR7 role, 145–147 features of cells, 143–145 secondary lymphoid organs, 145 tables of types and receptors, 61–62, 65, 116–117 therapeutic applications and targeting, 91–92 Chromatin structure DNA methylation of CpG motifs hypomethylation and transcription, 184–186 Ig␬ locus methylation, 185

331

INDEX

methyl cytosine binding protein function, 183–184 methyltransferases, 184 ␤-globin locus transcription cis-acting elements as assembly platforms, 182–183 histone acetylation, 187–188 linking model, 193 looping model, 192 model for V(D)J recombination, 179 organization and expression, 182 heterochromatin versus euchromatin, 181 interchromatin granules and splicing factors, 181 nucleosome structure, 179 transcription site visualization, 182 V(D)J recombination DNase sensitivity and recombination, 186–188 high-mobility group protein effects on RAG cleavage, 188–189 histone acetylation, 188 nucleosome remodeling complexes, 189–190 promoter exposure, 189 recombination signal sequence accessibility modeling, 213–215 sterile transcripts, 190–191 c-Maf, T helper cell differentiation role, 241 Colitis, eosinophils, 301 CpG motifs methylation hypomethylation and transcription, 184–186 Ig␬ locus methylation, 185 methyl cytosine binding protein function, 183–184 methyltransferases, 184 Toll-like receptor recognition, 33–34 CREB, see Cyclic AMP response element-binding protein Cyclic AMP response element-binding protein (CREB), lipopolysaccharide signaling in macrophages, 19

D DC, see Dendritic cell Defensins

CCR6 interaction with ␤-defensin, 138–139 types, 138 Dendritic cell (DC) activation and maturation, 125, 137 chemokine and receptor expression antigen uptake and migration of immature cells MIP-3␣/CCR6 interaction, 137–138 overview, 136–137 receptor expression, 137 CCR6, 88–89 CCR7, 88–89 chemokine production, 89–90 receptor expression in maturation, 88–89 T cell interactions and chemokines, 139–140 Dif, Toll signaling, 3 DNA microarray, differentially-expressed genes in T cell subsets humans, 252 mice, 252–253 overview, 250–251 table of genes, 251 Dorsal, Toll activation, 2–3

E E2A, V(D)J recombination regulation, 201–203 Early B cell factor (EBF), V(D)J recombination regulation, 206 EBF, see Early B cell factor ECSIT, interleukin-1 receptor signaling, 5–6 18W, Toll homology, 3 Endotoxemia, ABCD chemokine role, 147–148 Eosinophil cytokine production, 291–292 functional overview, 291–292 gastrointestinal eosinophils developmental biology, 316–317 disease roles asthma, 319–320 colitis, 301 colonic cell migration, 310 eotaxin regulation of lamina propria cells in allergy, 303–304, 317

332

INDEX

Eosinophil (continued) esophageal cell migration, 310–311 experimental eosinophilic gastroenteritis, 302–303 food allergy, 300 gastroenteritis, 300–301 gastroesophageal reflux, 301 helminth infection and cell recruitment, 311–315 inflammation role, 302, 314–315 inflammatory bowel disease, 301 interleukin-5 regulation role in allergy, 304–307, 317 levels of cells, 292–293, 299 Peyer’s patch migration in allergy, 307–310 T cell overexpression effects on lamina propria eosinophils, 305–309 eotaxin role in homing, 296–297 hematopoietins and accumulation granulocyte-macrophage colony-stimulating factor, 298 interleukin-3, 298 interleukin-5, 298 immunoglobulin E-mediated reactions, 299–300 inflammatory mediators and accumulation, 296 interepithelial cells, 294 lamina propria, 294 mouse versus humans, 293–294 perinatal mice and endogenous flora, 294–296 Peyer’s patch, 294 T cell interactions, 298–299, 315–316 secondary granule contents, 291 Eotaxin eosinophil homing role, 296–297 regulation of lamina propria eosinophils in allergy, 303–304, 317 ETS proteins, V(D)J recombination regulation, 203–204

F Food allergy, gastrointestinal eosinophils, 300

G Gastroenteritis, eosinophils, 300–301 Gastroesophageal reflux, eosinophils, 301 GATA-3, T helper cell differentiation role, 241 ␤-Globin locus transcription, see Chromatin structure Glycosylation, see Major histocompatibility complex molecules GM-CSF, see Granulocyte-macrophage colony-stimulating factor G proteins, lipopolysaccharide signaling in macrophages, 15–16 Granulocyte-macrophage colony-stimulating factor (GM-CSF), gastrointestinal eosinophil accumulation role, 298

H Helminth infection, eosinophil recruitment, 311–315 Hematopoietic stem cell chemokine and receptor expression within primary lymphoid organs, 121–123 differentiation, 119–120 migration during embryogenesis integrins, 119 overview, 117–118 stem cell rolling, 118–119 thymus homing, 119–121 Human immunodeficiency virus (HIV), ABCD chemokine role in infection, 151

I ICOS, T helper cell function, 248–249 Ig␤, V(D)J recombination regulation, 208 Ikaros, V(D)J recombination regulation, 208 Immunoglobulin chain rearrangement, see V(D)J recombination Innate immunity specificity, 1 Toll receptor role in Drosophila, 2–4 Interleukin-1, T helper cell differentiation role, 240 Interleukin-1 receptor ECSIT in signaling, 5–6 IRAK deletion studies, 6–7

INDEX

MyD88 deletion studies, 6, 23 p62 in signaling, 6 phosphorylative signaling, 4, 6 signal transducing complex, 4 TAK1 in signaling, 4–5 toll-like receptor signaling homology, 9 Toll signaling homology, 7 TRAF6 in signaling, 4–7 Interleukin-3, gastrointestinal eosinophil accumulation role, 298 Interleukin-4, T helper cell differentiation role, 239–240 Interleukin-5 eosinophil regulation role in allergy, 304–307, 317 gastrointestinal eosinophil accumulation role, 298 Interleukin-7 receptor, V(D)J recombination regulation, 207 Interleukin-12, T helper cell differentiation role, 239–240 Interleukin-18, T helper cell function, 240, 249–250 IRAK interleukin-1 receptor signaling role, 6–7 lipopolysaccharide signaling role, 23

L Lamina propria, gastrointestinal eosinophils normal tissue, 294 T cell overexpression effects, 305–309 LBP lipopolysaccharide binding, 10–11 signaling, see Lipopolysaccharide Leukocyte migration billiard theory of chemoattraction, 117–118 chemokine direction, see Chemokines compartmental homing within secondary lymphoid organs B cell-rich areas, 130 microarchitecture formation role lymphotoxins and receptors, 131–133 tumor necrosis factor and receptor, 131–133 T cell-rich areas, 130 lymphocyte recirculation, 73, 111

333

lymphoid cell movements into secondary lymphoid organs during T cell-dependent, antigen-specific immune response B cell activation and follicle formation, 126 circulation, 123–124, 128 dendritic cell activation and maturation, 125 T cell activation, 127 lymphopoiesis, 73 steps, 57–58 T cell subtypes, 73–74 Lipopolysaccharide (LPS) binding molecules CD11b/CD18, 11–12 CD14, 10 LBP, 10–11 scavenger receptor, 12 components, 9 internalization in macrophages, 24–25 IRAK role in signaling, 23 macrophage stimulation, 9–10 MyD88 role in signaling, 22–24 signal transduction by LPS/LBP/CD14 ternary complex AP-1, 19–20 ceramide, 16–17 cyclic AMP response element-binding protein, 19 G proteins, 15–16 mitogen-activated protein kinase, 14–15 NF-IL6, 19 nuclear factor-␬B, 18–19 p70 S6 kinase, 17 peroxisome proliferator-activated receptor ␥ antagonism, 20 phosphatidylinositol 3-kinase, 14 phospholipase A2, 15 phospholipase C, 13 phospholipase D, 15 protein kinase A, 16 protein kinase C, 13–14 protein tyrosine kinases, 13 Rho, 17 Sp1, 20 species differences in response, 25 Taxol mimetic activity, 26 TLR2 signaling, 29–30 TLR4 signaling, 21–24 tolerance mechanisms

334

INDEX

Lipopolysaccharide (LPS) (continued) cytokine roles, 27 nuclear factor-␬B activation defects, 28 prostaglandin E2 role, 27–28 signaling pathway downregulation, 28 LPS, see Lipopolysaccharide Lymph nodes, chemokine and receptor expression, 77–80 Lymphoma, ABCD chemokine role, 150 Lymphotoxin, lymphocyte compartmental homing role within secondary lymphoid organs, 131–133

M Major histocompatibility complex (MHC) molecules length by class, 267 posttranslational modification effects on presentation and T cell recognition acetylation, 269–270, 275 binding studies glycopeptides, 272–273 phosphopeptides, 273 deamidation, 269 glycosylation Apa-derived peptides, 271 linkages, 268–269 processing inhibition, 271 subcellular distribution, 269 terminal modifications of class I molecules, 275, 277 immunological relevance, 284–286 mass spectrometry characterization, 285–286 overview, 267–268 phosphorylation, 270–271 presentation effects vivo, 274–277 protease blocking effects, 270–271 T cell recognition studies cysteinyl residue recognition, 281 epitope analogs, 277 glycopeptide complexes with H-2Db, 279 glycopeptide complexes with H-2Kb, 279, 281 hybridoma studies, 277–278

phosphopeptide studies, 280–281 Tn-modified analogs, 278–279 in vitro, 274 in vivo, 276, 281–284 T cell responses against modified peptides bee venom phospholipase A2, 283 biliary cirrhosis and ␣-ketoglutarate dehydrogenase peptide modification, 283 celiac disease, 282–283 collagen-induced arthritis, 282 TAP transport effects, 271 types of modifications, 268 presentation pathways class I, 270 class II, 270 MAPK, see Mitogen-activated protein kinase MHC molecules, see Major histocompatibility complex molecules Mitogen-activated protein kinase (MAPK), lipopolysaccharide signaling in macrophages, 14–15 Mucosa-associated lymphoid tissue chemokine and receptor expression CCR6 expression by dendritic cells, 86 fractalkine, 87 LARC, 86 MEC/CCL28, 87 TECK/CCL25, 86 gastrointestinal eosinophils, see Eosinophil overview, 85 ␮E3-binding proteins, V(D)J recombination regulation, 204–205 MyD88 interleukin-1 receptor signaling, deletion studies, 6, 23 lipopolysaccharide signaling role, 22–24

N NF-IL6, lipopolysaccharide signaling in macrophages, 19 NF-␬B, see Nuclear factor-␬B Nuclear factor-␬B (NF-␬B) activation defects in lipopolysaccharide tolerance, 28 lipopolysaccharide signaling in macrophages, 18–19

335

INDEX

O Oct1, V(D)J recombination regulation, 205–206 Oct2, V(D)J recombination regulation, 205–206

P p62, interleukin-1 receptor signaling, 6 p70 S6 kinase, lipopolysaccharide signaling in macrophages, 17 Pattern-recognition receptor (PRR) Drosophila, see Toll receptor innate immunity concept, 1 PD␤1 element, immunoglobulin heavy chain gene assembly role, 195–196 PDQ52 element, immunoglobulin heavy chain gene assembly role, 194–195, 209 Peroxisome proliferator-activated receptor ␥ (PPAR␥ ), lipopolysaccharide signaling antagonism in macrophages, 20 Peyer’s patch chemokine and receptor expression, 77–80 eosinophil migration in allergy, 307–310 normal tissue gasrointestinal cells, 294 Phosphatidylinositol 3-kinase (PI3K), lipopolysaccharide signaling in macrophages, 14 Phospholipase A2 (PLA2) lipopolysaccharide signaling in macrophages, 15 T cell responses against modified bee venom peptides, 283 Phospholipase C (PLC), lipopolysaccharide signaling in macrophages, 13 Phospholipase D (PLD), lipopolysaccharide signaling in macrophages, 15 PI3K, see Phosphatidylinositol 3-kinase PKA, see Protein kinase A PKC, see Protein kinase C PLA2, see Phospholipase A2 PLC, see Phospholipase C PLD, see Phospholipase D PPAR␥ , see Peroxisome proliferator-activated receptor ␥ Prostaglandin E2, lipopolysaccharide tolerance role, 27–28

Protein kinase A (PKA), lipopolysaccharide signaling in macrophages, 16 Protein kinase C (PKC), lipopolysaccharide signaling in macrophages, 13–14 PRR, see Pattern-recognition receptor PU.1, V(D)J recombination regulation, 203–204

R RAG proteins, see V(D)J recombination Relish, Toll signaling, 3 Rheumatoid arthritis, T cell responses against modified peptides in collagen-induced arthritis, 282 Rho, lipopolysaccharide signaling in macrophages, 17

S Scavenger receptor, lipopolysaccharide binding, 12 SDF-1, hematopoietic development role within lymphoid organs, 121–122 Sepsis, ABCD chemokine role, 148 Sox-4, V(D)J recombination regulation, 208–209 Sp1, lipopolysaccharide signaling in macrophages, 20 Spatzel, Toll interaction, 2 Spleen, chemokine and receptor expression, 77–80 ST2, see T1 Stat6, T helper cell differentiation role, 241

T T1, T helper cell function, 249 TAK1, interleukin-1 receptor signaling, 4–5 Taxol binding proteins, 26–27 lipopolysaccharide mimetic activity, 26 TLR4 binding, 26 TEA element, immunoglobulin heavy chain gene assembly role, 196 Tetraspans, T helper cell function, 250 T cell ␣␤ T cell recognition of antigens, 284 activation, 127

336

INDEX

T cell (continued) antigen recognition, see Major histocompatibility complex molecules B cell encounters and chemokines, 141–143 CD8+ cells autoimmune diabetes role, 236 cytokine production, 236 DNA microarray studies of differentially-expressed genes humans, 252 mice, 252–253 overview, 250–251 table of genes, 251 subsets, 236 virus response, 236–237 compartmental homing within secondary lymphoid organs microarchitecture formation role lymphotoxins and receptors, 131–133 tumor necrosis factor and receptor, 131–133 T cell-rich areas, 130 effector subsets, 233–234 eosinophil interactions, 298–299, 315–316 gastrointestinal cell characteristics, 318–319 ␥ ␦ T cell function, 238 memory/effector cell migration, see also T helper cell CCR4 role, 146–147 CCR6 role, 146–147 CCR7 role, 145–147 features of cells, 143–145 secondary lymphoid organs, 145 NK1 cell function, 237–238 overexpression effects on lamina propria eosinophils, 305–309 V(D)J rearrangements, see V(D)J recombination T helper cell antibody production role, 238–239 chemokine and receptor expression CCR3, 82, 245–246 CCR4, 82–83, 246 CCR5, 81–82, 244–245 CCR7, 83–84, 244 CCR8, 83 CXCR3, 81–82, 244 CXCR5, 84–85, 239, 247–248 follicular T helper cell migration, 247–248 inhibitors of receptors, 246–247

intervention therapy, 92 overview, 242–244 receptor expression by cell type, 80–81 Th1 responses, 244–245 Th1/Th2 polarization role, 90, 140–141, 246 Th2 responses, 245–246 classification by cytokine production, 80, 91, 140, 234–235 differentiation factors antigen dose, 240 interleukin-1, 240 interleukin-4, 239–240 interleukin-12, 239–240 interleukin-18, 240 transcription factors c-Maf, 241 GATA-3, 241 identification, 240–241 Stat6, 241 Th1 factors, 241–242 DNA microarray studies of differentially-expressed genes humans, 252 mice, 252–253 overview, 250–251 table of genes, 251 functions of secreted cytokines, 234–236 molecules distinguishing effector function ICOS, 248–249 interleukin-18 and receptor, 249–250 T1, 249 tetraspans, 250 Thymus chemokine and receptor expression, 75–77, 121–123 hematopoietic stem cell homing, 119–121 TECK and thymocyte migration, 122–123 TLRs, see Toll-like receptors TNF, see Tumor necrosis factor Toll Dif signaling, 3 domains, 2 Dorsal activation, 2–3 18W homology, 3 receptor human homologs, see Toll-like receptors innate immunity role in Drosophila, 2–4 interleukin-1 receptor signaling homology, 7

INDEX

Relish signaling, 3 Spatzel interaction, 2 Toll-like receptors (TLRs) classification and discovery, 7–8, 35 dendritic cell expression of TLR3, 8 gene loci, 7–8 host resistance role in microbial infection, 34–35 interleukin-1 receptor signaling homology, 9 microbial cell wall component recognition CpG motifs, 33–34 Gram-positive bacteria, 30–31 heat shock protein response, 32–33 lipoproteins, 2 mycobacteria, 31 zymosan, 32 regulation of expression, 9 TLR2 lipopolysaccharide signaling, 29–30 TLR4 lipopolysaccharide signaling, 21–24 Taxol binding, 26 TRAF6, interleukin-1 receptor signaling, 4–7 Tumor necrosis factor (TNF), lymphocyte compartmental homing role within secondary lymphoid organs, 131–133

V V(D)J recombination antigen receptor loci structure, 170, 172 transcriptional control elements and sterile transcripts, 172–174 chromatin structure and accessibility DNA methylation of CpG motifs hypomethylation and transcription, 184–186 Ig␬ locus methylation, 185 methyl cytosine binding protein function, 183–184 methyltransferases, 184 DNase sensitivity and recombination, 186–188 ␤-globin locus transcription cis-acting elements as assembly platforms, 182–183 histone acetylation, 187–188 linking model, 193 looping model, 192

337 model for V(D)J recombination, 179 organization and expression, 182 heterochromatin versus euchromatin, 181 high-mobility group protein effects on RAG cleavage, 188–189 histone acetylation, 188 interchromatin granules and splicing factors, 181 nucleosome remodeling complexes, 189–190 structure, 179 promoter exposure, 189 sterile transcripts, 190–191 transcription site visualization, 182 developmental pattern of recombination events, 172 DNA recognition and cleavage by RAG proteins, 174–176 end processing and joining, 176–177 errors in lymphoid malignancy, 170 immunoglobulin heavy chain gene assembly and cis-acting control elements D-to-J rearrangement, 194–196, 199–200 KI/KII region, 197 MARs knockout effects, 194, 196–197 ordered rearrangement and allelic exclusion, 199–201 overview, 193–194 PD␤1 element, 195–196 PDQ52 element, 194–195, 209 TEA element, 196 V-to-DJ recombination, 197–200 V-to-J recombination, 196–197 overview, 169 RAG protein expression regulation, 177–179 recombination signal sequences accessibility modeling, 213–215 chromatin unfolding for access, 181, 189 recognition, 172 restrictions on productive interactions, 211–213 substrate matching, 213 synapsis of endogenous sequences, mechanisms, 209–211 transcription and recombination dependence, 191 12/23 rule, 212 regulation BSAP, 206–207 E2A, 201–203

338 V(D)J recombination (continued) early B cell factor, 206 ETS proteins, 203–204 Ig␤, 208 Ikaros, 208 interleukin-7 receptor, 207 ␮E3-binding proteins, 204–205 Oct1, 205–206 Oct2, 205–206

INDEX

overview, 170 PU.1, 203–204 Sox-4, 208–209 T cell development, 120, 169

Z Zymosan, Toll-like receptor recognition, 32

CONTENTS OF RECENT VOLUMES

Volume 74

¨ BARBEL RAUPACH, AND STEFAN H. E. KAUFMANN

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

The Cytoskeleton in Lymphocyte Signaling A. BAUCH, F. W. ALT, G. R. CRABTREE, AND S. B. SNAPPER

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

TGF-␤ Signaling by Smad Proteins KOHEI MIYAZONO, PETER TEN DIJKE, AND CARL-HENRIK HELDIN

Receptor Editing in B Cells DAVID NEMAZEE

MHC Class II-Restricted Antigen Processing and Presentation JEAN PIETERS

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

T-Cell Receptor Crossreactivity and Autoimmune Disease HARVEY CANTOR

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

Strategies for Immunotherapy of Cancer CORNELIS J. M. MELIEY, RENE E. M. TOES, JAN PAUL MEDEMA, SJOERD H. VAN DER BURG, FERRY OSSENDORP, AND RIENK OFFRINGA Tyrosine Kinase Activation in the Decision between Growth, Differentiation, and Death Responses Initiated from the B Cell Antigen Receptor ROBERT C. HSUEH AND RICHARD H. SCHEUERMANN

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

Volume 75 The 3′ IgH Regulatory Region: A Complex Structure in a Search for a Function AHMED AMINE KHAMLICHI, ERIC PINAUD, CATHERINE DECOURT,

Exploiting the Immune System: Toward New Vaccines against Intracellular Bacteria ¨ JURGEN HESS, ULRICH SCHAIBLE,

339

340

CONTENTS OF RECENT VOLUMES

CHRISTINE CHAUVEAU, AND MICHEL COGNE´ INDEX

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

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction

S. CELESTE POSEY MORLEY AND BARBARA E. BIERER Raft Membrane Domains and Immunoreceptor Functions THOMAS HARDER Human Basophils: Mediator Release and Cytokine Production JOHN T. SCHROEDER , DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN Btk and BLNK in B Cell Development SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2 s MAKOTO MURAKAMI AND ICHIRO KUDO The Antiviral Activity of Antibodies in Vitro and in Vivo PAUL W. H. I. PARREN AND DENNIS R. BURTON Mouse Models of Allergic Airway Disease CLARE M. LLOYD, JOSE-ANGEL GONZALO, ANTHONY J. COYLE, AND JOSE-CARLOS GUTIERREZ-RAMOS Selected Comparison of Immune and Nervous System Development JEROLD CHUN INDEX

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